The glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse. / text

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.