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The role and application of polymers in different parts of an LMB are presented, including anode, cathode, electrolyte and anode/electrolyte interphases.The 20 pickup is available across 10 trim levels and offers three engine options, the 3.6-liter Pentastar, 3.0-liter Eco Diesel, and 5.7-liter Hemi V8. The Hemi puts out 395 horsepower and can tow up to 10,200 pounds. Ram hasn’t had a major redesign since 2009, so the 2017 model rests on old architecture. As a result, Rams of this year lack key safety features found in competitors, like lane-keep assist, automatic braking, and more. With the Hemi V8, it’s one of the most powerful pickups in its class. While some competitors best it in certain areas, none can match its low entry price. Owners and critics alike give the Ram high praise, especially concerning performance and comfort. Poorly designed and bad quality, made to fail right after the warranty ends. The rust started to pop up on the hood from under the paint before 5 years, water is dripping into the cabin since new, one door not closing properly since new. DEF tank is so improperly designed that liquid will crystallize and block the level device. Dealer recommendation: replace the tank (DEF tank price: over $2400). You see a nice truck but the quality is very low, not worth the price. The mirrors fold in and has a lot of power with the Hemi engine. Customer service is biased, "out of warranty, we cannot help". Great sporty look, reliable with a decent gas mileage for a full size truck. Warranty is 60,000Km or 3 years like any poorly made car. I got 1 problem with this truck, apart from the fender flairs falling off. Talk text radio, most is located on steering wheel or voice activated. Has a backup camera and all the sensors on all sides. Remote start and heated seats and steering wheel, which is great in the winter time. If you did not have problems during warranty you will sure have right after. The navagation is just useless.90% of the time it does not understand a word you say, no matter how fast or slow you speak, even if you spell the words, it comes up with some address in lally land. My passenger side head gasket failed at 71,000mi, causing irreparable damage to the motor. Decent gas mileage for a truck, definitely better on long trips. Hydraulic lift shocks that raise and lower the truck making it easier to get in. If you try to type the address in ,it goes crazy altogether, example, if i try to type in 333 of an address, its already giveing me a whole different address after i type in just one 3, and will not let you type in further than one letter or number. At $6500 for a new motor installed from the dealer, I now have a very nice lawn ornament. It handles great on the road in all weather and turns. This is my first truck that I have owned and primarily drive it has good power and handles nice. Its the most annoying navagation system i ever encounted, A complete useless piece of junk. Lots of interior leg room comfortably seats 5 adults. It is a bigger truck so I feel safe driving it with my kids. Seats are comfortable and it has a pretty decent sound system. Was a difficult when grandbaby was in small car seat but much easier now that it's not in the middle. Not having a cover on the bed for more storage and safekeeping of items if you are shopping at multiple stores. Gas mileage is not so good but that's expected with the larger V8 engine. Heated seats and steering wheel automatically come on when temperatures are below forty degrees. I like having a truck a d feeling safer but it's hard to park and I have a side job of doing deliveries and it's not the easiest vehicle for the hat. This truck does not have the steps on the tailgate. Speakers take up all of the storage underneath the rear seat. The back seat doesn't have much leg room luckily my kids are still fairly small so don't need much. The tires are the 20 in rims so it may cost a little more to put tires on.. The 3.6-liter Pentastar V6 has been out since 2011, so seven or so years of road testing. That’s a lot of time for problems to appear, but the 3.6 has held strong. In fact, the National Highway Traffic and Safety Administration (NHTSA) has yet to issue any recalls involving the 3.6-liter in the 2017 Ram. Depending on how much “work” you actually do with the work-capable 3.6-liter Pentastar, 200k or more is a reachable goal with regular maintenance. As for potency, the 3.6-liter Pentastar is surprising and puts out an impressive 305 horsepower and 269 lb.-ft. What does all this mean to the average truck shopper? A 2017 Ram with the Pentastar V6 is still a capable truck. When properly equipped, it can tow up to a max of 7,600 pounds. As for fuel efficiency while not towing, expect about 17 miles per gallon in the city and 25 at highway speeds under optimal conditions. Yes, Ram was the first to finally do it, starting in 2014 in the form of the 3.0-liter Eco Diesel. As expected, fuel economy was impressive, at 20 mpg around town and 27 on the highway with RWD models. However, the 3.0-liter Ecodiesel has had a pair of recalls issued: Thankfully, if you’re buying from a dealer, any recalls should already be performed. How much can a 20 tow with the 3.0-liter Eco Diesel? Yes, we priced a stock 20 Tradesman with the 3.6-liter Pentastar against an identical unit with the 3.0-liter Eco Diesel. Around 9,200 pounds depending on the package, much thanks to its 240 horsepower and impressive 420 lb.-ft. We found the diesel to cost an extra $1,800 on average. Rams equipped with the 395-horsepower 5.7-liter Hemi V8 are absolutely the most fun to drive. of torque, the Hemi is also the most powerful and can tow 10,200 pounds when properly equipped. The 5.7-liter initially launched on Ram models back in 2003, so in 2021 it’s had about 14 years of reliability history for you to consider. The NHTSA has so far issued zero recalls specifically related to the 5.7-liter in a 20 pickup. What’s more, the 5.7-liter Hemi engine uses a cylinder deactivation system that can effectively turn a V8 into a four-cylinder when the engine is not under strain. In 2WD the 5.7-liter still manages a respectable 15 city and 22 highway mpg. Just one transmission is standard/available on all 2017 1500s, regardless of engine, an eight-speed automatic that debuted on Ram Pickups back in 2013. So far, one transmission-related recall has been issued: The NHTSA reports 313 registered complaints with the 2017 Ram. The bulk consists of 64 related to the engine, 57 the powertrain, 40 the electrical system, 33 to airbags, and 30 to structure. While this might seem like a lot, 20 models were even worse. Ram 1500s are problematic, and owning one means you’ll likely end up at a service center now and then. Your best bet, and a major factor when buying a used pickup, have a mechanic look for signs that suggest the truck has had its maintenance schedule followed. If you do end up buying, be sure to follow it yourself for the best experience. As for the potentially hazardous issues, when enough of them coincide, a recall gets issued. The 2017 Ram had 10 recalls issued, of which we’ve already mentioned two for the engine and one for the transmission. Below you’ll find a few of the more common 20 recalls: As for safety, the Ram 1500 is a safe vehicle, with the NHTSA giving it a four out of five-star rating overall. The model’s weakest areas include a four-star frontal-impact rating and four-star rollover risk. Another site that tests a vehicle’s crash test ratings, the Insurance Institute for Highway Safety, gave the 2017 Ram a mostly positive “crashworthiness” score. Where it struggled was crash avoidance and mitigation, meaning its ability to avoid a crash altogether. No, Ram 1500s in this year lacked safety items like automatic braking and lane-keep assist. The Ram goes toe-to-toe with most competitors in its class, including the mighty F-150. But should you actually buy a 2017 Ram over another used pickup? Here, we compare the Ram 1500 to the Ford F-150, Toyota Tundra, and Chevrolet Silverado. The 2017 Toyota Tundra shares something with the 2017 Ram, both rest on dated platforms. So besides a few upgrades/updates here and there, a 2017 model looks very similar. Only two engines are available on the 2017 Tundra, the base 4.6-liter V8 or the more potent 5.7-liter V8. Regardless, even the latter doesn’t best the Hemi’s horsepower, falling shy by 14. However, Tundra’s 5.7-liter does offer 300 more pounds of maxing towing when properly equipped. Using KBB, we found the 5.7-liter to cost about $1,200 more than the 4.6-liter engine. The 20 series is known for being a long-lasting half-ton truck but there are a few problems that do seem to be commonplace amongst the lineup. The first and most common problem is issues with the TIPM. This stands for totally integrated power module and it is the computer system that runs the entire truck. These have been known to fail across many Dodge, Jeep, Ram, and Chrysler vehicles and the 1500 series is no exception. The second most common problem with the Ram 1500 series is problems with the cams and lifter arms. This is a known problem across the HEMI V8 and it can lead to the lifters being stuck open too long which can lead to the engine not running properly. These problems have been mitigated in more recent years though through different engine revisions. The final common problem with the Ram 1500 series is that there are some interior issues. The dashboards and interior trim pieces have been known to crack on these trucks, even when they are fairly new. This has resulted in Ram replacing some dashes under warranty since the materials just could not hold up. A 20 series should have no problem going over 250,000 miles and there are a lot of earlier Ram 1500 series trucks that are still on the market with over 350,000 miles. These are built to be long-lasting professional-grade trucks and Ram builds them with that in mind when it comes to longevity. The most important thing you can do if you want your Ram 1500 series to last a long time is to take care of it. These trucks are tough can take some abuse, but you still need to be on top of your maintenance if you want your engine to last. It is also a good idea to make sure to check for any problems regularly so that you can fix them before they develop into larger issues. The Ram 1500 series is a great line of trucks that competes with a lot of other full-size offerings from Chevrolet and Ford. The Ram can hold its own in the competition as well since these can last a very long time. The 20 series is estimated to depreciate about 42% over 5 years. This is a higher rate of depreciation compared to some of the other half-ton trucks on the market, but it still depreciates less than sedans or SUVs that are in the same price range. The Ram 1500 series does depreciate more than the other American trucks primarily because it is known to be the least reliable overall. These have had issues in the past with transmissions, and the TIPM problems are well-known throughout the truck community. That leads to these having a lower resale value, despite being as capable as the other trucks. If you're in the market for a full-size truck then a Ram 1500 series can be a great bargain. These are usually less expensive than the competition on the used market, and they also have a more modern and comfortable interior. If you are in the market for a used truck you should check it out and compare it to the other options to see what works best for you. The 20 series does not have the same widespread transmission issues that were found in the earlier versions of the Ram 1500 series trucks. Ram, and Dodge, had garnered a reputation in the past for having transmissions that would not make it to 100,000 miles, but they have improved their designs over the past 20 years to make a much more reliable transmission. Both the 6-speed and 8-speed automatic transmissions in the 2017 Ram are known to be decently reliable, but a bit complex. These have extra computer systems on them that can become problematic if there are any issues with the TIPM or the transmission control module. If you notice that your vehicle is hunting for the right gear then you should have it checked out as soon as possible since it is usually an electronic fault. The Ram 1500 series does not have a lot of reported complaints about mechanical issues with the transmissions. The gears and associated hardware in these trucks seem to be able to hold up against the stresses of driving and towing. Choosing the right 20 series truck can be difficult since they are offered in many different trim configurations. It will take some getting used to if you are not used to driving a big truck. Generally, the Express trim is considered to be a worthwhile entry point for someone in the market for a Ram 1500. And the paint job on this truck has been the most durable by far. It is a truly great vehicle to drive once you get used to it. This has some added luxury and better styling than the entry-level tradesman work truck which is the most affordable choice. The exhaust manifold has been repaired at 75,000 miles. Our truck is my families main vehicle and we drive it a lot. I love this truck as it is a great size, has 4 doors and full back seat. It has a wonderful sound system and I subscribe to Sirius XM radio. If you like going off-road and know that you will spend your time out on the trails then the Rebel is a great trim option. The backup camera is so helpful and I really depend on it. This comes standard with the 5.7L HEMI V8 and a new suspension kit that will give you better clearance and travel for the more difficult off-road adventures you plan to go on. The mirrors pull in so that helps in tight parking spaces. If you want the ultimate in luxury then the Ram 1500 Limited is the best option for you. The 20 Lone Star edition is a very comfortable, dependable vehicle with a number of features that makes it convenient and fun to drive. This comes with a four-corner air suspension which helps it ride more like a luxury sedan than a truck around town. I bought my ram 1500 on the used truck market in 2018. I have never had any mechanical issues with this truck. This trim also has the best interior options available. I have Bluetooth, navigation, CD player, Sirius radio. Sometimes difficult to see at the front corners of the windshield. Since my purchase of the truck, I have had zero issues with it. It has been a pleasure to drive and I would recommend it to anyone. The 5.7l hemi v8 does everything I need it to and the 4x4 works great as well. I really do love my truck, but it is almost too basic. None of which are really much of a problem, but some things I dislike nonetheless. I went with the bench front seat for added seating and comfort. Trim is too wide between windshield and side windows. Oil changes and a set of tires is the only maintenance items I have had to do on my truck. Fantastic towing capabilities without the cost of diesel fuel. I will say there have been some improvements from the 2014 — the electronic shifting knob no longer locks up on me at random times, better headlights, and just an all round smoother running truck. I also ride dirt bikes so plenty of room in the bed and great towing capabilities are a must. I have had my truck a little over a year and have had no problems minus the recall that was taken care of right away at my convenience. I searched long and hard to find the exact truck I wanted. Crew cab feels large enough for 5 full grown adults to travel comfortably. I have a 2017Ram Eco Diesel with less than 25000 miles - no accidents, garage kept. However, I still get recalls quite a bit for its age ( two of which have been a cruise control problem where the vehicle will not stop accelerating and airbags not inflating ) but I have never experienced the issues the recalls are on. Spare tire is difficult to get to, especially in bad weather. I am extremely happy with my purchase and wouldn't think twice about buying another ram product when the time comes. Could have a few more glitzy features such as a larger screen for the radio & a rear back up camera, maybe sensors too, but it is only one step above the base model, and for the price, i'd say it is good enough. Mpg is low with the v8, currently around 13.6 of mostly in city driving. Recently had not only the radio but also the backup camera go bad. It hauls my small horse trailer wonderfully and gets decent gas mileage for daily driving. The versatility of a truck is a must have for every household imo. Very suspicious both electronics go out almost simultaneously. Contacted Chrysler customer service and was informed I could have bought extended warranty for electronics when I purchased the vehicle…. If I expect items to not even last this short a period of time I would not buy them… Shame on Fiat Chrysler for not standing behind their product. My passenger side head gasket failed at 71,000mi, causing irreparable damage to the motor. I will remember this when I purchase my next vehicle… At $6500 for a new motor installed from the dealer, I now have a very nice lawn ornament. The 5.7l hemi v8 does everything I need it to and the 4x4 works great as well. Fantastic towing capabilities without the cost of diesel fuel. Crew cab feels large enough for 5 full grown adults to travel comfortably. It's practical for transporting large items and has enough space in the cab to carry 5 people comfortably while getting 18 mpg with a powerful 5.7l v8. The ram 1500 is a dependable vehicle that has had few issues. The only issue I have is the truck rusts very easily. About the only thing that I would like better is if there was less plastic in the interior. Plus it even has a button where if I ever need to get help. The primer isn't the greatest so if you use your bed, I suggest getting a liner or spraying it. When I bought it I didn't realize it was 2wd and had no hitch so now I am stuck with a large car when I need a truck. The vinyl package holds up well and I enjoy the exhaust noise. Great services to the family for the last 3 years just the recommended oil changes and wiper blades and other minor things. Powerful motor paired with an eight speed automatic transmission. The ram is a great truck pulls our camper across the state and back very comfortable and great ride excellent in 4 wheel drive in the snowy North Country. Vindas Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Oslo, Norway Uni Environment, Uni Research AS, Bergen, Norway Marco A. Vindas Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Oslo, Norway Uni Environment, Uni Research AS, Bergen, Norway It is well-established that sustained exercise training can enhance brain plasticity and boost cognitive performance in mammals, but this phenomenon has not received much attention in fish. The aim of this study was to determine whether sustained swimming exercise can enhance brain plasticity in juvenile Atlantic salmon. Brain plasticity was assessed by both mapping the whole telencephalon transcriptome and conducting telencephalic region-specific microdissections on target genes. We found that 1772 transcripts were differentially expressed between the exercise and control groups. Gene ontology (GO) analysis identified 195 and 272 GO categories with a significant overrepresentation of up- or downregulated transcripts, respectively. A multitude of these GO categories was associated with neuronal excitability, neuronal signalling, cell proliferation and neurite outgrowth (i.e. Additionally, we found an increase in proliferating cell nuclear antigen (pcna) after both three and eight weeks of exercise in the equivalent to the hippocampus in fish. Furthermore, the expression of the neural plasticity markers synaptotagmin (syt) and brain-derived neurotrophic factor (bdnf) were also increased due to exercise in the equivalent to the lateral septum in fish. In conclusion, this is the first time that swimming exercise has been directly linked to increased telencephalic neurogenesis and neural plasticity in a teleost, and our results pave the way for future studies on exercise-induced neuroplasticity in fish. There is ample evidence in the mammalian literature that exercise training leads to increased neurogenesis and synaptic plasticity and that this is associated with increased cognition. Specifically, running exercise has been shown to improve the cognitive performance of rodents in spatial tasks [1,2]. This effect is strongly associated with increased neurogenesis and synaptic plasticity in the hippocampus, particularly in the dentate gyrus, mediated by an increased abundance of growth factors, neurotransmitters and neurotrophic factors, although the exact mechanisms are yet to be elucidated [2–4]. Even though the link between exercise, neural plasticity and cognition is now well-described in mammals, this phenomenon has not received much attention in other vertebrates, such as fish. In this context, fish species are a promising model to study neurobiological mechanisms, particularly those associated with neurogenesis, since fish display neurogenesis in a multitude of proliferation zones throughout their entire lives and rates of cell proliferation in the teleost brain are one to two orders of magnitude higher than in the mammalian brain [5]. These higher cell proliferation rates, besides imparting remarkable neural plasticity, also contribute to the fact that upon neural damage, teleost fish species have an incredible capacity for regeneration of the central nervous system [6]. Notably, a pilot study conducted by Luchiari & Chacon [7] demonstrated that exhaustive swimming exercise in zebrafish (Danio rerio) improved their learning performance in a conditioning test within several days. Furthermore, a 10-day swim training regime promoted the expression of neurogenesis markers in the brain of zebrafish larvae [8]. In contrast to mammals, fish lack a six-layered pallium, but nonetheless, they are able to display a number of higher cognitive functions, which are mainly under forebrain telencephalic control [9–11]. Importantly, the fish telencephalon contains neural populations and networks associated with emotional and relational memory, learning and stress-reactivity, thus driving processes which show functional resemblances to processes which are under limbic system control in mammals [12–15]. Therefore, we hypothesize that increased exercise training will enhance telencephalic neurogenesis and neural plasticity in fish. The aim of this study was to assess whether sustained swimming exercise can promote forebrain neurogenesis and neuroplasticity in Atlantic salmon (Salmo salar, L.). We chose salmon since this species is characterized by sustained periods of increased swimming exercise (i.e. migrations to and from seawater to freshwater spawning grounds [16]), and swimming exercise has already been shown to have beneficial effects in salmon, including increased growth rates [17] and stress alleviation [18]. We subjected fish to an eight-week sustained swimming exercise regime and assessed their expression of neural plasticity and neurogenesis markers at three and eight weeks. We here report that swimming exercise leads to the upregulation of key neuroplasticity- and neurogenesis-related genes in telencephalic areas of the salmon brain, similarly to effects reported in mammalian studies. These results highlight a promising new model for the study of swimming enhanced neurogenesis/neuroplasticity mechanisms and their link to enhanced cognition. This experiment was performed in accordance with Dutch law for experimentation and procedures on live animals. Experimental fish were hatchery-reared Atlantic salmon juveniles, which were first-generation offspring from wild-caught parents from the river Imsa, in southwestern Norway. The experimental protocol was approved by the Animal Experimental Committee (DEC) of Wageningen University and Research (case no. Eggs hatched in late January 2017 and fish were reared under standard hatchery conditions at the Norwegian Institute for Nature Research (NINA) Research Station at Ims, Norway, in water from the adjacent river Imsa. At eight months old, fish were transported from the Ims hatchery to the aquaculture research facilities at Wageningen University and Research (WUR), the Netherlands. After 18 days of acclimatization at the WUR experimental facilities, fish were tagged intraperitoneally with passive integrated transponders tags (Trovan ID100A/1.4 mini transponders) and the fish were then left to recover for an additional week before the experiment started. Fish were 9-months-old at the start of the experiment and 11-months-old at final sampling. Fish were exercised for a total of eight weeks on a volitional training regime (i.e. fish could, to a certain degree, choose their preferred swimming speed). At the start of the exercise regime, fish measured 123 ± 5 mm (fork length; FL) and weighed 20.8 ± 3.6 g (mean ± s.d.). The experimental set-up consisted of two standard cylindrical 800-l holding tanks holding 110 fish each (density = 2.6 kg m throughout the tank. The selected flow rates were the maximum speeds that could be achieved in the standard hatchery tanks and were well within the aerobic scope of salmon [19], as well as within the preferred range of flow rates of Atlantic salmon in natural habitats [20]. Both tanks were covered with mesh and half of the tank was covered with black foil to provide shelter. Light intensity at the water surface was approximately 45 lux. Exercised fish showed no sign of fatigue and generally positioned themselves facing the current, while occasionally drifting down with the current. The light cycle was maintained at 12 L : 12 D throughout the experiment. Water temperature was maintained at 14.9 ± 0.45°C and nutrient levels were 0.06 ± 0.05 mg NH (mean ± s.d.). Fish were fed commercial pellets (Nutra Parr, Skretting, Stavanger, Norway) by hand, twice per day until satiation and water flow was stopped during feeding to provide equal feeding opportunities for both exercised and sedentary fish. All fish were measured and weighed after the swimming treatment and specific growth rates were calculated as follows: Fish were sampled at two time points: after three and eight weeks for microdissection and at eight weeks only for RNAseq analysis. Fish were randomly collected from their holding tanks and quickly anaesthetized in 2-phenoxyethanol (VWR #26244.290, 1.3 ml l). Opercular movement ceased completely within 30 s, after which weight and length were recorded. Immediately after, fish were decapitated and brains were sampled in two ways: (i) the jaw and gills were trimmed before fish heads were sealed in a plastic bag, snap-frozen on dry ice and stored at −80°C until processing or (ii) brains were dissected out and the telencephalon was extracted out and placed overnight in RNAlater (Invitrogen AM7024) at 4°C. The following day, surplus RNAlater was removed and samples were stored at −80°C. Frozen trimmed skulls of 10 fish per treatment and per time period were sectioned (100 µm thick) transversely in a cryostat (Leica CM 3050) at −22°C. Sections were thaw-mounted onto glass slides (VWR 631-151) and subsequently stored at −80°C. Microdissections were performed on frozen sections kept on a cooling plate (−14°C) as described by Vindas et al. Three subregions of the telencephalon were microdissected: the dorsolateral pallium (Dl), the dorsomedial pallium (Dm) and the ventral part of the ventral telencephalon (Vv). On average, per individual, a total of 21 punches were taken for the Dl and the Dm and 11 for the Vv. Microdissected tissue was injected into RLT buffer (RNeasy Plus Micro Kit, Qiagen 74034) and immediately frozen at −80°C until RNA extraction, which was conducted within 3 days after microdissection. Relative transcript abundance of brain-derived neurotrophic factor (bdnf), neural differentiating factor (neurod), synaptotagmin (syt) and proliferating cellular nuclear antigen (pcna) in microdissected areas was measured using real-time PCR (q PCR). Microdissected tissue was thawed, vortexed for 30 s, centrifuged at 13 400×g for 5 min and total RNA was subsequently extracted using the RNeasy Micro Plus Kit (Qiagen, ID 74034), according to the manufacturer's instructions. RNA concentrations were measured using nanodrop and the quality of extracted RNA was checked on a subset of samples using a Bioanalyzer RNA 6000 Pico Kit (Agilent 2100): RNA integrity numbers (RIN) were 8.5 ± 0.8 (mean ± s.d.). Reverse transcription was performed using an i Script c DNA Synthesis Kit (Bio-Rad 1708891) according to the manufacturer's instructions, using 90 ng of total RNA as template in a total reaction volume of 20 µl. The four target genes, as well as three reference genes (elongation factor 1αa (ef1αa), ribosomal protein s20 (s20) and hypoxanthine phosphoribosyltransferase 1 (hprt1)) were selected for q PCR. Previously published primer sequences (electronic supplementary material, table S1) were retrieved from the National Center for Biotechnology Information (NCBI: Calibration curves were run for all primer pairs (electronic supplementary material, table S2) and q PCR products were sequenced to confirm the specificity of the primers. The stability of the three reference genes ef1αa, s20 and hprt1 was evaluated (following protocol by Silver et al. [22]), after which s20 was selected as the most stable reference gene. Real-time PCR was carried out in duplicate using a Roche Light Cycler 96 (Roche Diagnostics, Penzberg, Germany) and accompanying software (version The reaction volume was 10 µl including 5 µl Light Cycler Green I Master (04887352001, Roche Diagnostics Gmb H, Mannheim, Germany), 1 µl of each forward and reverse primer (1 n M final concentration for each primer) and 3 µl of c DNA (diluted 1 : 5). Cycling conditions were 10 min at 95°C, followed by 40 cycles of 10 s at 95°C, 10 s at 60°C and 8 s at 72°C, followed by a melting curve analysis. Samples which had Cq values greater than 35 and/or technical replicates with an s.d. difference greater than 0.1 were eliminated following the methodology of q PCR analysis by Bustin et al. A calibrator, made by pooling aliquots of c DNA of all samples, was included in triplicate in all plates to allow for comparison of Cq values between plates. Expression values were calculated according to Vandesompele et al. [24], and are expressed as relative to the expression of the reference gene s20. Gene expression levels of the three-week control fish were normalized to 1, and data are presented as normalized values to this treatment control average (fold-change). The telencephalon samples (n = 4 per treatment) were homogenized using a Tissue Ruptor (Qiagen, Venlo, The Netherlands) and total RNA was extracted using the mi RNeasy mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. Integrity and concentration of the RNA were checked on a Bioanalyzer 2100 total RNA Nano series II chip (Agilent, Amstelveen, The Netherlands) and the median RIN value was 9.0. Illumina RNA-seq libraries were prepared from 0.5 µg total RNA using the Illumina Tru Seq Stranded m RNA Library Prep kit according to the manufacturer's instructions (Illumina, San Diego, USA). All RNA-seq libraries (150–750 bp inserts) were sequenced on an Illumina Hi Seq2500 sequencer as 1 × 50 nucleotides single-end reads according to the manufacturer's protocol. Image analysis and base calling were done using the Illumina pipeline. The reads were aligned to the latest version of the Atlantic salmon genome reference (ICSASG v. 1.2 [27] was used to remove secondary alignments, i.e. 2, NCBI Ref Seq GCF_000233375.1; [25]) using Top Hat v. alignments that meet Top Hat's reporting criteria but are less likely to be correct than simultaneously reported primary alignments. Alignments to annotated exons were counted and summarized at the gene level using HTSeq-count v. 0.10.0 using the ‘intersection-nonempty’ setting [28]. Raw read counts for 48 436 protein-coding genes were analysed in R v. 3.4.4 (R Development Core Team 2016) using the edge R package v. Initially, read counts were normalized using the TMM method and a multi-dimensional scaling (MDS) plot was generated to identify outliers. After outlier removal, the read counts were normalized again, and differential expression between the four treatment groups was calculated using edge R's recommended quasi-likelihood F-test for generalized linear models. MDS plots and differential expression were only calculated for genes with at least 10 aligning reads in each sample. For downstream analyses, reads per kilobase million (RPKM) expression values (normalized between samples and corrected for transcript length) were exported from edge R. Transcripts with a false discovery rate (FDR) less than 0.01 were considered to be significantly differentially expressed between treatments. 4.9.0 ( was used to visualize expression profiles in heat maps. Ref Seq.db/), and overrepresentation of ‘Biological Process’ categories was assessed using the R package GOseq [30], using the ‘Wallenius’ method and including correction for transcript length. Gene ontology (GO) annotations for the ICSASG_v2 assembly were retrieved using the Ssa. Body mass at the start of the experiment was compared using a Student's t-test. Two-way analysis of variance (ANOVA) was used to compare body mass and SGR, with treatment (training versus control) and time (three and eight weeks) as independent variables. The gene expression data included treatment, time and sex as explanatory variables. The most parsimonious model (with the lowest corrected Akaike Information Criterion score; AICc) was a model with only treatment and time as explanatory variable, and this model was subsequently used. Models were assessed by their capacity to explain the variability and interaction effects between treatment and conditions were accepted or rejected according to total model ‘lack of fit’ probabilities (provided by the ANOVA model). Differences between all groups were assessed by Tukey–Kramer honestly significant difference post hoc test. Before final acceptance of the model, diagnostic residual plots were examined to ensure that no systematic patterns occurred in the errors (e.g. fitted values versus observed values and q–q plots). Data outliers were determined by the χ = 2.54, p = 0.11] on body mass, with exercised fish having a higher body mass at eight weeks (p = 0.002), but not at three weeks (p = 0.99), compared to controls. In addition, there was a significant treatment [F Body mass and specific growth rate (SGR) for exercised (E) and control (C) fish at three and eight weeks. Repeated-measures ANOVA statistics are given within each panel. Small letters symbolize statistical differences obtained with Tukey post hoc test. = 3.35, p = 0.08] in the relative gene expression of syt in the Dl, with control fish at three weeks showing a higher expression, compared to all other groups (figure 2a). There were no effects of time [F Mean (±s.e.m.) relative m RNA expression (to the s20 reference gene) of the neuroplasticity markers synaptotagmin (syt) and brain-derived neurotrophic factor (bdnf) in the dorsolateral pallium (Dl), dorsomedial pallium (Dm) and the ventral part of the ventral telencephalon (Vv) of exercised (E) and control (C) fish at three and eight weeks. Small letters symbolize statistical differences obtained with Tukey post hoc test. Mean (±s.e.m.) relative m RNA expression (to the s20 reference gene) of the neurogenesis marker proliferating cell nuclear antigen (pcna) and the cell differentiation marker neurotrophic factor d (neurod) in the dorsolateral pallium (Dl), dorsomedial pallium (Dm) and the ventral part of the ventral telencephalon (Vv) of exercised (E) and control (C) fish at three and eight weeks. In order to determine whether exercise affects brain plasticity at the cellular level, we measured gene expression levels in the telencephalon of five fish per group using Illumina RNA-seq. We obtained between 10.1 and 39.1 million reads per sample (median 17 million), of which 90.6–95.8% (median 95.4%) was aligned to the salmon genome, of which, 63.2–79.1% (median 76.9%) could be attributed to a protein-coding gene. An MDS plot of all samples (figure 4) shows a clear clustering of each experimental group, indicating robust gene expression changes correlated with the treatments. We therefore analysed differential expression of 27 171 genes (which does not include genes below a very low expression threshold of 10 reads per sample; figure 5). The contrast between exercise and control groups yielded 1772 genes differentially expressed using a FDR cut-off of 1%, of which 923 had significantly higher expression in swimmers (electronic supplementary material, table S3), and 849 had significantly higher expression in control fish (electronic supplementary material, table S4). A selection of these genes is presented in table 1. Forebrain gene expression in exercised versus control salmon. Depicted are expression values of 27 171 genes, normalized for between-sample differences in sequencing depth and within-sample transcript length differences (RPKM, reads per kilobase per million). Genes highlighted in red are significantly differentially expressed between exercised and control groups. Selection of significantly (false discovery rate (FDR) less than 0.01) differentially expressed genes in exercised fish, compared to unexercised controls. Expression is given as fold-change (FC) difference in exercised:control individuals. To summarize which biological processes are over- or under-expressed in the swimmer group, we performed a GO category overrepresentation test on the sets of significantly differentially expressed genes between exercised fish and their controls. In total, 194 (electronic supplementary material, table S5) and 271 (electronic supplementary material, table S6) GO categories showed a significant overrepresentation of upregulated and downregulated genes, respectively (p Heat map depicting expression of differentially expressed genes within gene ontology (GO) categories related to neuroplasticity. In order to make the absolute expression levels and amplitudes of expression changes comparable between genes, for every gene the original read per kilobase million (RPKM) values were converted to z-scores (i.e. Hierarchical clustering based on Pearson correlation was used to arrange genes by similarity in expression pattern. We here report that sustained swimming exercise increases the expression of neuroplasticity- and cell proliferation-related genes in the telencephalon transcriptome of juvenile Atlantic salmon. Specifically, we report an upregulation of synaptic plasticity and neurogenesis, as well as downregulation of apoptosis genes in the whole telencephalon in response to exercise. In addition, there were region-specific differences in the expression of the neurogenesis marker pcna at both three and eight weeks, with higher expression in exercised fish in the Dl, which is the functional equivalent of the mammalian hippocampus. Furthermore, the expression of the neural plasticity markers syt and bdnf was also increased following exercise in the Vv, which is the functional equivalent to the mammalian lateral septum. Interestingly, exercised-induced increased neural plasticity in the lateral septum area has not been reported in mammalian studies and allows for novel target systems in the study of mechanisms associated with swimming, neural remodelling and cognition in fish as a model for other vertebrate systems. The neurotrophin BDNF is a well-characterized neural growth factor which is important for synaptic plasticity and neural survival [4,31,32]. In exercised mammals, BDNF shows a robust upregulation in the dentate gyrus of the hippocampus [33,34], as well as in other brain regions, such as the amygdala [35]. Furthermore, BDNF plays a key role in activating the signal transduction pathways which drive increased neural plasticity [33,36]. Interestingly, our results on region-specific gene expression in the Dl, Dm and Vv show that exercised fish show an upregulation of bdnf only in the Vv. BDNF synaptic signalling is mainly mediated by the Trk B receptor tyrosine kinase. The binding of BDNF to the Trk B receptor triggers the autophosphorylation of tyrosine and leads to the activation of signalling pathways [37]. Therefore, in order to elucidate further how the exercise-mediated increase in Bdnf may be affecting signalling pathways in fish, future studies should include the expression of both Bdnf and Trk B. Interestingly, we also found in the Vv an exercise-induced increase in synaptotagmin (syt). Synaptotagmin is a synaptic vesicle protein that is selectively required as a Ca sensor in the regulation of neurotransmitter release [38]. These results suggest that the Vv in fish is a target for exercise-induced changes in neuroplasticity and this may give exercised fish an advantage in stress coping and goal-oriented behaviour, an exciting possibility that should be further investigated. Curiously, we found that control fish at three weeks had the highest expression of syt in the Dl. The reasons for this difference are not clear to us, particularly since this group was exposed to standard rearing conditions and this effect is not present at eight weeks or in any other brain area or marker. Exercised fish showed significantly enhanced growth rates after eight weeks of training, compared to unexercised controls. Thus, our observation of enhanced growth in exercised fish suggests that our experimental treatment had beneficial effects on the physiology of the fish and did not lead to chronic stress. It can be argued that due to our experimental set-up, it is not possible to control for any potential tank effects affecting the results obtained in growth and gene expression. However, the result of enhanced growth due to increased swimming exercise in this study is in agreement with previous studies on salmonids (e.g. Furthermore, preliminary data from a pilot experiment we have conducted in a different experimental set-up shows that the telencephalic gene expression profile of fish subjected to a forced-exercise training regime is similar to the one reported in this study. After eight weeks of swimming, 1772 transcripts in the telencephalon were differentially expressed in exercised fish compared to unexercised controls. GO analysis attributed these differences in transcript abundance to increased expression of genes associated with processes relating to neural plasticity, such as dendritic spine development, synaptic plasticity and cell proliferation, as well as downregulation of genes associated with apoptosis in exercised individuals, compared to control fish. This is in agreement with mammalian studies, which report that exercise stimulates the forebrain, particularly the hippocampus, through the increased abundance of neurotrophins such as BDNF, as well as stimulation of memory and learning-related processes such as long-term potentiation [39,40]. Recent studies have proposed the molecular pathways underlying exercise-induced neurogenesis and synaptic plasticity (reviewed by Lista & Sorrentino [36]). In summary, physical activity in mammals first leads to an increased abundance of neurotrophins such as BDNF and insulin-like growth factor (IGF). Subsequently, BDNF can directly promote neurogenesis, or it may activate signal transduction pathways through signalling molecules, such as calcium/calmodulin-dependent protein kinase II (CAMK-II), mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and c AMP response element binding (CREB) protein, which in turn stimulate neural processes such as synaptogenesis and LTP. Furthermore, synaptogenesis is enhanced by synaptic trafficking molecules such as synaptotagmin and syntaxin, which are promoted through CAMK-II after activation by BDNF or IGF. Notably, our forebrain transcriptome gene expression profile analysis in exercised fish uncovered an upregulation of several genes within these pathways, such as synaptotagmin, syntaxin CAMK-II, MAPK, PKC and CREB, as well as two IGF receptor-related transcripts. [33] reported that running exercise activates the mammalian hippocampal glutamatergic system and suppresses the gamma-aminobutyric acid (GABA)ergic system. Similarly, exercised fish in our experiment showed increased expression of several glutamate receptor transcripts and reduced expression of several GABA receptor transcripts, although effects on the GABAergic system are somewhat ambivalent, as we observed concurrent upregulation of several GABA receptor subunit transcripts in exercised individuals. In summary, there appear to be several parallels between the teleostean and mammalian neural response to exercise in processes regarding synaptic trafficking, signal transduction and the glutamatergic and GABAergic systems. These findings suggest that many of the molecular pathways which have been proposed to underlie exercise-induced neuroplasticity are conserved between mammals and teleost fish. The transcriptome of exercised fish revealed a significant overrepresentation of downregulated genes in several GO categories related to apoptosis. This is an interesting observation, as mammalian studies have revealed that, besides promoting neurogenesis, exercise also increases cell survival [41] and can inhibit neuronal apoptosis, particularly in ageing animals [42] or individuals with traumatic brain injury [43]. Our observation that swimming exercise may also affect neuronal apoptosis in fish suggests that fish models may be particularly amenable in the study of exercise-induced neural repair in damaged animals, as fish with neural damage show an incredible capacity for neural regeneration in the central nervous system [6]. A better understanding of the effects of swimming exercise on neuroplasticity in fish species may have further ramifications for human disease research, as an important application of mammalian exercise-induced neuroplasticity is its potential to prevent cognitive decline, particularly in the context of ageing and neurodegenerative diseases (reviewed by Ma et al. Increased neurogenesis in response to exercise in mammals has been reported in the hippocampus and is associated with increased capacity for spatial tasks [2–4]. In agreement with these results, we found an increased pcna expression (a commonly used marker for neurogenesis, e.g. [44]) in the Dl, which is functionally equivalent to the mammalian hippocampus [13,45] and plays an important role in spatial memory in fish [46,47]. It is of particular interest that Luchiari & Chacon [7] report an improvement in learning in goal-oriented behaviour (i.e. classical conditioning) in zebrafish subjected to increased swimming exercise. Therefore, future studies should combine behaviour and brain region-specific studies in order to elucidate exercise-induced effects in fish. Interestingly, an increase in the expression of the neurogenesis marker pcna was not accompanied by increased expression of the cell differentiation marker neurod. In fact, we found that neurod expression decreased over time in the Dm of both groups, with the lowest values found at eight weeks. Neurod is a transcription factor involved in the differentiation of cells into maturation [48]. were provided by COST Action FA1304 ‘Swimming of fish and implications for migration and aquaculture’ (FITFISH) and the Research Council of Norway under the HAVBRUK programme, project no. Published by the Royal Society under the terms of the Creative Commons Attribution License which permits unrestricted use, provided the original author and source are credited.© 2020 The Authors. The lack of expression of this gene at the sampled timepoints suggests that exercise does not lead to increased cell differentiation, at least within the timeframe and within the forebrain areas that we studied. designed the study, processed microdissection samples, conducted all molecular biology analyses, analysed final datasets and drafted the manuscript. Published by the Royal Society under the terms of the Creative Commons Attribution License which permits unrestricted use, provided the original author and source are credited. In other words, the exercise-induced neurogenesis indicated by increased pcna expression may represent a recent increase in new-born cells which have not yet gone through differentiation and maturation. All authors gave final approval for publication.) and under the Marie Sklodowska-Curie Actions: Innovative Training Network ‘IMPRESS’, grant agreement no. Importantly, while expression of neuroplasticity-associated genes are commonly used as markers for neuroplasticity (for reviews, see Zupanc & Lamprecht [49]), it is important to note that m RNA gene expression does not always mirror protein levels. conducted all RNAseq data analysis and helped draft the manuscript. We therefore emphasize that future research on exercised-induced neurogenesis in fish species should corroborate the findings of this study using additional techniques for visualizing neurogenesis, such as 5-ethynyl-2′-deoxyuridine (Ed U) labelling and Pcna, Neuro D, Bdnf and Trk B immunohistochemistry. In conclusion, we report that juvenile fish show an exercise-induced increased expression of neuroplasticity and neurogenesis markers in the forebrain. coordinated study, helped with sample collection and helped draft the manuscript. We are among the first to study the effects of exercise on neuroplasticity in fish and our results uncover several parallels with mammalian studies, such as increased pcna expression in functional equivalent of the hippocampus in fish (i.e. designed and coordinated the study, helped with biometric data analysis, helped draft the manuscript. the Dl) and increased expression of neuroplasticity, synaptic trafficking and signal transduction in the telencephalon, with special emphasis in the fish's lateral septum functional equivalent (i.e. Future studies should study the link between exercised-induced neural plasticity and cognition in fish. In addition, histological studies looking at protein abundance of neurogenesis and neural plasticity markers should further elucidate the region-specific effects of exercise training in all regions of the fish brain. designed and conducted the study, conducted sampling and processed samples used in RNAseq, participated in biometrical data analysis and helped with RNAseq data analysis, helped draft the manuscript. These endeavours will help shed light on possible applications of increased swimming training in husbandry practices aimed at increasing animal welfare (aquaculture), as well as validating fish as an ideal model in the study of vertebrate neural mechanisms, including the functional relationship between exercise and neurodegenerative diseases. This experiment was performed in accordance with Dutch law for experimentation and procedures on live animals. 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