A colony of Mediterranean field crickets, Gryllus bimaculatus (originally supplied by Dr. Ron Hoy, Cornell University), was maintained on a 12:12 light/dark cycle at 28°C and 40–60% relative humidity. Crickets were fed commercial cat chow and drinking water ad libitum. Moist soil was offered for egg laying. Only crickets from this breeding colony were used for the described experiments.
Denervation and tissue collection
All crickets used in this study were treated identically throughout the experiment, with the exception of the type of surgery. Surgeries and tissue collections were performed side by side. Tissue was collected from crickets in the antepenultimate instar stage, which is the stage at which wing buds are first evident. Unilateral denervation of the central auditory neurons was accomplished by amputating the right prothoracic leg (foreleg) at or above the tibial femoral joint. Control crickets used for SSH were amputated at the right tarsal-tibial joint, leaving the developing auditory organ intact. All crickets used in this study were cooled to 4°C prior to amputation and tissue collection. After surgeries were performed, crickets were grouped by condition (control or denervate) and housed for three days in small, shoebox sized containers with adequate food, water and hiding places. Three days post-amputation, the prothoracic ganglia were dissected in RNase-free PBS, frozen immediately in liquid nitrogen, and stored at −80°C until processing. Control and denervated samples were amputated and dissected in parallel.
For backfill experiments, two types of denervations were performed. In the first, chronically amputated crickets were used to generate the schematic in . These crickets had legs removed initially during the first nymphal instar. These crickets were checked every few days and any regenerating blastema were removed. Once these animals matured into adults, AN-2 was backfilled (see below). The second type of backfill experiment was used to demonstrate the anatomical changes three days after denervation in antepenultimate instar nymphs ().
Biocytin backfills of AN-2 and microscopy
Anesthetized crickets were immobilized by pinning and their thoracic cavity was opened. Approximately half to three-quarters of the axons in the appropriate neck connective were removed, leaving intact the medial-ventral portion containing the axon for ascending-neuron 2 (AN-2). AN-2 was backfilled in the animal by retrograde uptake of 4% biocytin (Sigma) dissolved in 50 mM NaHCO3. Animals were maintained at 4°C for 18–22 hours. Ganglia were dissected and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hartfield, PA, USA), then treated with 0.5% triton and a 1:400 dilution of Alexafluor 488 streptavidin (Invitrogen). Tissue was rinsed, dehydrated, and mounted in methyl salicylate. Images were collected using a Zeiss LSM 510 META and a 40× Plan Neofluor objective (1.3 NA, Zeiss, Thornwood, NY, USA). All images shown are projections of multiple optical sections, and the number of sections for each presented image was included in the figure legends. The midline was determined using three criteria, low power images were first used to roughly find the midline as defined by the point between the anterior and posterior intraganglionic connectives. Then, this general location was influenced by the characteristic point between the major L-shaped dendrite and the connective leading to the soma. Finally, viewing images at high gain made the autofluorescent cells of the midline evident, and the midline location was confirmed.
Confocal images were also used to create the schematic in . Briefly, one example of a control AN-2 and one example of a chronically denervated AN-2 were created from projections of multiple optical sections (151 and 141 optical sections respectively), printed, and placed on a light box. Individual processes were then traced and a rough schematic outline of the ganglia was placed appropriately around each traced neuron. A backfill of nerve 5 was imaged with the confocal microscope and, as above, used to trace the extent of the auditory neuropil. Though the individual (HWH) doing the tracing was not informed of the identify of each cell, the anatomical differences are such that the tracing could not realistically be performed blindly.
Suppression subtractive hybridization
Total RNA was purified, in parallel, from 10 control prothoracic ganglia (right tibia removed three days prior) and 11 denervated prothoracic ganglia (unilaterally amputated above tibial/femoral joint three days prior) using RNApure Reagent (GenHunter, Nashville, TN, USA) according to the manufacturer's protocol, and the subtractive hybridization was performed (Clontech). Briefly, amplified double-stranded cDNA was prepared for each condition from 300 ng total RNA as described in the SMART PCR cDNA Synthesis Kit (Clontech). First strand cDNA was diluted five-fold, and 1 μl of the dilution was used for PCR amplification (18 cycles). SMART-amplified cDNA was digested by RsaI endonuclease. Suppression subtractive hybridization was performed in both directions (forward subtraction: 3-day denervate ganglia minus control ganglia, which will be referred to as denervate-enriched; and reverse subtraction: control ganglia minus 3-day denervate ganglia, which will be referred to as control-enriched). For each direction, two tester populations were created by ligating different suppression adaptors (Adaptor 1 or 2R). Each tester population was hybridized with 30-fold excess driver cDNA, and the two tester populations were hybridized together. Subtracted cDNA was amplified by primary PCR (25 cycles) and secondary (nested) PCR (10 cycles). The subtracted cDNA samples obtained by secondary PCR (control-enriched cDNA and 3-day denervate-enriched cDNA) were used for library construction. 40 ng of purified cDNA was cloned into the pAtlas vector and transformed into E. coli.
Differential screening of subtracted libraries
288 randomly picked white colonies from the control-enriched library and 288 randomly picked white colonies from the 3-day denervate-enriched library were used for differential screening. E. coli cultures were grown in 96-well format in 100 μl Luria broth with ampicillin (75 μg/ml) media for six hours at 37° C. Plasmid inserts were amplified with F1S and R1S primers (5'-AGTACGCTCAAGACGACAGAA-3' and 5'-AAAGCAGTGGTAACAACGCAG-3', respectively), and 100 ng of PCR-amplified insert was arrayed onto duplicate nylon membranes and hybridized with 32P-labeled subtracted control and subtracted 3-day denervate cDNA probes. Blots were imaged with a phosphoimager, and clones were considered positive if there was at least a two-fold enhancement of probe binding as compared to its relevant duplicate.
Sequence analysis of candidates
Plasmids from 120 differentially expressed clones were purified and the inserts were sequenced using M13dir or M13rev plasmid primers. Sequence results were analyzed using BLAST web service at NCBI using the tblastx feature (http://blast.ncbi.nlm.nih.gov/Blast.cgi
accessed November, 2008). A threshold E value of 0.001 was used to determine the identity. Those candidates with larger E values were labeled as “no significant hits” and listed separately (). When possible, a candidate's identity was defined as that hit with the lowest E-value, from the class insecta, and that was annotated ().
Virtual Northern blots
A portion of the original, unsubtracted SMART- control and denervate cDNAs (used above for SSH) were resolved on agarose gels and transferred to Hybond-N membranes (GE Healthcare Biosciences Corp, Piscataway, NJ, USA). Membranes were hybridized with 32P-labeled probes prepared from six control-enriched clones randomly selected from the first blot (C1E9, C1F4, C1H1, C1B3, C1C8, C1E1).
Tissue was dissected and pooled from an independent group of 33 control (tibia removed) and 32, 3-day denervate prothoracic ganglia (leg below tibial/femoral joint removed). Crickets for this experiment were handled in an identical manner as for the SSH experiments. RNA was purified using RNApure Reagent (GenHunter), DNaseI-treated with the MessageClean Kit (GenHunter), and quantified by an ND-1000 (nanodrop) Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). All samples were processed in parallel. Single-stranded cDNA was made from 3 μg total RNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). To check for genomic contamination, a “no-RT” control was included for each sample. The first-strand synthesis reaction was diluted in order to allow reproducible and accurate aliquots (5 μl) to be removed for subsequent PCR reactions (Pernas-Alonso et al., 1999
). Gene specific primers () for eight candidates as well as for Gryllus bimaculatus
β-actin (accession #DQ630919) were designed using Primer 3 (Rozen and Skaletsky, 2000
). Pilot experiments indicated that these were good primer pairs to use because single well-defined bands were amplified. For each primer pair, a cycle series was run on control cDNA in order to determine the optimal cycle number, defined as producing a product strong enough to detect on an agarose gel, but still in the pre-plateau, exponential range. Multiplex PCR reactions, using candidate-specific primers and β-actin-specific primers, were run in triplicate at the optimal cycle number on four different cDNA templates: 1) control, 2) denervates, 3) control “no-RT,” and 4) denervate “no-RT.” Amplified products were run on a 2% agarose gel with ethidium bromide and digitally photographed using a Bio Doc-It Imaging System transilluminator (Ultra-violet Products, Upland, CA, USA). Digital images were saved for quantification.
Quantification of SQ-RT-PCR
Band intensity was measured using LabWorks software (Ultra-Violet Products). Background-subtracted intensity values were obtained for each band, and either used as is, or were then normalized based on the measured β-actin band background-subtracted intensities for each lane. The normalized control values were defined as one, and the denervate values were expressed as a proportion of control for each primer set. The non-normalized data are included and are presented in the same manner. Percent error was calculated for each triplicate by dividing the SEM by the average. Standard error propagation was applied. A one-sided Fisher's exact test was used to assess significance.