Massimo A. Hilliard

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca2+ responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper‐ and hypo‐osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC‐1, a G‐protein γ‐subunit expressed specifically in chemosensory neurons. We hypothesize that G‐protein γ‐subunit heterogeneity provides a mechanism for repellent‐specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

C. elegans Responds to Chemical Repellents by Integrating Sensory Inputs from the Head and the Tail

The phasmids are bilateral sensory organs located in the tail of Caenorhabditis elegans and other nematodes. The similar structures of the phasmids and the amphid chemosensory organs in the head have long suggested a chemosensory function for the phasmids. However, the PHA and PHB phasmid neurons are not required for chemotaxis or for dauer formation, and no direct proof of a chemosensory function of the phasmids has been obtained. C. elegans avoids toxic chemicals by reversing its movement, and this behavior is mediated by sensory neurons of the amphid, particularly, the ASH neurons. Here we show that the PHA and PHB phasmid neurons function as chemosensory cells that negatively modulate reversals to repellents. The antagonistic activity of head and tail sensory neurons is integrated to generate appropriate escape behaviors: detection of a repellent by head neurons mediates reversals, which are suppressed by antagonistic inputs from tail neurons. Our results suggest that C. elegans senses repellents by defining a head-to-tail spatial map of the chemical environment.

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans.

An animals ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water‐soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein α subunits GPA‐3 and ODR‐3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr‐3 can only be appreciated in the absence of gpa‐3. We identified and cloned a new gene, qui‐1, necessary for quinine and SDS avoidance. qui‐1 codes for a novel protein with WD‐40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.

A Core Metabolic Enzyme Mediates Resistance to Phosphine Gas

Dissecting Phosphine Resistance Worldwide populations of pest insects—such as the lesser grain borer, Rhyzopertha dominica, and the rust-red flour beetle, Tribolium castaneum—have become highly resistant to the fumigant phosphine, providing a potential threat to global food security. The nematode, Caenorhabditis elegans is vulnerable to phosphine, but phosphine-resistant strains are known. Schlipalius et al. (p. 807) show that mutations in the delta-1-pyrroline-5-carboxylate dehydrogenase and dihydrolipoamide dehydrogenase (dld-1) genes both give rise to phosphine resistance in C. elegans. Phosphine resistance mutants in R. dominica, and T. castaneum also map to the dld-1 gene, which codes for a core metabolic enzyme. These mutants are, however, hypersensitive to arsenic, mimics of which might thus synergize with phosphine. Mutations in a lipoic acid metabolism enzyme confer resistance to phosphine but also result in sensitivity to arsenite. Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein–protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Axonal degeneration and regeneration: a mechanistic tug-of-war

Axonal degeneration is a common hallmark of both nerve injury and many neurodegenerative conditions, including motor neuron disease, glaucoma, and Parkinson’s, Alzheimer’s, and Huntington’s diseases. Degeneration of the axonal compartment is distinct from neuronal cell death, and often precedes or is associated with the appearance of the symptoms of the disease. A complementary process is the regeneration of the axon, which is commonly observed following nerve injury in many invertebrate neurons and in a number of vertebrate neurons of the PNS. Important discoveries, together with innovative imaging techniques, are now paving the way towards a better understanding of the dynamics and molecular mechanisms underlying these two processes. In this study, I will discuss these recent findings, focusing on the balance between axonal degeneration and regeneration.

Big ideas for small brains: what can psychiatry learn from worms, flies, bees and fish?

While the research community has accepted the value of rodent models as informative research platforms, there is less awareness of the utility of other small vertebrate and invertebrate animal models. Neuroscience is increasingly turning to smaller, non-rodent models to understand mechanisms related to neuropsychiatric disorders. Although they can never replace clinical research, there is much to be learnt from ‘small brains’. In particular, these species can offer flexible genetic ‘tool kits’ that can be used to explore the expression and function of candidate genes in different brain regions. Very small animals also offer efficiencies with respect to high-throughput screening programs. This review provides a concise overview of the utility of models based on worm, fruit fly, honeybee and zebrafish. Although these species may have small brains, they offer the neuropsychiatric research community opportunities to explore some of the most important research questions in our field.

Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons

Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In Caenorhabditis elegans following laser‐induced axotomy, the axon still attached to the cell body is able to regrow and reconnect with its separated distal fragment. Here we show that reconnection of separated axon fragments during regeneration of C. elegans mechanosensory neurons occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re‐establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. We also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re‐growing axon recognizing its own separated distal fragment. Thus, efficient axonal regeneration can occur by selective reconnection and fusion of separated axonal fragments beyond an injury site, with restoration of the damaged neuronal tract. Developmental Dynamics 240:1365–1372, 2011.

EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway

Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion. PSR-1 functions cell-autonomously in the regrowing neuron and, instead of acting in its canonical signalling pathway, acts in a parallel phagocytic pathway that includes the transthyretin protein TTR-52, as well as CED-7, NRF-5 and CED-6 (refs 9, 10, 11, 12). We show that TTR-52 binds to PS exposed on the injured axon, and can restore fusion several hours after injury. We propose that PS functions as a ‘save-me’ signal for the distal fragment, allowing conserved apoptotic cell clearance molecules to function in re-establishing axonal integrity during regeneration of the nervous system.

Rapid and Permanent Neuronal Inactivation In Vivo via Subcellular Generation of Reactive Oxygen with the Use of KillerRed

Inactivation of selected neurons in vivo can define their contribution to specific developmental outcomes, circuit functions, and behaviors. Here, we show that the optogenetic tool KillerRed selectively, rapidly, and permanently inactivates different classes of neurons in C. elegans in response to a single light stimulus, through the generation of reactive oxygen species (ROS). Ablation scales from individual neurons in single animals to multiple neurons in populations and can be applied to freely behaving animals. Using spatially restricted illumination, we demonstrate that localized KillerRed activation in either the cell body or the axon triggers neuronal degeneration and death of the targeted cell. Finally, targeting KillerRed to mitochondria results in organelle fragmentation without killing the cell, in contrast to the cell death observed when KillerRed is targeted to the plasma membrane. We expect this genetic tool to have wide-ranging applications in studies of circuit function and subcellular responses to ROS.