Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene

We have investigated the mechanism that patterns dopamine expression among Caenorhabditis elegans male ray sensory neurons. Dopamine is expressed by the A-type sensory neurons in three out of the nine pairs of rays. We used expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway, the DBL-1 pathway, dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability.     

The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine

The functional role of the ABC transporter PGP-2 from the nematode Caenorhabditis elegans has been studied by combining phenotype analyses of pgp-2 deletion mutants or pgp-2 RNAi treated worms with reporter gene studies using a pgp-2::GFP construct. pgp-2 mutants showed a strong reduction of lipid stores. In addition, we found that in the case of the pgp-2 mutant or after pgp-2 RNAi the worms were unable to perform pinocytosis and to acidify intestinal lysosomes. Especially under cholesterol-restricted conditions, the viability of the mutant was reduced. Surprisingly, the chemosensory AWA neurons in the head region were identified as expression sites by reporter gene studies. These neurons are known to be involved in attraction behaviour towards odorants associated with potential food bacteria. Our results imply that PGP-2 is involved in a signalling process that connects sensory inputs to intestinal functions, possibly by influencing acidification of intestinal lysosomes, which in turn may affect pinocytosis and lipid storage.     

Homeotic genes influence the axonal pathway of a Drosophila embryonic sensory neuron

Each abdominal hemisegment of the Drosophila embryo has two sensory neurons intimately associated with a tracheal branch. During embryogenesis, the axons of these sensory neurons, termed the v'td2 neurons, enter the CNS and grow toward the brain with a distinctive pathway change in the third thoracic neuromere. We show that the axons use guidance cues that are under control of the bithorax gene complex (BX-C). Pathway defects in mutants suggest that a drop in Ultrabithorax expression permits the pathway change in the T3 neuromere, while combined Ultrabithorax and abdominal-A expression represses it in the abdominal neuromeres. We propose that the axons do not respond to a particular segmental identity in forming the pathway change; rather they respond to pathfinding cues that come about as a result of a drop in BX-C expression along the antero-posterior axis of the CNS.     

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.     

A genetic programme for neuronal connectivity

What is the nature of the genetic programme that allows neurons to extend their axons and connect to other neurons with a high degree of specificity? Work on the sensory neurons of the fly has shown how the control of neuronal identity is embedded in the general developmental programme of the organism. The ongoing analysis of pathfinding mutants suggests plausible mechanisms for the translation of neuronal identity into axonal behaviour.     

Drosophila Fatty Acid Taste Signals through the PLC Pathway in Sugar-Sensing Neurons

Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.     

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aβ-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ～50% of the nodose neurons. GFRα2 was expressed in ～50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.     

Molecular neurogenetics of chemotaxis and thermotaxis in the nematode Caenorhabditis elegans

Chemotaxis and thermotaxis in Caenorhabditis elegans are based on the chemical senses (smell and taste) and the thermal sense, respectively, which are important for the life of the animal. Laser ablation experiments have allowed identification of sensory neurons and some interneurons required for these senses. Many mutants that exhibit various abnormalies have been isolated and analyzed. These studies have predicted novel signaling pathways whose components include a putative odorant specific transmembrane receptor (ODR-10) and a cyclic nucleotide-gated channel (TAX-4/TAX-2) functioning in taste and thermosensation as well as in smell. The emerging picture of the mechanisms of sensory transduction in C. elegans seems to be basically similar to what is known of visual and olfactory sensory transduction in vertebrates. Thus, molecular and cellular analyses of chemotaxis and thermotaxis in C. elegans have proved useful and will continue to provide significant implications for the molecular basis of sensory systems in higher animals.     

Mechanosensitive enteric neurons in the myenteric plexus of the mouse intestine

Background

Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.

Methodology/Principal Findings

We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca⁺⁺/high Mg⁺⁺. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.

Conclusions/Significance

We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.     

Identification of the AFD neuron as the site of action of the CREB protein in Caenorhabditis elegans thermotaxis

Behaviour is a consequence of computation in neural circuits composed of massive synaptic connections among sensory neurons and interneurons. The cyclic AMP response element-binding protein (CREB) responsible for learning and memory is expressed in almost all neurons. Nevertheless, we find that the Caenorhabditis elegans CREB orthologue, CRH-1, is only required in the single bilateral thermosensory neuron AFD, for a memory-related behaviour. Restoration of CRH-1 in AFD of CREB-depleted crh-1 mutants rescues its thermotactic defect, whereas restorations in other neurons do not. In calcium-imaging analyses, the AFD neurons of CREB-depleted crh-1 mutants exhibit an abnormal response to temperature increase. We present a new platform for analysing the mechanism of behavioural memory at single-cellular resolution within the neural circuit.     

Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice

To investigate the role of brain-derived neurotrophic factor (BDNF) in differentiation of cranial sensory neurons in vivo, we analyzed development of nodose (NG), petrosal (PG), and vestibular (VG) ganglion cells in genetically engineered mice carrying null mutations in the genes encoding BDNF and the proapoptotic Bcl-2 homolog Bax. In bax(-/-) mutants, ganglion cell numbers were increased significantly compared to wild-type animals, indicating that naturally occurring cell death in these ganglia is regulated by Bax signaling. Analysis of bdnf(-/-)bax(-/-) mutants revealed that, although the Bax null mutation completely rescued cell loss in the absence of BDNF, it did not rescue the lethality of the BDNF null phenotype. Moreover, despite rescue of BDNF-dependent neurons by the bax null mutation, sensory target innervation was abnormal in double null mutants. Vagal sensory innervation to baroreceptor regions of the cardiac outflow tract was completely absent, and the density of vestibular sensory innervation to the cristae organs was markedly decreased, compared to wild-type controls. Moreover, vestibular afferents failed to selectively innervate their hair cell targets within the cristae organs in the double mutants. These innervation failures occurred despite successful navigation of sensory fibers to the peripheral field, demonstrating that BDNF is required locally for afferent ingrowth into target tissues. In addition, the bax null mutation failed to rescue expression of the dopaminergic phenotype in a subset of NG and PG neurons. These data demonstrate that BDNF signaling is required not only to support survival of cranial sensory neurons, but also to regulate local growth of afferent fibers into target tissues and, in some cells, transmitter phenotypic expression is required.     

The homeobox gene Gsh2 is required for retinoid production in the embryonic mouse telencephalon

We have examined the role of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer DARPP-32 neurons than wild types. We show that the expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, we found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Previously, we had shown that removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. As RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, we examined whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, our results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon.     

Organization of motor neurons innervating the proboscis musculature in Drosophila melanogaster meigen (diptera : Drosophilidae)

The present study addresses the question as to how the motor neurons involved in feeding in Drosophila melanogaster Meigen (Diptera : Drosophilidae) are organized. The motor neurons have been visualized both by Golgi-silver impregnation and by intramuscular injection of horseradish peroxidase, and analyzed in light of the existing information on taste sensory system and the feeding behaviour. The motor neurons have been broadly classified into the following types: labial nerve motor neurons, pharyngeal nerve motor neurons, and accessory pharyngeal nerve motor neurons, depending on the nerve through which their axons exit. The arborization of all the motor neurons is confined to the suboesophageal ganglion (SOG). All of them have predominantly ipsilateral and some contralateral arborizations. Their dendrites predominantly occupy the ventral region of the neuropil of the SOG and partially overlap the taste sensory projections, thereby providing an opportunity for interaction with the taste sensory input. The pharyngeal motor neurons arborize more extensively in the ventral tritocerebram, anteroventral. and mid-ventral neuropil, whereas the dendritic fields of labial motor neurons are confined to the mid-ventral neuropil. There is a functional segregation in motor neuron organization: cibarial muscles involved in sucking are innervated by pharyngeal motor neurons, while the proboscis muscles involved in positioning, of the proboscis are innervated by labial motor neurons. We have also observed projections of the stomodaeal nerve in the tritocerebrum.     

The wiring of Grueneberg ganglion axons is dependent on neuropilin 1

The Grueneberg ganglion is a specialized olfactory sensor. In mice, its activation induces freezing behavior. The topographical map corresponding to the central projections of its sensory axons is poorly defined, as well as the guidance molecules involved in its establishment. We took a transgenic approach to label exclusively Grueneberg sensory neurons and their axonal projections. We observed that a stereotyped convergence map in a series of coalescent neuropil-rich structures is already present at birth. These structures are part of a peculiar and complex neuronal circuit, composed of a chain of glomeruli organized in a necklace pattern that entirely surrounds the trunk of the olfactory bulb. We found that the necklace chain is composed of two different sets of glomeruli: one exclusively innervated by Grueneberg ganglion neurons, the other by axonal inputs from the main olfactory neuroepithelium. Combining the transgenic Grueneberg reporter mouse with a conditional null genetic approach, we then show that the axonal wiring of Grueneberg neurons is dependent on neuropilin 1 expression. Neuropilin 1-deficient Grueneberg axonal projections lose their strict and characteristic avoidance of vomeronasal glomeruli, glomeruli that are innervated by secondary neurons expressing the repulsive guidance cue and main neuropilin 1 ligand Sema3a. Taken together, our observations represent a first step in the understanding of the circuitry and the coding strategy used by the Grueneberg system.