A quantitative proteomic analysis of long-term memory

Three sets of animals were used for this study, an operantly conditioned (LTM) group, a yoked control group, and a naïve group. Lymnaea are bi-modal breathers able to obtain their oxygen requirements cutaneously via diffusion across the skin, or aerially via the pneumostome (an opening into the lung). This allows for the aerial respiratory behaviour to be conditioned without compromising the animals survival [15]. This is done by placing individually labelled animals in water made hypoxic by bubbling N₂ through for 20 minutes prior to training, in order to increase the aerial respiratory drive. Upon commencement of training the snails receive a gentle tactile stimulus to the pneumostome area each time aerial respiration is attempted. With training the animals learn to withhold this behaviour thus decreasing the number of attempted pneumostome openings. The LTM animals received three 45 min reinforcement sessions. Two 45 min training sessions on the first day separated by an hour, and a third 45 min training/memory test session on the second day. The yoked animals received the same hypoxia exposure and number of stimuli as the LTM group, except in a non-contingent manner (animals only receive stimulation if pneumostome is closed). Naïve animals received no treatment.

Animals were dissected one hour after the completion of the final training or control session. The animals were incapacitated by submersion in ice-cold saline, with the dissections also carried out in ice-cold saline. The central ganglia of the animals were quickly dissected out. Next the buccal ganglia were dissected away from the central ganglia followed by the removal of the left pedal (L.Pe.G), cerebral (L.Ce.G), and pleural ganglia (L.Pl.G) (Figure 2). This was performed in order to better isolate the brain regions containing the central pattern generator which controls aerial respiration [10], in an attempt to enhance the signal to noise ratio (cells where memory is stored to non-memory cells) of the samples. Upon completion of dissection the ganglia were placed in eppendorf tubes, and immediately snap frozen and stored at -80°C until use.

0.5 mL of solubilization buffer (Urea-Thiourea-CHAPS buffer containing, 40 mM Tris, 5 M Urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, and a Protein inhibitor cocktail) was added to each sample, and the samples were homogenised on ice using the loose pestle of a Dounce homogeniser. The samples were then mixed on a rotary mixer for 30 minutes and then centrifuged at 10 000 g for 10 minutes. The supernatant was transferred to a new tube and this was centrifuged for 45 minutes at 150 000 g to sediment any un-dissolved material. The supernatant was transferred to a new tube, and protein concentration was determined using the EZQ assay (Invitrogen). The samples were stored at -20 until used. The samples were then processed as follows for mass spectrometry analysis.

Protein samples were analyzed via an ultra-performance liquid chromatography tandem mass spectrometry (nano UPLC-MS/MS) protocol as previously described [16, 17]. Briefly, the methanol-chloroform precipitated samples were subjected to in-solution tryptic digestion. Samples were then desalted using Sepak C₁₈ columns (Waters, Milford, MA, USA). Next lyophilized peptides were resuspended (97% H2O, 3% acetonitrile, 0.1% formic acid) and subjected to the nano UPLC-MS/MS analysis using a Waters Nano-Acquity UPLC system with a 75-um inner diameter × 25 cm UPLC column and a 90 min gradient of 2-45% solvent B (solvent A: 99.9% H₂O, 0.1% formic acid; solvent B: 99.9% acetonitrile, 0.1% formic acid; final flow rate 250 nl/min, 7000 psi), coupled to a Waters QTOF-premier tandem mass spectrometer (Waters, Milford, MA, USA). Data was acquired in high-definition MSE mode (low collision energy 4 eV, high collision energy ramping from 15 to 40 eV, switching every 1.5 seconds) and processed with ProteinLynx Global Server (PLGS version 2.2.5, Waters, Milford, MA, USA) to reconstruct MS/MS spectra by combining all masses with identical retention times. MS/MS spectra (peaklists) were searched against the Swiss Prot database using PLGS. Each sample was analysed in triplicate runs and were spiked with an alpha-enolase tryptic digest (125 fmol) as an internal standard. Quantitative changes in protein abundance, based on peptide ion peak intensities were analyzed using the Waters Expression Analysis Software (WEPS), which is part of the PLGS package.

For the western blot analysis, the CNS of another set of animals were dissected out and snap frozen after treatment as described above (naïve n = 8, yoked n = 8, and trained n = 8). 0.5 mL of Nonidet-P40 (NP-40) buffer (containing, 20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mM PMSF, and a Pearce Halt protease inhibitor cocktail) was added to each sample, and the samples were homogenised on ice using the loose pestle of the glass homogeniser. The samples were then mixed on a rotary mixer for 30 minutes and then centrifuged at 10 000 g for 10 minutes. The supernatant was transferred to a new tube and this was centrifuged for 45 minutes at 150 000 g to sediment any insoluble material. Protein levels were standardised and samples were resolved using 10% SDS-PAGE gel. Proteins were transferred overnight at 4°C onto PVDF membranes. Membranes were blocked in 5% non-fat milk/TBST (20 mM Tris-HCl, 0.15 M NaCl, 01% TWEEN 20), and incubated with polyclonal anti-ADCY8 (1:5000) or anti-β-actin (1:10000) (Abcam, Cambridge, MA, USA). Membranes were then washed and incubated with an HRP-conjugated secondary antibody (1:10000). Western blots were visualized using ECL plus (GE healthcare, Piscataway, NJ, USA). Membranes were scanned using a Molecular Dynamics Storm 860 phosphoimager, and densiometric analysis was completed using ImageJ software.

Three groups of animals were used in this study; an operantly conditioned group (n = 8), a yoked control group (n = 8), and a naïve group (n = 8). The operantly conditioned animals received two 45-minute training sessions (TS1 and TS2) separated by an hour on day 1. We tested for memory 24 h later (MT). We define memory to be present if the number of attempted pneuomostome openings in the MT session is significantly less than that in TS1, and not significantly greater than that of TS2. As expected the operantly trained animals showed a significant reduction in the number of attempted pneumostome openings in the last session (MT) as compared to the first (TS1) (ANOVA F _(2,7) = 45.24, p < 0.001), and thus we conclude that they successfully formed LTM (Figure 1). This data is consistent with previously published findings using this training regime [18]. Yoked control snails did not exhibit LTM (data not shown).

Following behavioural testing for LTM, all snails (including the two control groups) had their nervous systems dissected out (Figure 2) and processed for proteomic screening as described in the methods. Briefly the snail brains were homogenized, proteins were extracted, subjected to a trypsin digestion and analyzed by inline liquid chromatography and mass spectrometry. Hundreds of proteins were positively identified in each sample (LTM = 503, Naïve = 506, and Yoked = 518). From these identified proteins there were found to be a large number that were significantly different between the LTM group and the controls (yoked and naïve). To better focus the presentation of the data we are reporting only those proteins that were found to be significantly different (increased/decreased, present/absent) in the LTM group as compared to both the naïve and yoked control groups. That is any protein found to be significantly different in the LTM group, showed a significant difference from both the naïve and the yoked groups for the identical protein, and in the same direction. Using these criteria there were 8 proteins that were found to be significantly more abundant in the LTM trained animal sample as compared to the controls, and 13 proteins which were significantly less abundant (Table 1). 19 proteins were found to be unique to the LTM trained animal sample, while 12 proteins were detected in both control samples but not in the LTM sample (Table 2). Included in the list of proteins that were up regulated in LTM animals were adenylate cyclase type 8 (ADCY8) and mitogen activated protein kinase kinase kinase 1 (MEKK1) whose chromatograms are shown in figure 3. These two proteins have previously been associated with the memory formation process. ADCY8 is membrane bound enzyme that catalyses the formation of cyclic AMP (cAMP) in response to stimulus evoked entry of calcium into neurons. ADCY8 knockout mice show deficits for memory retention in object recognition and passive avoidance tasks [19], as well as deficits in the formation of mossy fiber long-term potentiation (LTP) in the hippocampus [20]. MEKK1 is a serine/threonine kinase, and a member of the MAPK signal transduction pathway. MEKK1 has been shown to be an activator of both the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the c-Jun N-terminal kinase 2 (JNK 2) pathways [21], with ERK activity being necessary for the induction of LTP [22], and the formation of various kinds of memories [23‐25].

To validate the results of the quantitative MS experiments, we performed a set of western-blot experiments on one of the proteins found to be up-regulated in the LTM sample; ADCY8. For this experiment three new sets of samples were prepared, as previously described, and four separate gels and blots were done to allow for quantification. As shown in figure 4, animals trained to form a LTM showed a significant increase in the amount of ADCY8 as compared to naïve animals. While the western blot results show a similar pattern of results when compared to the UPLC-MS/MS data, they differed slightly as the change in ADCY8 expression between yoked and trained animals did not reach statistical significance by western blot analysis. This difference may be attributed to the much higher precision and sensitivity of measurement made by the UPLC MS/MS method.