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2.2.1 Nematode Strains and Maintenance

The wild-type strain used in this work was the strain Bristol N2. Other strains used were LX645 [dop-1(vs100); [88], LX703 [dop-3(vs106); [88], OS2649 [hlh-17(ns204); [30], and OS2929 [hlh-17(ns204); hlh-31(ns217); hlh-32 (ns223) ; [30]. Standard methods used for culturing C. elegans were as described by Lewis and Fleming, 1995. Strains were maintained at 20°C and all assays were at 22°C.

2.2.2 Behavioral Assays

For all assays except gustatory plasticity assays, cultures were synchronized by hypochlorite as previously described [87]. Measurements of rates of egg-laying behavior in response to DA and 5HT were as previously described [89]. Newly hatched L1 animals were allowed to feed on OP50 for 72 hours prior to the assay. Individual animals were then rinsed

briefly in M9 and transferred into a well of a 48-well microtiter plate containing 50 µL of M9 buffer +/- the appropriate drug. DA was used in these assays at 3 mg/ml; serotonin (5HT) was used at 5 mg/ml.

Immobilization assays were used as measurements of resistance to exogenous DA as described previously [88]. Young adult animals were transferred to NGM plates containing 0, 5, 10, or 20 mM DA and incubated at 22°C for 40 minutes. Animals were scored by visual

inspection as being mobile if they initiated spontaneous body bends within 20 seconds. Animals that did not move spontaneously, but responded to gentle prodding with a platinum wire were scored as immobile; those that did not respond to prodding were scored as paralyzed.

Basal slowing response was measured using young adults as previously described [88]. Prior to the assay, animals were washed in sterile deionized water, placed on assay plates either with or without a fresh lawn of OP50, and allowed to recover for 2 minutes. The number of body bends made by each animal (n=10 animals/strain/feeding condition) was counted for three consecutive 20-second intervals.

Gustatory plasticity was measured as the response to 25 mM NaCl after pre-exposure to 100 mM NaCl and was performed essentially as described previously [90, 91]. Mixed-staged cultures of C. elegans were enriched for gravid adults by incubating in ice-cold chemotaxis (CTX) buffer (minus NaCl) and aspirating away animals that were not heavy enough to sink to the bottom. Naïve animals were rinsed three times in CTX buffer (without NaCl). Pre-exposure to 100 mM NaCl was performed in CTX buffer for 20 minutes in the absence of food. Animals were scored after 30 minutes on chemotaxis plates, and the chemotaxis index was calculated using the formula (A-C)/(A+C) where A represents the number of animals in the quadrant containing NaCl, and C represents the number of animals in the quadrant without NaCl [91].

Statistical significance was calculated using single factor ANOVA, with Bonferroni correction. As described previously [92], significance was determined by comparing the response of mutant animals to the response of wild-type animals under the same conditions. Error bars represent the 95% confidence interval.

2.2.3 Analysis of Gene Expression

The extraction of total RNA from L1 stage populations was performed using the RNeasy Plus Micro Kit (Qiagen, Inc.) essentially as directed by the manufacturer for purifying RNA from animals and cells. Worms were collected from two 100 mM NGM-agar plates, rinsed twice in sterile, deionized water, and pelleted by centrifugation at 1,000 rpm for 1 min in a 15 ml conical tube. The samples were transferred to 1. 5 ml microcentrifuge tubes, and frozen on dry ice. Samples were subjected to two additional freeze/thaw cycles, resuspended in 0.1 ml of STE buffer (0.5% SDS, 5% 2-mercaptoethanol, 10 mM EDTA, 10 mM Tris-Cl pH 7.5) containing 0.5 mg/ml proteinase K, and then incubated at 55°C for 30 minutes. After incubation, the samples were mixed with 0.35 ml of RTL buffer (provided in RNeasy Plus Micro Kit, contents

proprietary; Qiagen #1053393) and homogenized by two passages through a Qiashredder column (Qiagen, Inc) and were then processed as directed by the manufacturer. For the final elution step, the total RNA was recovered in 20 µL of sterile, nuclease-free water. RNA purity and

concentration was determined by UV spectroscopy at 260, 280, and 230 nm. RNA samples were only used if 260/230 ratios were equal to or greater than 1.7. RNA quality was determined by denaturing gel electrophoresis. Finally, we tested each RNA for genomic DNA contamination by performing PCR in the absence of reverse transcription with the endogenous control primer pairs. cDNA synthesis reactions were performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), as directed by the manufacturer, with each 20 µL

reaction containing 2.0 µg of total RNA. Real-time PCR was performed on the 7500 Fast Real- Time PCR System® using either the Fast Sybr Green or the Taqman Universal Master Mix (Applied Biosystems) and the appropriate gene specific primer/probes listed in Table 1. Each reaction was performed in quadruplicate, and three biological replicates were tested for each gene. Assays were performed using relative quantitation, with normalization against two different endogenous control genes. The endogenous control genes were pmp-3and cdc-42 for the Sybr green reactions and were pmp-3 and rrc-1 for the Taqman reactions. Relative fold changes in expression were determined by setting the wild-type to an arbitrary level of one. In the final determination of genes whose expression was significantly altered by loss-of hlh-17, we considered only those genes whose expression changed for an average of at least 1.5 fold over the course of three experiments and that showed a P-value less than 0.05 when evaluated by single factor ANOVA.