Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh. Our objective was to determine whether elimination of ACh synthesis during embryogenesis alters prenatal viability, intestinal function, the neurotransmitter complement, and the microbiome. Conditional deletion of choline acetyltransferase ( ChAT), the ACh synthetic enzyme, in neural crest-derived neurons ( ChAT-Null) was performed. Survival, ChAT activity, gut motility, and the microbiome were studied. ChAT was conditionally deleted in ENS neural crest-derived cells. Despite ChAT absence, mice were born live and survived the first 2 wk. They failed to gain significant weight in the third postnatal week, dying between postnatal d 18 and 30. Small intestinal transit of carmine red was 50% slower in ChAT-Nulls vs. WT and ChAT- Het. The colons of many neonatal ChAT-Null mice contained compacted feces, suggesting dysmotility. Microbiome analysis revealed dysbiosis in ChAT-Null mice. Developmental deletion of ChAT activity in enteric neurons results in proximal gastrointestinal tract dysmotility, critically diminished colonic transit, failure to thrive, intestinal dysbiosis, and death. ACh is necessary for sustained gut motility and survival of neonatal mice after weaning.-Johnson, C. D., Barlow-Anacker, A. J., Pierre, J. F., Touw, K., Erickson, C. S., Furness, J. B., Epstein, M. L., Gosain, A. Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Tubby mice 1 and individuals with Bardet-Biedl syndrome 2 have defects in ciliated neuron function and obesity, suggesting an as-yet unknown metabolic signaling axis from ciliated neurons to fat storage tissues. Here we show coordinate regulation of Caenorhabditis elegans fat storage by orthologues of these genes acting in ciliated neurons and by a 3-ketoacyl-coA thiolase (encoded by kat-1 ) that acts in fat storage tissue. A genetic screen for markedly enhanced fat storage in tub-1 mutants led to the isolation only of kat-1 alleles, which impair fatty acid β-oxidation. kat-1 acts in the intestine, the major C. elegans fat storage tissue, and is transcriptionally upregulated in animals with high fat storage. A genetic screen for synergistic increase in fat storage of a kat-1 mutant identified bbs-1 . bbs-1 acts in 15 ciliated neurons that are poised to sense external and internal nutrient levels, supporting a model in which bbs-1 and tub-1 in ciliated neurons form part of an ancient, conserved neuroendocrine axis. This pathway also includes genes encoding intraflagellar transport proteins and cyclic nucleotide gated channels, demonstrating that C. elegans fat storage is under polygenic control.

Background: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development.Results: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type.Conclusions: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.

Emotions coordinate our behavior and physiological states during survival-salient events and pleasurable interactions. Even though we are often consciously aware of our current emotional state, such as anger or happiness, the mechanisms giving ...Emotions are often felt in the body, and somatosensory feedback has been proposed to trigger conscious emotional experiences. Here we reveal maps of bodily sensations associated with different emotions using a unique topographical self-report method. In ...

Use of the human genome sequence in disease therapy will require efficient identification of disease-causing and disease-associated genes with functions that are amenable to pharmacological manipulation. The validation and development of such genes as therapeutic targets requires information about both the genes' functions and the biochemical pathways in which they participate. One powerful means of obtaining such information is the study of homologous genes in model organisms amenable to laboratory manipulation. Among model organisms the nematode Caenorhabditis elegans offers several advantages, including well-established techniques for genetic and experimental manipulation and the first completed genome sequence for a multicellular organism. Molecular genetic experiments using C. elegans can contribute at several levels to drug discovery programs, from elucidation of genetic functions and pathways to the validation of candidate targets. Additionally, the ease of culture allows adaptation of the nematode for use in high-throughput chemical screens for the identification of lead compounds in drug development.

The human PTEN tumor suppressor gene is mutated in a wide variety of sporadic tumors. To determine the function of PTEN in vivo we have studied a PTEN homolog in Caenorhabditis elegans. We have generated a strong loss-of-function allele of the PTEN homolog and shown that the deficient strain is unable to enter dauer diapause. An insulin-like phosphatidylinositol 3-OH kinase (PI3'K) signaling pathway regulates dauer-stage entry. Mutations in either the daf-2 insulin receptor-like (IRL) gene or the age-1 encoded PI3'K catalytic subunit homolog cause constitutive dauer formation and also affect the life span, brood size, and metabolism of nondauer animals. Strikingly, loss-of-function mutations in the age-1 PI3'K and daf-2 IRL genes are suppressed by loss-of-function mutations in the PTEN homolog. We establish that the PTEN homolog is encoded by daf-18, a previously uncloned gene that has been shown to interact genetically with the DAF-2 IRL AGE-1 PI3'K signaling pathway. This interaction provides clear genetic evidence that PTEN acts to antagonize PI3'K function in vivo. Given the conservation of the PI3'K signaling pathway between C. elegans and mammals, the analysis of daf-18 PTEN mutant nematodes should shed light on the role of human PTEN in the etiology of metabolic disease, aging, and cancer.

We have used immunocytochemical techniques to investigate the distribution of serotonin-like immunoreactivity in the nematode Ascaris suum. Antisera raised against serotonin (5-hydroxytryptamine, 5-HT) conjugated to bovine serum albumin (BSA) labelled a pair of neurons in the pharynx of both sexes and five cells in the ventral cord of the male tail. The labelling was blocked by 5-HT or by 5-HT conjugated to BSA. The 5-HT-immunoreactive cells in the pharynx resemble neurosecretory cells and are probably homologous to the neurosecretory motor neurons (NSM) in Caenorhabditis elegans; the cells in the male tail appear to be motor neurons that are homologous to CP neurons in C, elegans. Other cells that stain with 5-HT antisera have been observed in C. elegans but are not seen in Ascaris. (C) 1996 Wiley-Liss, Inc.

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.

Choline acetyltransferase (ChAT; EC 2.3.1.6) and acetylcholinesterase (AChE; EC 3.1.1.7) activities were measured in cynomolgus monkey ciliary muscle 1 month and 6 or more months after ciliary ganglionectomy (CG) or post-ganglionic ciliary neurectomy (PCN). ChAT activity was undetectable and AChE activity was elevated 1 month after CG or PCN, while both averaged about 30% of normal levels 6 or more months after denervation. Four out of six eyes reinnervated by functional criteria 6-12 months after CG or PCN. In one of the two remaining eyes permanently denervated, ChAT was absent from the ciliary muscle. In the other, ChAT activity was about 50% of normal, similar to the reinnervated eyes, but the regenerated cholinergic nerves were not approximated to the ciliary muscle fibers.

In C. elegans, the newly identified ace-3 is the third gene affecting acetylcholinesterase (ACNE) activity. ace-3 II specifically affects class C ACNE and is unlinked to ace-1 X or ace-2 I, which affect the other two AChE classes (A and B, respectively). Strains homozygous for an ace-3 mutation have no apparent behavioral or developmental defect; ace-1 ace-3 and ace-2 ace-3 double mutants are also nearly wild type. In contrast, ace-1 ace-2 ace-3 triple mutant animals are paralyzed and developmentally arrested; their embryonic development is relatively unimpaired, but they are unable to grow beyond the hatching stage. Based on the analysis of genetic mosaics, we conclude that in the absence of ace-2 and ace-3 function, the expression of ace-1(+) in muscle cells, but not in neurons, is essential for postembryonic viability.

Monoclonal antibodies (mAbs) to chick choline acetyltransferase (ChAT) were obtained from mouse-hybridoma cultures after immunization with partially purified enzyme isolated from optic lobes. Antibodies that bound active enzyme were detected in 11 hybridoma cultures. The mAbs showed cross-reactivity to ChAT from quail and beef but not to ChAT from several other species. An affinity column prepared with one of the mAbs was used to purify ChAT to apparent homogeneity. Polyclonal antiserum to mAb affinity-purified ChAT was produced in a rabbit. This antiserum inhibited chick ChAT activity and quantitatively precipitated ChAT activity from solution. On immunoblots, the antiserum stained ChAT and two other proteins. After preadsorption of the antiserum with effluent from the mAb affinity column, the antiserum became monospecific for ChAT. This antiserum was useful for immunocytochemical localization of ChAT, it selectively stained neuronal cell bodies in chick spinal cord and rat brain at locations known to contain cholinergic neurons.

Abstract: Extracts of the nematode Caenorhabditis elegans contain five molecular forms of acetylcholinesterase (AChE) activity that can be separated by a combination of selective solubilization, velocity sedimentation, and ion‐exchange chromatography. These are called form IA (5.2s), form IB (4.9.s), form II (6.7s), form III (11.3s), and form IV (13.0s). All except form III are present in significant amounts in rapidly prepared extracts and are probably native; form III is probably derived autolytically from form IV. Most of forms IA and IB can be solubilized by repeated extractions without detergent, whereas forms II, III, and IV require detergent for effective solubilization and may therefore be membrane‐bound. High salt concentrations are not required for, and do not aid in, the solubilization of these forms. For all forms, molecular weights and frictional ratios have been estimated by a combination of gel permeation chromatography and velocity sedimentations in both H2O and D2O. The molecular weight estimates range from 83,000 to 357,000 and only form II shows extensive asymmetry. The separated forms have been characterized with respect to substrate affinity, substrate specificity, inhibitor sensitivity, thermal inactivation, and detergent sensitivity. Judging by these properties, C. elegans is like other invertebrates in that none of its cholinesterase forms resembles either the “true” or the “pseudo” cholinesterase of vertebrates. However, internal comparison of the C. elegans forms clearly distinguishes forms IA, III, and IV as a group from forms IB and II; the former are therefore designated “class A” forms, the latter “class B” forms. Genetic evidence indicates that separate genes control class A and class B forms, and that these two classes overlap functionally. Several factors, including kinetic properties, molecular asymmetry, molecular size, and solubility, all suggest that a molecular model of the multiple cholinesterase forms observed in vertebrate electric organs probably does not apply in C. elegans. Potential functional roles and subunit structures of the multiple AChE forms within each C. elegans class are discussed.

The similarities between the tail of asymmetric acetylcholinesterase (AcChE) and collagen prompted us to investigate if asymmetric AcChE, like collagen, can interact with fibronectin. Gradient centrifugation studies revealed that asymmetric, but not globular, AcChE bound to fibronectin and could be cross-linked covalently to fibronectin by plasma transglutaminase. The interaction of asymmetric AcChE with fibronectin paralleled the interaction of fibronectin with collagen. These results raise the possibility that fibronectin may be involved in attaching asymmetric AcChE to cell surfaces.