Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map.

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene.

Mutations of the caenorhabditis elegans brain-specific inorganic phosphate transporter eat-4 affect habituation of the tap-withdrawal response without affecting the response itself.

The studies reported here were designed to investigate the role of the mutation eat-4 in the response to tap and in habituation in the nematode Caenorhabditis elegans. In C. elegans eat-4 has been found to affect a number of glutamatergic pathways. It has been hypothesized to positively regulate glutaminase activity and therefore glutamatergic neurotransmission. In the eat-4(ky5) loss-of-function worms, there is presumably insufficient glutamate available for sustained transmission. In the experiments reported here eat-4 worms showed no differences from wild-type in the magnitude of response to a single tap, indicating that the neural circuit underlying the response was intact and functional in the mutant worms. However, when eat-4 worms were given repeated taps the resulting habituation was different from that seen in wild-type worms: eat-4 worms habituate more rapidly and recover more slowly than wild-type worms at all interstimulus intervals tested. In addition, eat-4 worms do not show dishabituation. The same transgene rescues pharyngeal activity defects and both the habituation and dishabituation deficits seen in the eat-4 worms. Our results suggest that neurotransmitter regulation plays a role in habituation and may play a role in dishabituation.

CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans.

Forward genetic screens using novel assays of nematode chemotaxis to soluble compounds identified three independent transposon-insertion mutations in the gene encoding the Caenorhabditis elegans dynein heavy chain (DHC) 1b isoform. These disruptions were mapped and cloned using a newly developed PCR-based transposon display. The mutations were demonstrated to be allelic to the che-3 genetic locus. This isoform of dynein shows temporally and spatially restricted expression in ciliated sensory neurons, and mutants show progressive developmental defects of the chemosensory cilia. These results are consistent with a role for this motor protein in the process of intraflagellar transport; DHC 1b acts in concert with a number of other proteins to establish and maintain the structural integrity of the ciliated sensory endings in C. elegans.

Developmental analysis of habituation in the Nematode C. elegans.

Habituation and spontaneous recovery from habituation to tap were studied across development in C. elegans. Unlike adult worms, larval worms do not consistently swim backwards to tap, but reverse half of the time and accelerate forward half of the time. In adult worms, the tap response is produced by the integration of two competing circuits: The head touch circuit, mediated by ALM and AVM sensory neurons, produces backward movement (reversals); the tail touch circuit, mediated by PLM neurons, produces forward movement (accelerations). Because the response type changes over development, habituation of each of the subcircuits was studied separately. Habituation of the head touch circuit was studied by laser ablating PLM, and habituation of the tail circuit was studied by ablating ALM. Worms were tested at six stages of development at either 10- or 60-s interstimulus intervals. All stages of development showed normal habituation and spontaneous recovery at both interstimulus intervals.

Effects of tap withdrawal response habituation on other withdrawal behaviors: the localization of habituation in the nematode Caenorhabditis elegans.

Four experiments were conducted to identify the possible loci of habituation of the nematode tap withdrawal response (TWR) by characterizing the effects of TWR habituation on other nonmechanosensory withdrawal behaviors that are mediated by overlapping sets of neurons. Experiments 1-2 established behavioral and anatomical relationships between spontaneous and tap-induced backward locomotion in the worm. Experiment 3 demonstrated that habituation of the TWR affected neither the magnitude nor frequency of spontaneous reversal activity. Experiment 4 extended this result to an evoked response: Habituation of the TWR had no effect on reversals evoked by a thermal stimulus. These studies, which show that the loci of change associated with habituation of the TWR are presynaptic to the interneurons and motor neurons that control locomotion, probably distributed among the mechanosensory neurons, illustrate that a complete understanding of plasticity requires a knowledge of both the anatomical and molecular substrates of change.

The integration of antagonistic reflexes revealed by laser ablation of identified neurons determines habituation kinetics of the Caenorhabditis elegans tap withdrawal response.

Previously, we described the circuitry that underlies the tap withdrawal response of the nematode Caenorhabditis elegans. In response to a light mechanosensory stimulus a worm will withdraw, usually by initiating backward locomotion, but occasionally with increased forward locomotion. The form of an animal's response is a product of the balance between two antagonistic reflexes: backward locomotion (reversals) triggered by anterior mechanosensory input and forward locomotion (accelerations) triggered by posterior mechanosensory input. During habituation of this reflex, the frequency of forward and backward locomotion in response to tap is modulated by both experience and interstimulus interval; reversals are more frequent early in a habituation series and at longer Inter stimulus intervals. Single-cell laser microsurgery was used to study each of the subcomponents of the intact behavior during habituation training. Groups of intact or laser-ablated worms were habituated at either a 10-s or a 60-s inter stimulus interval and the kinetics of habituation in each group was analyzed. We demonstrate that each component of the behavior habituates and does so with kinetics that are consistent with the decrement observed in the intact animal.

Recovery from habituation in Caenorhabditis elegans is dependent on interstimulus interval and not habituation kinetics.

The habituation of the tap withdrawal reflex of Caenorhabditis elegans was assessed to determine whether the kinetics of recovery from habituation were dependent on the interstimulus interval (ISI) used during habituation training, or alternately, on the rate and asymptotic level of habituation produced at a given ISI. Two groups of intact animals were trained at either a 10-s (CON10) or a 60-s (CON60) ISI. Laser ablation was used to alter the habituation kinetics in one further group of animals (PLM10), independent of ISI. Although the PLM10 animals trained at a 10-s ISI habituated like CON60 worms, the recovery from habituation of the PLM10 animals very closely resembled the recovery of the CON10 worms. Thus recovery kinetics are dictated by consequences of a given ISI, which do not impact upon habituation rate and asymptote. This suggests the recruitment of multiple ISI-dependent processes during habituation in C. elegans.

A dynamic network simulation of the nematode tap withdrawal circuit: predictions concerning synaptic function using behavioral criteria.

The nematode tap withdrawal reflex demonstrates several forms of behavioral plasticity. Although the neural connectivity that supports this behavior is identified (Integration of mechanosensory stimuli in Caenorhabditis elegans, Wicks and Rankin, 1995, J Neurosci 15:2434-2444), the neurotransmitter phenotypes, and hence whether the synapses in the circuit are excitatory or inhibitory, remain uncharacterized. Here we use a novel strategy to predict the polarity configuration, i.e., the array of excitatory and inhibitory connections, of the nematode tap withdrawal circuit using an anatomically and physiologically justifiable dynamic network simulation of that circuit. The output of the modeled circuit was optimized to the behavior of animals, which possessed circuits altered by surgical ablation by exhaustively enumerating an array of synaptic signs that constituted the modeled circuit. All possible polarity configurations were then compared, and a statistical analysis was used to determine whether, for a given synaptic class, a particular polarity was associated with a good fit to behavioral data. The results from four related experiments were used to predict the polarities of seven of the nine cell classes of the tap withdrawal circuit. In addition, the model was used to assess possible roles for two novel mechanosensory integration neurons: DVA and PVD.

Integration of mechanosensory stimuli in Caenorhabditis elegans.

The tap withdrawal reflex in Caenorhabditis elegans demonstrates various forms of nonassociative learning. A first step in determining the cellular mechanisms of this learning is to identify the neuronal circuitry that underlies this reflex. Studies by Chalfie et al. (1985) have defined the touch-circuit that mediates the response to a stimulus related to tap--a light touch. We used the touch circuit as a starting point in the identification of the tap withdrawal circuitry. Here we report the effects of lesions of identified neurons on the tap withdrawal reflex. Ablations of the sensory neurons and interneurons of the touch circuit produce effects on the tap withdrawal response that generally confirm and expand upon the roles of these cells in mechanosensory integration as proposed by Chalfie et al. (1985). However, no role for the LUA interneurons could be identified in the production of the tap withdrawal response. Furthermore, the effects of ablating some neurons outside the touch circuit suggest roles for two of these cells in the integration of the tap withdrawal response. Ablation of either the midline neuron DVA or the PVD neurons resulted in a decrease in both the frequency and magnitude of reversals that were elicited by tap. Additionally, the ablation of either cell decreased the magnitude of accelerations produced by animals in response to tap.

Effect of formulation on the pharmacokinetics and efficacy of doramectin.

The pharmacokinetics of doramectin, a novel avermectin, were evaluated following parenteral administration in a range of oil-based formulations in an attempt to optimise the formulation. Therapeutic and persistent efficacies against Cooperia oncophora were also evaluated. This approach led to the identification of formulations based upon sesame oil and ethyl oleate which gave more prolonged doramectin plasma concentrations with no loss in therapeutic efficacy and improved persistent efficacy following subcutaneous administration to cattle at a dosage of 200 micrograms kg-1. The importance of using both pharmacokinetic and efficacy end points to distinguish between formulations is discussed. All formulations were well tolerated as evidenced by the absence of any reaction to injection either in the form of behavioural responses, injection site swelling or postmortem lesions. Sesame oil with ethyl oleate was the best parenteral vehicle tested for doramectin, allowing the expression of a high level of therapeutic and persistent efficacy and offering the benefit of excellent injection site toleration.

Doramectin--a potent novel endectocide.

Doramectin, 25-cyclohexyl-5-O-demethyl-25-de(l-methylpropyl)avermectin A1a, was selected as the best of a series of novel avermectins prepared by mutational biosynthesis. The primary evaluation of its in vivo antiparasitic activity was carried out using a rat Trichostrongylus colubriformis model and a rabbit Psoroptes cuniculi model. In each case the new avermectin performed favourably relative to dihydroavermectin B1a (DHAVM), the major component of ivermectin. Doramectin was extensively evaluated in cattle using an experimental micelle formulation, proving highly effective in cattle infected with Ostertagia ostertagi, Cooperia oncophora and Dictyocaulus viviparus when administered subcutaneously at 200 micrograms kg-1. The plasma pharmacokinetic characteristics of doramectin in cattle following intravenous administration revealed a plasma half-life of approximately 89 h. In the micelle formulation, doramectin administered subcutaneously at 400 micrograms kg-1 provided persistent activity against infection of cattle with C. oncophora and O. ostertagi for at least 8 and 12 days respectively.

The use of polyamide coatings for selective adsorption control on activated charcoal.

Polymer-coated, activated charcoal granules have found considerable use for the direct detoxification of blood in cases of uraemia and drug overdose. Although polymer coating materials are presently selected for their biocompatibility, more selective polymers could be used to increase the adsorption capacity for specific drugs and toxins. To gain an understanding of the fundamental factors influencing these adsorbent systems, we have investigated a possible selective coating material, nylon 6 and studied its influence on adsorption rates of simple model compounds when applied as a thin coat to activated charcoal granules. Thermodynamic studies have shown that phenolic compounds interact with the polymer by a hydrogen bonding mechanism, whereas nonphenolic compounds probably bind less strongly due to Van der Waals type interactions. Kinetic studies have shown that the selectivity of charcoal granules for phenolic compounds was increased by coating the granules with a thin layer of nylon 6. The increase in selectivity is probably a result of the different binding mechanism between solute and the polymer. These studies have shown that possible selective coatings may be evaluated more effectively on the basis of simple preliminary drug-plastics interaction studies.