The hippocampus supports a cognitive map of space and is critical for encoding declarative memory . Recent studies have implicated hippocampal subfield CA2 in social and contextual memory but how it does so remains unknown. Here we find that in adult male rats, presentation of a social stimulus (novel or familiar rat) or a novel object induces global remapping of place fields in CA2 with no effect on neuronal firing rate or immediate early gene expression. This remapping did not occur in CA1, suggesting this effect is specific for CA2. Thus, modification of existing spatial representations might be a potential mechanism by which CA2 encodes social and novel contextual information. Recent work has implicated hippocampal subfield CA2 in encoding social and contextual memory yet the neural mechanisms are not known. Here, Alexander and colleagues demonstrate that, compared to CA1 neurons, CA2 neurons modify their place fields when presented with social or novel stimuli. We present evidence that CA2 place fields globally remap in response to social and novel contextual experiences. Interestingly, firing rates and IEG expression were unaffected by social experience. Thus, CA2 neurons have the capacity to update their spatial representations online in response to social or contextual changes in the environment, possibly as a means to encode social and novel contextual information. These results suggest that CA2 place cells may link social and novel contextual information with representations of space.In this study, we use P=0.0019, one-tailed unpaired Mann–Whitney test, n=61 CA2, 31 CA1 neurons; P<0.0001, one-tailed unpaired Mann–Whitney test, n=61 CA2, 31 CA1 neurons; P<0.001, one-tailed unpaired Mann–Whitney test, n=61 CA2, 31 CA1 neurons; P>0.05, one-tailed unpaired Mann–Whitney test, n=61 CA2, 31 CA1 neurons; P<0.001; CA1: P<0.001, n=61 CA2, 31 CA1 neurons, Wilcoxon signed-rank test; P<0.0001; CA1: P<0.0001, n=61 CA2, 31 CA1 neurons, Wilcoxon signed-rank test; First, we assessed the spatial properties of CA2 and CA1 neurons using single unit recordings from awake, behaving adult male rats, while animals explored a familiar open field arena . Spatial6activity-regulated cytoskeleton-associated protein (Arc), early growth response 1  and purkinje cell protein 4 (Pcp4) within the same sections. CA2 was defined by detection of Pcp4, the expression of which delineates the CA1/CA2 and CA2/CA3 borders , n=5–10 rats per condition; Arc transcription at 15 min, also returning to baseline by 30 min, similar to previous findings20Egr1 transcription foci was significantly increased in CA1 at 5, 15 and 30 min . Separate cohorts of adult male rats explored a familiar open field arena for either 3 or 10 min and were returned to their home cage for the remaining time until they were killed at 5 min (for the 3-min exploration condition), or 15 or 30 min (for the 10-min exploration conditions). Multiplexed FISH was used to probe for  borders . We also borders 3419. IEGd in CA2 .P>0.05 for novel and familiar, Wilcoxon matched-pairs signed-rank test; n=55 neurons for novel, n=49 neurons for familiar; see Arc foci after social exploration (familiar and novel) at the peak time point (15 min) was similar to the percentage after exploration of the context alone  was also similar to the percentage after exploration alone ; thus, about 60% of Arc+ neurons were Calb1+  than we could detect in CA2. Kohara greatest . Across nditions , the avein vivo before and during exposure to (1) a familiar male rat (prior cage mate), (2) a novel male rat or (3) a control condition with no social exposure to control for the passage of timeP<0.01, Kruskal–Wallis test with Holm–Bonferroni correction, control: n=31 neurons, familiar social: n=37, novel social: n=54; P>0.05, Kruskal–Wallis test, control: n=31 neurons, familiar social: n=37, novel social: n=54; 6P<0.01 unpaired Mann–Whitney test, CA2: n=31 neurons, CA1: n=28; Given that we were unable to detect changes in CA1 or CA2 activity levels during social stimulation, we considered the possibility that non-spatial information such as social stimuli is encoded by modifications in representations of space (place fields) in CA2. To test this possibility, we monitored activity of individual CA2 and CA1 neurons P=0.0661, Kruskal–Wallis test, control: n=28 neurons, familiar social: n=59, novel social: n=57; P>0.05, Kruskal–Wallis test, control: n=28 neurons, familiar social: n=59, novel social: n=57; As CA1 is the primary target of CA2 neurons, we expected some effect of social stimuli on CA1 spatial representations. CA1 spatial correlation values across the three conditions  were not significantly different from each other but did show a trend in that direction =39.49, two-way ANOVA, control: n=31 CA2 and 28 CA1 neurons, familiar social: n=37 CA2 and 59 CA1 neurons, novel social: n=54 CA2 and 57 CA1 neurons) and a main effect of exposure =6.512) but no significant interaction  and novel animal (P<0.0001) exposure but not during control exposure (P>0.05).Comparing CA2 and CA1 spatial correlations across the three conditions showed a main effect of brain area , but CA1 neurons showed no significant changes in spatial correlation values for social conditions  we obtained strikingly similar results  and 5. In=349): effect of area, F=449.5, P<0.001; effect of time, F=38.1, P<0.001). A significant effect of time was also found for CA2 and CA1 cells tested separately : F=15.2, P<0.001; CA1 (n=253): F=11.9, P=0.001; P<0.001; effect of condition, F=1.9, P=0.2; interaction, F=1.6, P=0.2; n-values same as above). Because of these effects, the correlations for temporally adjacent sessions were tested separately from A1–A4 correlations, and CA2 and CA1 cells were also assessed separately.Consistent with previous reports6n=11 neurons, familiar animal: n=47, familiar toy: n=38), spatial correlations for temporally adjacent sessions were significantly lower in the social stimulation  condition compared with the control and toy (familiar object) conditions =8.5, P<0.001; Bonferroni post hoc tests: P=0.04 for control versus familiar animal and P=0.001 for familiar animal versus familiar object; post hoc test: P=1.0). A1–A2 spatial correlations were significantly different across conditions =6.1, P=0.003; n-values same as listed above), whereas A2–A3 correlations were not significantly different =2.7, P=0.07; n-values as above). These results suggest that CA2 place cells remapped in response to social stimulation, and that stable maps were formed in CA2 to represent social stimulation. Spatial correlations for A3–A4 were also significantly different across conditions =8.6, P<0.001; n-values as above). For A1–A4 spatial correlations, which compare two no-stimulus conditions that flank sessions containing either no stimulus or a familiar animal or object, spatial correlations were significantly lower in the social stimulation condition than in the other conditions =7.4, P=0.001; Bonferroni post hoc tests: P=0.05 for control versus familiar animal and P=0.002 for familiar animal versus familiar object; n-values as above). Taken together, these results suggest that CA2 place cells remapped in response to social stimulation and did not revert back to the original A1 maps during the A4 session. We did not detect a bias in the direction of place field shifts for any of the stimuli . Unlike CA2, CA1 place cells did not show significant remapping in response to social stimulation =0.6, P=0.6, control: n=26 neurons, familiar animal: n=134, familiar toy: n=93; For CA2 neurons , Arc foci in CA2 were smaller under all conditions and time points tested. Single-molecule FISH is a very sensitive technique that affords substantially greater spatial resolution than traditional FISH methods. Thus, our observation that Arc foci were smaller in CA2 than in CA1 is indicative of lower rates of Arc transcription, consistent with the overall lower levels of Arc detected in CA2. This is probably because of CA2 neurons' robust ability to buffer and extrude calciumArc transcription2930Our results also demonstrated that Arc foci in CA2 at the 5-min time point than seen in CA1. We note that this may be due to CA2 having fewer and larger neurons that are more dispersed than those in CA1, so this percentage does not reflect the overall number of Arc foci+ neurons. However, one intriguing possibility is that transcriptional responses are indeed faster for a greater proportion of CA2 neurons than CA1 neurons. When taken together with the lower amount of Arc transcription in CA2, and that the percentage of Arc foci in CA1 surpasses the percentage of Arc foci in CA2 at the 15-min time point, a second scenario is that the higher percentage of Arc foci in CA2 at 5 min simply reflects earlier activation of more CA2 neurons. This may partly be explained by the stronger activation of CA2 by ECII than CA1 by ECIIIArc+ foci at the 5-min time point32Another interesting finding from our study was the higher percentage of neurons with Arc foci in CA2 and CA1 in the control condition compared with the familiar and novel social conditions at 5 and 15 min. This is likely to have resulted from the social condition rats traversing the side of the arena with the social stimulus more frequently, at the expense of time spent in the rest of the arena, with the rats thus visiting fewer place fields. Notably, at the 15-min time point when rats had 10 min to explore the arena with the social stimulus (versus 3 min at the 5-min time point), the percentage of cells with Arc foci in the social conditions was more similar to that of the control condition.We also observed a greater percentage of neurons that were positive for Calb1-expressing neurons and deep, Calb1-lacking neurons. Recent reports have shown using recordings from rat hippocampal slices in vitro that these populations are modulated via different and opposing inputs. Stimulation of CA3 evokes a predominantly excitatory response in superficial CA1 neurons and predominantly inhibitory response in deep CA1 neurons. Conversely, stimulation of CA2 evokes an inhibitory response in superficial CA1 and a biphasic excitatory response followed by an inhibitory response in deep CA1 . Rats were housed under a 12:12 light/dark cycle with access to food and water ad libitum. The experiments were conducted according to the guidelines of the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals under a protocol approved by the University of Texas at Austin Institutional Animal Care and Use Committee.For the studies in in vivo electrophysiology experiments presented in −1) and xylazine (10 mg kg−1), and placed in a stereotaxic device. Four to six metal ground and anchor screws were secured in the cranium. A total of eight tetrodes bundled in medical grade tubing were implanted into either CA2  or CA1 , except for one animal in which two bundles of four tetrodes each were implanted into CA2 and the contralateral CA1. Tetrodes were constructed from 12.7 μm polyimide-coated nickel chromium (Rediohm-800) wire , which were connected to a printed circuit board  and miniature connector . On the day of surgery, electrode tips were cut to the appropriate length and plated with gold to reduce electrode impedances to between 150 and 300 kΩ at 1 kHz by passing current though the wires, while the tips were immersed in gold solution . Implanted electrodes were secured with dental acrylic. Following surgical implantation, animals were treated with carprofen (5 mg kg−1) to alleviate postsurgical pain.For For the experiments in −1) to alleviate postsurgical pain. Implanted rats were singly housed in cages (40 cm × 40 cm × 40 cm) constructed from clear acrylic and containing enrichment materials .In the first rat, the stereotaxic coordinates (in mm) were −5.0 anteroposterior and 5.0 mediolateral; seven tetrodes hit CA1, one tetrode hit CA2 and three tetrodes hit CA3 (one tetrode did not reach its target). In the second rat, the coordinates were −5.0 anteroposterior and 4.4 mediolateral; six tetrodes hit CA1, one tetrode hit CA2 and five tetrodes hit CA3. In the third rat, the coordinates were −3.8 anteroposterior and 3.2 mediolateral; two tetrodes hit CA1, one tetrode hit CA2 and four tetrodes hit CA3 (five tetrodes did not reach their targets). In the fourth rat, the coordinates were −3.8 anteroposterior and 3.2 mediolateral; four tetrodes hit CA1, two tetrodes hit CA3 and one tetrode hit DG (five tetrodes did not reach their targets). The coordinates provided above did not exactly match with tetrode locations, because the diameter of the tetrode bundles was ∼1 mm. Tetrode placements were verified histologically at the end of each experiment. CA2 tetrodes were localized to the intermediate hippocampus in the first rat and the dorsal hippocampus in the second and third rats. CA1 tetrodes were localized to the dorsal hippocampus in all of the rats. Immediately following drive implantation, animals were treated with carprofen (5 mg kgFor experiments presented in For the experiments in Rats were familiarized to the empty testing enclosure for at least 3 days (three 20-min sessions per day) before behavioural experiments began. Rats were then familiarized to the social stimulation condition  and position data were stored with the neural data. When comparing neuronal firing properties before and during exposure to social or novel object stimuli or control exposures with no stimuli presented, the two conditions occurred within a single recording session. Each data file that contained the two conditions were spike-sorted and files were separated into the two conditions only after the sorting was complete, thereby minimizing the possibility of misidentifying neurons due to multiple recording files. Following recordings, neurons were sorted into individual units by tetrode mode-based cluster analysis in feature space using Offline Sorter software . Autocorrelation and cross-correlation functions of spike times were used as separation tools. Only units with clear refractory periods and well-defined cluster boundaries were included in the analyses53For experiments presented in For the experiments in Following recordings, neurons were sorted into single units offline using graphical cluster-cutting software . Clustering was performed manually in two-dimensional projections of the multidimensional parameter space . Autocorrelation and cross-correlation functions were used as additional separation tools. Each accepted single unit was required to exhibit well-defined cluster boundaries and a clear refractory period. Putative pyramidal cells were distinguished from putative interneurons on the basis of spike width, average firing rate and the presence of bursts. Putative interneurons, detected using these criteria, were excluded from further analyses.For experiments presented in 55For the experiments in For experiments presented in n) is different for spatial verses social analyses. All graphs show mean and s.e.m. Data were analysed using Python 2.7 and Graphpad PRISM 6 software, and significance was determined using an α-level of 0.05.Of the total 252 CA2 neurons and 181 CA1 neurons, 181 CA2 and 145 CA1 neurons were used for analysis of changes in firing rate and spatial properties in response to social object of control exposure. Of the 181 CA2 neurons used for the social and object experiments, 31 were used for control exposure, 49 were used for familiar social exposure (37 for remapping), 55 were used for novel social exposure (54 for remapping) and 46 were used for object exposure. Of the 145 CA1 neurons, 28 were used for control exposure, 59 were used for familiar social exposure and 58 were used for novel social exposure (57 for remapping). These data were collected on days after the spatial experiments above. As we cannot say that we recorded from identical neurons on subsequent days, it is possible that a different population of neurons were used for spatial analyses and social analyses. Thus, the number of cells  and CA2 neurons (n=61 neurons).where n=61 CA2 pyramidal cells and 31 CA1 pyramidal cells; Behavioural states were identified based on locomotor behaviour and the ratio of the power in theta band (5–11 Hz) to delta band (1–4 Hz) of LFPs, to eliminate any periods of sleep . In separate analyses, the same comparisons were made for only those cells that displayed place fields in the open-field arena .To test the effect of social exposure on average firing rates, locomotor/exploratory periods were compared before and during social stimulation. Comparisons of average firing rates between CA1 and CA2, and between pre-social and social (either familiar or novel) conditions, were made using Wilcoxon matched-pairs signed-rank tests. Comparisons were made for all neurons regardless of whether they displayed a place field in the open-field arena or for which video analysis rather than tracking data were used to determine behavioural state before and during exposures , novel social exposure (n=57 CA1 neurons and 54 CA2 neurons) and novel object exposure (n=46 CA2 neurons). Example tracking and occupancy data are presented in n=28 CA1 neurons and 31 CA2 neurons). Statistical significance was assessed using Kruskal–Wallis tests with Holm–Bonferroni corrections.Two methods the hippocampus uses to encode spatial information are global and rate remapping59elations  were cal10(1−x), with x representing the spatial correlation value, before performing repeated-measures ANOVA, two-way ANOVA and one-way ANOVA, as indicated in the P=0.9). Bonferroni tests were used for post hoc comparisons.For the experiments presented in To determine whether CA2 firing fields tended to shift in a particular direction , we calculated the shift of the centre of mass of the dominant place field  for each place cell. We then assessed whether the directions of place field centre of mass shifts for each condition were uniformly distributed using the Rayleigh test .n=5 rats per condition): context alone, context plus former male cage mate (familiar), context plus novel oestrus female (novel). A novel oestrus female was selected to maximize the subject rats' interest. Power analyses were done a priori, to determine animal numbers per condition . After a rat was habituated to the room for at least 1 h, it was placed in the arena and a familiar or novel rat was placed in the clear insert described above. For the context-only condition, nothing was placed into the insert. The rats were allowed to explore the context for either 3 min (5 min time point) or 10 min (15 and 30 min time points) and then placed back into their home cage until they were killed by rapid decapitation without anaesthesia at a total time of 5, 15 or 30 min from the onset of exploration. Each time point was performed with a separate cohort of animals and the 30-min time point was performed twice, because one brain was not usable for in situ during the first cohort (n=9). HCC animals were taken directly from their cage at different hours throughout the testing day and immediately killed (n=10). Brains were removed and flash frozen in isopentane cooled to −20 °C in a dry ice/ethanol bath.Rats were group housed for 1 week, to acclimate to their new environment, and then singly housed for the duration of the study. Rats were extensively handled on multiple days for 2 weeks before the start of the experiment, to control for stress during the task and to familiarize the rats to the experimenter. Rats were placed in the testing room for 1 h each day before the task began, to habituate to the testing room. After the 1-h habituation, individual rats were exposed to the arena described above for 10 min each day for 3 consecutive days, to habituate to the context. During habituation, the centre and four corners of the arena were baited with fruit-flavoured sucrose treats, to encourage exploration of the entire space. The arena was cleaned with 70% ethanol after each session. On the fourth day (test day), rats were randomized into three conditions using a random number generator . RNAscope technology allows for the detection of single RNA molecules by way of its zz oligo pair design and DNA-based amplification methodsPcp4 labelling and area CA1 was identified using its defined anatomical location. Each image was analysed in its unprocessed form and no thresholding was done before quantification. All statistical analyses were carried out using Graphpad PRISM 6 software and significance was determined using an α-level of 0.05.All images were acquired on a Zeiss 710 meta confocal microscope using a × 40 oil-immersion lens. The image acquisition and quantification were done under blind conditions. Our acquisition parameters were set using 3plexed negative controls  in each of the three channels  so that any signal above the level of background was acquired. Area CA2 borders were identified using z-stack using 1-μm steps. Images were processed using FIJI software (NIH v.1.47). Arc and Egr1 foci were counted in cells as previously describedArc or Egr1 foci when they: (1) showed at least one characteristic bright spot of fluorescence within the nuclei and (2) the bright spot appeared in at least three individual z-slices. The data are presented as average per cent of total nuclei with Arc or Egr1 foci±s.e.m. Owing to the difference in cell size and spacing across subregions, the CA2 region had ∼60 cells per image and CA1 had ∼90 cells per image. Data from the left and right hippocampi were not statistically different and were included in statistical analyses as one average per animal.One 212 μm × 212 μm region per subfield per hippocampus  was imaged in z-stack per hippocampus for the 15-min time point cohort only. Nuclei were designated as positive for Arc foci (as defined above) and/or Calb1 positive or negative. In addition, the total number of Calb1-positive and -negative cells were counted and the data are presented as average per cent Calb1+ or − cells expressing Arc foci. A total of 7,941 cells were included in the analyses. As proximal, centre and distal CA1 regions were included in these analyses, we report the percentage of cells with Arc foci from these three CA1 regions in Arc foci in proximal, centre and distal regions of CA1 were statistically different; however, as no significant differences were detected across conditions, the three CA1 regions were collapsed within each animal and graphs are presented as an average per cent across rats ±s.e.m. per condition . We also measured the average nuclear Arc fluorescence per Arc foci+cell using Zen Blue 2012 software (Carl Zeiss). The Arc foci+nuclei were identified and outlined using the DAPI channel with the contour tool and saved as a region of interest. The nuclear region of interest was used to measure the fluorescence intensity in the Arc channel for each Arc foci+nuclei and then classified as Calb1 positive or negative. The data are presented as average Arc nuclear fluorescence per cell for Arc+ cells only.Three 212 μm × 212 μm dorsal CA1 regions  were imaged in Forty-micrometre-thick vibratome-cut brain sections from perfused rats were rinsed in PBS and blocked for at least 1 h in 3% normal goat serum/0.01% Tween 20. Sections were incubated in the following primary antibodies: mouse anti-regulator of G-protein signalling 14  or mouse anti-STEP  and rabbit anti-PCP4 . Antibodies were diluted in blocking solution and sections were incubated for 24 h. After several rinses in PBS/Tween, sections were incubated in secondary antibodies  for 2 h. Finally, sections were washed in PBS/Tween and mounted under ProLong Gold Antifade fluorescence media with DAPI (Invitrogen).How to cite this article: Alexander, G. M. et al. Social and novel contexts modify hippocampal CA2 representations of space. Nat. Commun. 7:10300 doi: 10.1038/ncomms10300 (2016).Supplementary Figures 1-12, Supplementary Table 1, Supplementary Methods and Supplementary References


We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles.We generated a mouse with a CaV1.1 required for Ca2+ binding and/or permeation , we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca2+ stores during sustained activity. Decreases in these Ca2+-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca2+ binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers.Using mice with a pore mutation in Ca2+ binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.While not essential for excitation-contraction coupling, CaThe online version of this article (doi:10.1186/s13395-014-0027-1) contains supplementary material, which is available to authorized users. Although Ca2+ entry through CaV1.1 is not required for skeletal muscle ECC [V1.1 opens after the initial voltage-gated release event to allow Ca2+ to both bind and permeate the channel pore [2+ channels, CaV1.1 undergoes multiple conformational changes driven by membrane depolarization and/or Ca2+ movement (and/or binding) through the channel [V1.1 actually occurs after the opening of RyR1 [V1.1 has been suggested to facilitate RyR1 closing [2+ permeation via CaV1.1, this influx has been suggested to regulate clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Excitation-contraction coupling (ECC) in skeletal muscle involves a mechanical interaction between the L-type voltage-dependent Caannel CaV.1 and th channel ,4. The o of RyR1 , and CaV closing . Althougjunction -9 and mujunction .2+ binding and permeation through CaV1.1 contribute to the pathological development of several muscle diseases [V1.1 are associated with malignant hyperthermia (MH) in humans. The CaV1.1 mutations either enhance Ca2+ influx, increase the Ca2+ sensitivity of RyR1, and/or destabilize a closed state of RyR1. At least one of these MH mutations (R174W) ablates the L-type current and increases the sensitivity of RyR1 to caffeine, but does not alter ECC, demonstrating that the disease mechanism involves a distinct role for CaV1.1 [2+ permeation via CaV1.1 in muscle function than what is presently understood.Alterations in Cadiseases -14. Mutar CaV1.1 . Togethe2+ regulates multiple signaling pathways depending on the amplitude, frequency, duration, and location of the Ca2+ signal  due to its ability to cluster nearby and, upon Ca2+ binding, to interact with CaV1.x channels [V1.x channels prevail over other Ca2+ influx channels with respect to transducing excitation-transcription coupling (ETC) [et al. [V1.2 and phosphorylates the carboxy-terminal tail of the α1 subunit to increase Ca2+ influx. The interaction of CaMKII with CaV1.2, in turn, facilitates CaMKII auto-phosphorylation.In skeletal muscle, as in most cells, Casterblad ). CaMKIIchannels . CaV1.x ng (ETC) ,19. Hudm [et al.  demonstr2+ permeation through CaV1.1 in skeletal muscle function, we created a mouse with a Ca2+ binding and permeation defect in CaV1.1. We demonstrate that this CaV1.1-mediated pathway utilizes the flexibility of Ca2+ signaling  to regulate CaMKII and calcineurin activation, thereby enhancing SR Ca2+ store refilling and protein synthesis to modulate fatigue susceptibility, muscle size, and fiber type distribution.To directly delineate the role of CaN-benzyl-p-toluene sulfonamide (BTS) was purchased from Tocris Biosciences , latrunculin A from AdipoGen , and 4-chloro-m-cresol (4CmC) from Pfaltz & Bauer . The calcium dyes fura-2 AM and mag-fluo-4 AM were purchased from Invitrogen . Molecular weight marker for western blotting was purchased from GenDEPOT . Every other reagent including Insulin, KN-92, KN-93, and the myristoylated autocamtide 2-related inhibitory peptide (AIP) were purchased from Sigma-Aldrich .ad libitum. All procedures were approved by the Animal Care Committees at Baylor College of Medicine  and the University of Rochester .In this study, male mice between six and 12 weeks old were used ), unless otherwise indicated. All mice were housed at room temperature with a 12:12 hour light-dark cycle and provided with food and water V1.1. This selection marker-containing fragment was used to isolate a larger CaV1.1 genomic clone from a mouse 129 phage library via homologous recombination in Escherichia coli [The EK mutation was engineered on a genomic fragment of about 500 bp containing the exon-encoding residue E1014. A tetracycline resistance gene cassette  and a neomycin cassette  were inserted into the middle of the engineered fragment, flanked by about 250 bp homologies to Cahia coli . Severalhia coli .To expedite the transfer of the E1014K mutation from the 129SvEv mouse sub-strain background to a congenic C57BL/6 J background, speed congenics were used, in addition to conventional backcrossing. We identified 92 microsatellites of maximal base-pair length disparity between the 129SvEv and C57BL/6 J strains. These microsatellites were chosen to representatively span the entire genome and show distinct electrophoretic separation. Primer pairs were selected to PCR amplify the chosen microsatellites to be resolved by electrophoresis in Spreadex EL300 Wide Mini Gels . Finally, we compared 129SvEv and C57BL/6 J DNA standards to DNA from our backcrossed mice, and selected mice with the most sequence homology to C57BL/6 J DNA to be used in the next backcross. This method both ensured congenicity between the wild-type (WT) and EK mutant mice and provided a means to more quickly begin experimentation.Skeletal muscle fibers were isolated from the flexor digitorum brevis (FDB) muscle obtained from WT and EK mice as described .V1.1 currents (ICa) in FDB fibers isolated from WT and EK mice. FDB fibers were bathed in an external recording solution containing (in mM): 157 TEA-methanesulfonate, 2 CaCl2, 10 HEPES, 0.5 anthracene-9-carboxylic acid (9-AC), and 0.1 BTS, at pH 7.4, adjusted with TEA-OH. The patch pipette internal solution contained (in mM): 140 Cs-methanesulfonate, 10 HEPES, 20 Na-EGTA, and 4 MgCl2, at pH 7.4, adjusted with CsOH. All reagents here were purchased from Sigma Aldrich . The patch pipette resistance when placed in the external solution was between 0.6 and 1.0 Mohm. Fibers were voltage-clamped at a holding potential of −80 mV. Series resistance was compensated up to 80%. Data were sampled every 120 μs and filtered using a low pass Bessel filter  with a 2 kHz cut-off frequency. ICa was activated by 200 ms depolarizing pulses ranging from −40 mV to +60 mV in 10 mV increments delivered every 10 seconds. CaV1.1 current-voltage relationships (ICa-V) were obtained from peak currents measured during each depolarization normalized to cell capacitance and plotted against the corresponding test potential. ICa-V data were then fitted by the following modified Boltzman equation:max is the maximal L-channel conductance, V is test potential, Vrev is the L-channel reversal potential, V0.5 is the potential for half-maximal activation of Gmax, and kg is a slope factor.The whole-cell patch clamp technique was used to assess CaV1.1 currents were analyzed using Igor Pro 6  and Clampfit 9  software.Ca2, 1 MgCl2, and 10 HEPES, at pH 7.4. Fibers were then washed and incubated for 20 minutes in dye-free Ringer’s solution supplemented with 20 μM BTS, a skeletal muscle myosin inhibitor, to block contraction. Mag-fluo-4 AM-loaded FDB fibers were excited at 480 ± 15 nm and fluorescence emission detected at 535 ± 20 nm was collected at 10 kHz using a photomultiplier system. Electrical field stimulation  was elicited using a glass electrode placed adjacent to the cell of interest. Peak changes in mag-fluo-4 fluorescence for all 10 stimuli were measured as (Fmax-F0)/F0 and then averaged to generate a single peak value for each fiber. The rate of mag-fluo-4 fluorescence decay for the second, third, and fourth twitches for each fiber was fitted to a first order exponential function and the resulting amplitude and tau values were averaged.Acutely isolated FDB fibers were loaded for 20 minutes at room temperature with 4 μM mag-fluo-4 AM in a Kreb’s Ringer solution containing (in mM): 146 NaCl, 5 KCl, 2 CaCl2+ quench of fura-2 emission was measured in myotubes loaded with 5 μM fura-2 AM for 1 h at 37°C in Kreb’s Ringer solution. Briefly, prior to Mn2+ quench measurements, myotubes (primary cultured cells from muscle of mice) were treated with 100 μM ryanodine to block RyR1-mediated Ca2+ release during subsequent KCl application. Fura-2-loaded myotubes were excited at the experimentally determined fura-2 isosbestic point (362 nm) and emission monitored at 510 nm during perfusion of 50 mM KCl in the presence of 0.5 mM Mn2+. Maximum rates of fura-2 quench during KCl application were determined and evaluated for statistical significance.Mn2+ release during electrical stimulation, FDBs fiber were loaded with 5 μM of mag-fluo-4 AM for 30 minutes at room temperature in the presence of 20 μM of BTS. Loaded FDBs were placed on the stage of a confocal microscope with an adapted perfusion system (tyrode with 20 μM BTS at 0.5 mL/min) and imaged in line scan mode using the 20x objective (EC Plan-Neofluar) mounted in the confocal microscope , one line was acquired every 1.15 milliseconds (3.66 μsec/pixel time) using the 488 nm excitation laser and the LP 505 emission filter . FDBs were stimulated with 50 square electrical pulses (200 μsec duration) at 50 Hz and the produced florescence transients was normalized (F/F0) and plotted.To monitor Ca2+ store content, 1 mM of 4CmC was perfused at 3.25 ml/min, applied after 60 trains of electrical stimulation. Mag-fluo-4 fluorescence was collected at 20 Hz. Data were collected and analyzed using Metafluor version 6.2 software .Isolated FDB fibers were loaded with 5 μM of mag-fluo-4-AM for 30 minutes at room temperature, followed by washout with fresh DMEM . Electrical stimulation was performed using two platinum wires placed at each side of the fiber oriented longitudinally and fatigue was induced with uninterrupted application of electrical trains  for 300 seconds. For evaluation of RyR1-releasable SR Ca3VO4, 10 NaPyroPO4, 10 β-glycerophosphate, 10 NaF, PMSF, protease inhibitor cocktail (Santa Cruz), 1% NP40, 1% sodium deoxycholate, and 0.1% SDS . Equal amounts of total protein from whole muscle lysates were resolved by electrophoresis, transferred to PVDF  membrane and western blot analyses were performed using antibodies shown in Additional file Muscles were homogenized and lysed in ice-cold RIPA buffer consisting of (mM): 25 Tris pH 7.6, 150 NaCl, 1 Na2HPO4 and 81.4 mM NaH2PO4, pH7.2) for one hour at room temperature, washed twice with phosphate buffered saline (PBS) , permeabilized, and blocked in PBS containing 0.5% Triton X-100 and 5% BSA overnight at 4°C   according to the instruction of the user manual using an anti-goat MINUS PLA and anti-rabbit PLUS PLA probes and the orange detection reagent (Cy3) . Fibers were imaged with confocal microscopy . For analysis, Z stacks were projected and saved as a single image. The positive spots were counted with Image J , σ = 1, and same threshold per set adjusted at 15-30). The number of spots counted was normalized to the area of the fiber estimated from the width and length of the fiber in the image. For each set of experiments, the average counts of WT fibers (control) were set to 100% and the number of spots in each experimental condition was calculated as percentage change.We used proximity ligation assays (PLAs) to identify proteins that are within 40 Å of each other. PLA was performed on single FDB fibers plated on glass disks. Fibers were kept at 37°C in a 95% Oet al. [Eight-week-old WT and EK mice were fasted for 12 hours and then given an intraperitoneal injection of insulin (1 U/kg) diluted in saline. Control mice were injected with saline. After 7.5 minutes mice were sacrificed and muscles (soleus and EDL (Extensor Digitorum Longus)) were isolated, frozen in liquid nitrogen, and stored at −80°C until use. Muscle levels of pAkt1/2 and pGSK3β in the presence and absence of insulin were measured as described in Butler et al. .in vivo SUnSET technique [For measurement of protein synthesis we used an echnique ,25. BrieRas activity was measured using a Ras activity assay kit . Briefly, muscle was lysed in buffer and protein concentration was measured. Raf-Ras binding domain (RBD) beads (50 μl) were added to the muscle lysates  and the mixture was incubated at 4°C on a rotator for one hour. After incubation, Raf-RBD beads were pelleted by centrifugation at 5,000 × g at 4°C for one minute and washed with wash buffer. The bound active Ras was eluted in the two × sample buffer by boiling for three minutes. Eluted protein was run on 12% gel, transferred to PVDF membrane, and immunoblotted with Ras-specific antibody. The westerns were normalized to the amount of GAPDH in the muscle lysates (60 μg).2+-ATPase (SERCA) activity in tissue homogenates was performed as described [Sarco/endoplasmic reticulum Caescribed ,27.et al. [2/CO2, as above. After a 30 minute resting equilibration period, muscles to be stimulated underwent a fatigue protocol  for five minutes per muscle. At the completion of the stimulation protocol, rested and stimulated muscles were immediately frozen in liquid N2 and stored at −80°C. For muscles treated with KN-93, the drug was added to the chamber at the start of the 30 minutes equilibration period, at a final concentration of 5 μM.To assess stimulation-induced changes in signaling pathways we used the method of Sakamoto et al. . Intact 2/CO2. Muscles were tied with sutures at the musculotendinous junction and suspended between a force transducer and stationary anchor within a test chamber filled with warmed (35°C), oxygenated incubation medium. After a 20 minutes rest to allow mounted muscles to equilibrate, muscle optimal length (lo) was determined via single-twitch force generation measurements. Next, force frequency measurements were obtained at lo using frequencies from 15 to 300 Hz at 200 ms/train followed by a fatigue protocol performed over five minutes per muscle. The specific fatigue protocol for each muscle used was for the soleus: 15 Hz, 200 ms duration, one second intervals) and for EDL: 60 Hz, 200 ms duration, one second intervals. Muscle stimulation occurred within the test chamber using platinum electrodes attached to a Grass S48 stimulator and recorded within Chart5 (version 5.2) software .Intact soleus and EDL muscles were removed and immediately immersed in incubation medium comprised of KRS (oxygenated with a 95/5% mixture of O2b), MHCIIa , and/or MHCIIb  antibodies diluted 1:50 in PBS. After washing with PBS, muscle sections were incubated at room temperature for 90 minutes with isotype-specific AlexaFluor-594-conjugated goat anti-mouse IgG2b, AlexaFluor-488-conjugated goat anti-mouse IgG1, and AlexaFluor-594-conjugated goat anti-mouse IgGM secondary antibodies diluted 1:200. After three consecutive washes with PBS, muscle slides were mounted with VECTASHIELD mounting media . Images were taken under a fluorescence microscope . The relative numbers of the different fiber types were quantified and normalized by the total number of muscle fibers per field.Skeletal muscles (soleus and EDL) were dissected, embedded in OCT compound , and frozen in 2-methylbutane  precooled in liquid nitrogen. The frozen muscles were sectioned with 10-μm thickness using a SHANDON cryostat microtome . Immunofluorescent staining was performed using specific antibodies against myosin heavy chain I (MHCI), IIa (MHCIIa), and IIb (MHCIIb) . Briefly, sections were rehydrated with PBS for 10 minutes, followed by incubation at 4°C overnight with MHCI  calculations, 10-μm-thick frozen sections were obtained from the mid-belly area of the soleus and EDL muscles. Sections were immunostained for fiber type as described in the previous section, imaged at 10x magnification through an Olympus DP70 camera , and saved in .tif format. Saved images were then imported into Photoshop CSE version 10.0  for analysis. First, measurement scale was established by tracing a within-image scale bar (μm). Next, myofiber CSA was measured by tracing the external border of individual myofibers using the Magnetic Lasso tool. Myofibers exhibiting evidence of tears or processing artifacts were excluded from the analysis. Recorded measurements were then exported into Excel for analysis, with resulting CSA values reported in μmt-test. P <0.05 was considered to be statistically significant. *P <0.05, **P <0.01, and ***P <0.001 were used to indicate statistical significance.We performed a statistical analyses of two groups using the Student’s 2+ influx via CaV1.1, we created mice with a knock-in mutation (E1014K or EK) in the pore region of CaV1.1. The mutation of a glutamate to lysine residue in the repeat III pore region of CaV1.x abolishes both Ca2+ binding to this site [To explore the role of Cahis site  and diva2+ influx through CaV1.1, we compared whole-cell Ca2+ currents . The magnitude and decay kinetics of voltage-gated Ca2+ transients  elicited by a low frequency train of electrical stimulation (1 Hz) were not significantly different between FDB fibers from WT and EK mice  is unliand Na+;  relative2+ quench of fura-2 fluorescence assay to determine the effect of the EK mutation on excitation-coupled Ca2+ entry (ECCE) [V1.1 channels, providing a pathway for Mn2+ entry to quench fura-2 fluorescence  is absent in myotubes from EK mice  . In WT mr et al. . The EK 2+ transients during repetitive stimulation. During a single train of high frequency stimulation, the amplitude of the Ca2+ transients was lower in EK compared to WT fibers  of 4-chloro-m-cresol (4CmC) were significantly lower in EK compared to WT fibers .While the data in Figure 2+ indicator, fura-2 AM, to measure changes in cytosolic Ca2+ levels. Resting cytosolic Ca2+ levels did not differ between EK and WT fibers. However, following stimulation  [2+ transients  , and fous Figure H, and res Figure I in FDB 1 Figure J.2+ is required for mTORC1 activation of p70 ribosomal S6 kinase 1 (S6K1) [2+ permeation through CaV1.1 altered protein synthesis, we used the SUnSET technique [2+ permeation through CaV1.1, we examined the phosphorylation status of several key regulators of protein synthesis. We found that pS2448 mTOR, p-T37/T46-4EBP1/4EBP1, and p-S235/S236-S6/S6 were reduced in the soleus and EDL of insulin-treated EK mice compared to insulin-treated WT mice  upstream of mTORC2, PDK1, and ERK signaling, especially in the steps that lead to Ras activation.Ca1 (S6K1) -39. To dechnique  to asses2+ permeation and binding to CaV1.1 and activation of CaMKII in the regulation of these pathways, we isolated soleus and EDL muscle from WT and EK mice, electrically stimulated in the presence and absence of KN-93 (an inhibitor of CaMKII) and again analyzed the pathways described in Figure The data with and without insulin suggest that the effects of the EK mutation on insulin-stimulated protein synthesis involve both Akt phosphorylation (PDK1 and mTORC2) and ERK signaling. To assess Ras activation in EK and WT muscle, we pulled down activated Ras (GTP bound) with Raf-RBD-beads from soleus and EDL muscle and western blotted with RAS specific antibody to evaluate the amount of Ras that pulled down. As shown in Figure 2+ permeation though or binding to CaV1.1 activates CaMKII which, in turn, phosphorylates Raf to activate Ras/Raf and ERK1/2. ERK1/2 then activates mTORC1  was not different between muscles of young (between eight and 12 weeks of age) WT and EK mice  decrease of approximately 25% . However, we found no significant differences in grip strength, voluntary monitored wheel running, or endurance running in young (eight to 12 weeks of age) EK compared to WT mice, suggesting that the effects of the EK mutation on fiber type distribution, muscle contractility, and fatigue are relatively mild in young mice but increase in severity with age.The observed changes in activity dependent Cae Figure A and B, e Figure C and D. e Figure E and fure Figure F. Changee Figure G-J, but e Figure I. A decre Figure K and L. 2+ and in the presence of dihydropyridines were previously reported [2+ permeation through CaV1.1 in skeletal muscle has largely been ignored because Ca2+ influx through this channel is not required for ECC [2+ signaling in skeletal muscle is significantly altered by a permeation defect in CaV1.1 . This mutation resulted in concurrent inhibition of multiple Ca2+-sensitive pathways. These signaling changes translated to significant effects on muscle contractile function, providing compelling evidence that Ca2+ binding and/or permeation via CaV1.1 is important for maintaining optimal muscle physiology.Although acute changes in depolarization-induced contraction in low Careported , the rol for ECC . We demo2+ entering the muscle fiber through CaV1.1 is adequate to directly drive these changes in intracellular Ca2+ handling during repetitive stimulation. If so, previous studies have either underestimated the magnitude and relative contribution of Ca2+ influx through CaV1.1 during repetitive stimulation of muscle, or CaV1.1 Ca2+ binding and permeation activates a pathway (such as CaMKII) that promotes Ca2+ entry through a second Ca2+ influx channel . Another possibility is that Ca2+ binding to the selectivity filter triggers a conformational change in CaV1.1 that drives autophosphorylation of CaMKII bound to the channel (such as the C-terminus). CaMKII, activated by this mechanism, would then modulate multiple Ca2+ handling and downstream signaling pathways . The activation of these pathways would then alter numerous aspects of muscle physiology including fatigue, muscle fiber CSA, type II fiber type specification, and protein synthesis. Indeed, we demonstrate that autophosphorylation of CaMKII is decreased in the muscle of EK mice that have undergone repetitive stimulation, a condition that significantly enhances p-CaMKII levels in WT muscle.The question that arises is whether the amount of Ca2+-sensitive signaling pathways depends on the location, frequency, amplitude, and duration of the Ca2+ signal , since Ca2+ store refilling is decreased by CaMKII inhibitors in WT, but not EK fibers. However, at this point it is unclear whether the observed reduction in activity-dependent SR Ca2+ store content in FDB fibers from EK mice is due to loss of CaV1.1 Ca2+ influx or a downstream effect of CaMKII activation on SR Ca2+ reuptake and/or store-operated Ca2+ entry.The activation of different Casterblad ). ImportV1.1/CaMKII-dependent event that modulates insulin-mediated protein synthesis is upstream of PDK1 and/or mTORC2 activation, since phosphorylation of both Akt T308 (a PDK1 target) and Akt-S473 (an mTORC2 target) [2+-dependent regulation of the PI3K/Akt/mTOR and Ras/Raf/ERK pathways [One of the major findings in this study was a substantial decrease in insulin-mediated protein synthesis in EK muscle, as assessed by puromycin labelling and immunoblot analysis of the mTOR signaling pathway. The mTORC1 pathway activation in response to electrical stimulation is also decreased in EK compared to WT muscle. Decreased protein synthesis in response to growth factors such as insulin and repetitive stimulation may underlie the observed decrease in muscle fiber size in EK muscle. Activation of downstream substrates of mTORC1 was significantly decreased in EK muscle, as was activation of the upstream regulators of mTOR, Akt, and ERK1/2. We suggest that the Ca target) ,45 are dpathways -39.et al. [2+ influx associated with a deficiency in the β-subunit of CaV1.1 altered the formation of the neuromuscular junction. This may reflect a recently identified transcriptional role for the β1a-subunit [V1.1 Ca2+ currents, since mice with the EK mutation in CaV1.1 lack Ca2+ currents but do not exhibit alterations in neuromuscular junction preformatting (Additional file The phenotypic changes associated with the EK mutation do not arise from any obvious changes in the neuromuscular junction. Chen et al.  found th-subunit , as oppo2+ permeation through CaV1.1 is small, there is a significant impact of this pathway on muscle function. Changes include increased fatigue, decreased muscle fiber diameter, increased type IIb fiber specification, and decreased protein synthesis. Thus, our results indicate that small changes in CaV1.1 Ca2+ permeation, and CaMKII activation are amplified by changes in the activity of multiple critical downstream signaling pathways that impact a broad range of skeletal muscle functions. We propose that Ca2+ permeation through CaV1.1, while not required for ECC, modulates multiple downstream Ca2+-sensitive signaling pathways to improve muscle function.While the magnitude of Ca


In vitro studies have demonstrated that mouse fibroblast cell lines  that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication.As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes.in vivo for EIAV invasion and replication.Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential The online version of this article (doi:10.1186/s12977-015-0163-7) contains supplementary material, which is available to authorized users. Equine infectious anemia virus (EIAV) is the etiological agent for equine infectious anemia (EIA), a disease affecting equidae in most parts of the world. The infected animals, mainly horses and ponies, exhibit typical viremia, which is accompanied by fever, anemia, thrombocytopenia, edema, and weight loss. The virus maintains a certain level of stable replication in the peripheral blood and tissues, which are enriched with macrophages -3. EIA iTherefore, humanized mouse models, such as severe combined immunodeficient (SCID) mice implanted with human fetal thymus and liver  or Rag22ELR1, a member of the TNFR superfamily, has been identified as the sole receptor for EIAV . The posin vitro studies. To investigate this phenomenon, we generated transgenic mice carrying the ELR1 and eCT1 genes by microinjecting ELR1 and eCT1 recombinant plasmids together into fertilized oocytes from B6D2F1 (C57BL/6 × DBA/2F1) mice. This procedure was performed at the Liaoning Key Facility of Transgenic Laboratory Animals, China Medical University .NIH 3T3 cells (a cell line derived from mouse embryonic fibroblasts) that express the EIAV receptor ELR1 and eCT1 have been generated previously; these cells could support the productive replication of EIAV and the production of infectious virions at levels similar to those detected in the permissive equine dermal cell line (ED) . HoweverThe integration of exogenous ELR1 and eCT1 was identified in the genomes of four of six founder mice (from tail DNA samples) using polymerase chain reaction (PCR)  was performed using the primer sets ELR1QP-F/ELR1QP-R and eCT1QP-F/eCT1QP-R, as shown in Additional file in vitro, mouse embryonic fibroblasts (MEFs) and blood monocyte-derived macrophages (mMDMs) isolated from the ELR1- and eCT1-expressing transgenic mice and wild-type mice were prepared, as previously described [3 TCID50 of an EIAV pathogenic strain EIAVDLV34 (a donkey MDM-adapted virulent strain that was obtained from 34 passages that retained pathogenicity to horses), after detecting the ELR1 and eCT1 genes by PCR  using PCR 72 hours later, as previously described [env fragment in the culture supernatant of both the MEF and mMDM cultures from the transgenic mice, but not the wild-type mice  ELR1/eCT1 mice (n = 8) and wild-type mice (n = 8) with 104 TCID50 EIAVDLV34. Subsequently, using real-time RT-PCR, the copy number of the virus was detected in the plasma of peripheral blood obtained at week 0 , as well as at weeks 2 (W2) and 4 (W4) (post-injection using the primers EIAV-gag-F and EIAV-gag-R), as shown in Additional file The In addition, the virus distribution in the organs of the infected mice was investigated. Different from the relative expression levels of ELR1 and eCT1 mRNA and protein, which were most highly expressed in the intestine, the EIAV genomic RNA was detected in two secondary lymphoid organs, the spleen and lymph nodes, at significantly high copy numbers ; however, the viral RNA was only slightly above detectable levels (approximately 100 copies/μg) in the intestine, kidney, lung and liver  and immunohistochemistry (IHC), respectively. The EIAV-specific RNA probes used in FISH are shown in Additional file Furthermore, to provide direct evidence of EIAV infection and proliferation in ELR1/eCT1 mice, EIAV RNA and protein in the infected spleen and lymph nodes were examined using RNA-RNA fluorescence To investigate whether the proliferation of EIAV exerts a pathogenic effect on the involved organs, morphology changes in the tissues were examined by hematoxylin-eosin staining under optical microscopy. Significantly decreased numbers of lymphocytes were observed in the outer cortex or inner medulla region of the lymph nodes of ELR1/eCT1 mice compared with the organs from wild-type control mice. Furthermore, mononuclear phagocyte-like cells, with an extensive accumulation of deposited hemosiderin, were found clustering at many sites in the spleens of the transgenic mice Figure .Figure 4Previous studies of organs from horses acutely infected with EIAV demonstrated that the high levels of circulating viral load observed during acute EIA were associated with high levels of virus replication in macrophage-rich organs, including the spleen, lymph nodes, liver and kidney ,22,23. Oin vivo circulating virus-antibody complexes can bind to erythrocytes through the Fc receptor or the complement receptor; next, complement is activated through the classical pathway, resulting in the phagocytosis of the involved erythrocytes by macrophages and neutrophils and leading to hemolysis. Large amounts of hemosiderin are regularly observed in the spleens of acutely EIAV-infected horses [After infection by EIAV, d horses . In the d horses . Togethein vivo replication of EIAV, and we showed that ELR1 and eCT1 play a vital role in EIAV replication and production in vivo. Because it is more expensive to use horses in animal experiments and horse-related antibodies and reagents are not easily obtained, ELR1/eCT1 mice may provide an alternative in vivo infection model that is highly reproducible, inexpensive and widely available for evaluating the effectiveness of candidate EIAV vaccines and for studying the pathobiology of EIAV infection.In conclusion, we constructed for the first time a novel fully transgenic mouse (ELR1/eCT1 mice) that supports the Animal experiments were approved by Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and performed in accordance with animal ethics guidelines and approved protocols. The Animal Ethics Committee approval number was SYXK (Hei) 2011022.


Astrocytic glycogen degradation is an important factor in metabolic support of brain function, particularly during increased neuronal firing. In this context, glycogen is commonly thought of as a source for the provision of energy substrates, such as lactate, to neurons. However, the signalling pathways eliciting glycogen degradation inside astrocytes are themselves energy-demanding processes, a fact that has been emphasized in recent studies, demonstrating dependence of these signalling mechanisms on glycogenolytic ATP. Glycogen, the storage form of glucose in animals and fungi, provides cells with a readily accessible pool of metabolic energy. The complex polysaccharide was first isolated and described in the 19th Century by Bernard [As early as 1972, it was reported that brain glycogen, residing almost exclusively in astrocytes . Today, Astrocytic glycogen degradation is often regarded only in the light of either providing lactate as an energy substrate to neurons as part of the astrocyte–neuron lactate shuttle hypothesis ,5 or spaThe present review focuses on the most prominent glycogen-degrading signals in astrocytes, highlighting their metabolic demands and evidence for reliance on glycogenolytic ATP.Degradation and synthesis of glycogen are regulated in opposing ways, with phosphorylation and increased AMP concentration activating degradation and at the same time inhibiting synthesis . GP, theGP was first characterized in the 1930s by Cori et al. . A few y2+ or cAMP [2+ directly activates the GP-phosphorylating enzyme phosphorylase kinase (PK) by binding to its calmodulin subunit. PK is also activated by protein kinase A (PKA), which in turn is stimulated by cAMP , the enzyme generating cAMP, and phosphodiesterase (PDE), which catalyses cAMP degradation, can be positively or negatively regulated by Ca2+ [Moreover, cytosolic cAMP concentration is tightly coupled to Ca by Ca2+ –19.1-, α2-, β1- and β2-adrenergic receptors (ARs) [Although the somata of noradrenergic neurons are confined to two brain stem structures , their projections reach throughout the entire cortex and cerebellum, making noradrenaline (NA) an important and versatile modulator in various processes, such as circadian control or learning and memory ,21. Morers (ARs) . In factrs (ARs) .2+ or cAMP, or inhibit degradation by inhibiting cAMP production , which catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (IP3), the latter triggering release of Ca2+ from endoplasmic reticulum (ER) stores [Both β of cAMP ,30. α1-A) stores ,32.2-AR, which as a Gi-coupled receptor would be able to inhibit cAMP generation and therefore suppress glycogen degradation and stimulate glycogen synthesis, implying that the same messenger could induce mobilization and replenishment of astrocytic glycogen stores. This concept has been experimentally verified, showing that NA acting via α2-AR can increase astrocytic glycogen turnover [Interestingly, astrocytes can also express αturnover .2+ released from the ER upon IP3 signalling has to be removed from the cytosol by the actions of Ca2+-ATPases, such as plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).There are several ATP-requiring steps involved in adrenergic signalling, the most immediate being recovery of GTP. As G-protein-coupled receptors, ARs rely on provision of GTP upon ligand binding and signalling is terminated by hydrolysis of GTP to GDP. To regain GTP for new receptor activation, nucleoside diphosphate kinase (NDK) catalyses transfer of the γ-phosphate from ATP to GDP , therefo+, released during neuronal excitation, is taken up by astrocytes, triggering glycogen degradation [+ uptake is mainly facilitated by the Na+/K+-ATPase (NKA), and at very high extracellular K+ concentration also by the Na+–K+–Cl− co-transporter 1 (NKCC1) [+ a highly efficient process [Already 25 years ago, it was discovered that increased extracellular Kradation . Evidenc (NKCC1) ,36. NKCC process ,38.+ uptake can influence an array of ion exchangers and signalling pathways, downstream of which either Ca2+ or cAMP can be increased, activating glycogen degradation .3−)-sensitive soluble adenylate cyclase (sAC) [+ uptake, as Na+ extruded by increased NKA activity is taken back up through the Na+–HCO3− co-transporter (NBC), increasing cytosolic HCO3−. This study has demonstrated that cAMP generated by sAC, following this pathway, directly activates glycogen degradation.Astrocytes have recently been shown to express a bicarbonate (HCOse (sAC) . sAC is + uptake, it is evident that the process is heavily dependent on ATP supply. Furthermore, as in the case of NA signalling, cAMP generation and Ca2+ homoeostasis require additional ATP. Recently, it has been demonstrated that astrocytic K+ uptake is specifically dependent on ATP provided by glycogenolysis, as inhibition of GP abolished uptake, even in the presence of extracellular glucose [As NKA is the main facilitator of astrocytic K glucose ,39.One of the key functions of astrocytes in supporting neurotransmission is rapid uptake of glutamate (Glu) for swift termination of excitatory signalling. Glu is taken up in a sodium-dependent manner through the excitatory amino acid transporter 2 (EAAT2), following the sodium gradient created by activity of the NKA. Upon uptake, glutamine (Gln) synthetase catalyses ATP-dependent condensation of Glu and ammonia to Gln, which subsequently is released and taken up by neurons to be converted back into Glu . This Gl+ ions can activate the reverse mode of the sodium/calcium exchanger (NCX), leading to an influx of Ca2+ and activating glycogen degradation [Studies have demonstrated that uptake and recycling of Glu are glycogen-dependent, even in the presence of extracellular glucose . It has radation   channels [2+-binding EF-hand domains on its luminal side. Upon a decrease in Ca2+ concentrations inside the ER lumen, Ca2+ dissociates from STIM1, resulting in a conformational change of STIM1 that facilitates its accumulation in oligomers. These STIM1 aggregates migrate inside the ER membrane, towards sites close to SOCE channels in the plasma membrane, where they form discrete puncta [2+ from the extracellular space.The functional significance of the ER as a dynamic Catrocytes . Stromalachinery , whereaschannels  and Oraichannels . STIM1 ce puncta . Followi2+ through SOCE channels, the final step of SOCE is catalysed by SERCA, which pumps the Ca2+ ions into the ER, thus refilling stores [Upon entry of Cag stores .Currently, there is no consensus about the molecular identity of the SOCE-mediating proteins in astrocytes, as conflicting results show functional expression of either Orai or TRP channels . Thus it2+ entry through SOCE channels robustly stimulates glycogenolysis in both cerebellar and cortical murine astrocytes [Recently, we have shown that Catrocytes . This stInsight into the functional consequences of biochemical mechanisms requires an understanding of the regulatory pathways and dynamics that underlie them. The critical role occupied by glycogen in metabolic support of brain function is far from being completely understood. However, recent work has provided evidence that, in addition to the commonly discussed function of providing or sparing energy substrates for neurons, astrocytic glycogenolysis contributes to the ATP pool of astrocytes themselves, energetically supporting astrocytic signal transduction ,8.2+ and cAMP to GP activation upon a given stimulus.As no biological process is energy-neutral, any signalling pathway at some point will be dependent on provision of ATP. Therefore any signal leading to phosphorylation of GP will, at some point, be using ATP, producing AMP. Consequently, without careful experimental probing, there can be no clear distinction between the contribution of allosteric modification through AMP, and phosphorylation triggered by Ca


Cancer cells require nutrients and energy to adapt to increased biosynthetic activity and depend on mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. Whereas they exhibit a pronounced Warburg effect, their TCA cycle remains intact and becomes more dependent on glutamine metabolism through glutaminolysis. BesidesE Coli L-ase (Kidrolase®) and E. Chrysanthemi L-ase (Erwiniase®) are not mediated by the asparaginase activity of the enzyme. L-ases have also a glutaminase activity and transform extracellular glutamine into glutamate. Both L-ases induce dose and time-dependent mTORC1-inhibition which correlates with extra-cellular glutamine depletion[35] methionine incorporation into newly synthesized proteins, indicative of global protein synthesis inhibition. Finally, we showed that L-ase treatment acts through inhibition of leucine entry into the cells by depleting intracellular glutamine, thus preventing mTORC1 activation by Rheb at the lysosomal surface[The dependence of acute myeloid leukemia (AML) cells to glutamine is little studied. In a recent work, we have tested the effects of glutamine depletion in AML cells: leukemidepletion. Downstrl surface.E. Coli L-ase in refractory/relapsed AML in adults revealed a significantly increased CR rate with the association (40% vs. 24%)[in vitro sensitivity to L-ase very close to that of acute lymphoblastic leukemia cells. Further preclinical studies are thus warranted to characterize new chemotherapeutic combinations in association to L-ase in AML.Overall, those data open the way for new therapies targeting glutamine uptake in AML. L-ase has been previously tested in AML. A randomized study that compared the effects of high dose cytarabine with or without  vs. 24%). Blast cWe also identified potential mechanisms of resistance to L-ase treatment in AML. L-ase upregulates glutamine synthase (GS) protein expression and this process may limit the L-ase-induced anti-leukemic activity in AML. GS inhibition by the L-methionine-sulfoximine (MSO) may increase the sensitivity of AML cells to L-ase as described in ALL. Moreover, L-ase triggers an autophagic process, probably resulting from mTORC1 inhibition (mTORC1 inhibits autophagy by controlling the activity of Atg1/ULK and ATG13 proteins). Knockdown of either ATG5 or Beclin, two proteins controlling autophagy, increase apoptosis in L-ase-treated AML cells. The future testing of a combination of autophagy inhibitors and L-ase is thus warranted in AML.Another therapeutic strategy to exploit AML glutamine dependence may be to target the glutamine transporters. We showed that the knockdown of the high affinity transporter SLC1A5 has strong anti-leukemic effects in vitro and inhibits tumor formation in an AML mouse xenotransplantation model. HoweverWe only address here the role of glutamine uptake by AML cells on the control of mTORC1 activation. Studies are needed to examine its role in mitochondrial anaplerosis through glutaminolysis. The key gatekeeper of glutaminolysis is the enzyme glutaminase (GLS) which catalyzes the hydrolysis of glutamine to glutamate, which is then converted to the TCA cycle intermediate α-ketoglutarate (α-KG). Two different forms of GLS exist: the liver-type glutaminase  and the glutaminase 1 (GLS gene) which exists as two splice variants: the longer form called kidney-type glutaminase (KGA) and the shorter called glutaminase C (GAC). The twoTo conclude, through inhibition of mTORC1 activity and/or through suppression of the mitochondrial TCA cycle, glutamine metabolism constitutes an appealing target for treating this particularly poor prognosis disease.


This review focuses on the role of [Ca2+]i in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca2+]i is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca2+]i for the function of anti-cancer drugs is illuminated in this review as [Ca2+]i could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca2+]i could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma.Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca Paediatric cancers—like neuroblastoma—transpire during the neonatal epoch of an infant . NeuroblNeuroblastoma arises from undifferentiated simpatico-adrenal lineage cells which derive from neural crest cells . The reaThe International Neuroblastoma Risk Group Staging System (INRGSS) has introduced a standardized classification of the disease in order to guide clinical trials at different regions across the world ,12. INRGIn physiology terms, cancers could be described as the end-result of either of eight vital cellular modifications which include the (1) escape of cell from apoptosis, (2) development of an autocrine system (for synthesizing growth factors resulting in a higher rate of proliferation), (3) development of an insensitivity to anti-growth signals, (4) uncontrolled replication potential, (5) incessant angiogenesis, (6) ability for tissue invasion and metastasis, (7) reprogramming of cellular energetics and (8) escape from immune cells,15. Thus2+]i) is in prominence as it modulates the apoptotic or proliferative pathways of the cell. Several studies have conferred the impact of anti-cancer drugs on [Ca2+]i levels. In this review, the following chapters will highlight the essential role of [Ca2+]i in the development and treatment of neuroblastoma by interacting and exerting its effect on cell survival signalling, differentiation, proliferation and apoptosis i is discussed in 2+]i is not restricted to neuroblastoma alone, as its found to have an apoptotic effect on breast cancer cellline MCF7 cells i [2+]i concentration between 10 and 100nM which could rise up to 100 times upon Ca2+-entry from the extracellular space or its release from the internal Ca2+-stores [2+]i concentration and to secure clearly defined [Ca2+]i signals, calcium entry and extrusion mechanisms are tightly regulated i-concentration [2+]i is reduced by active transport mechanisms which move the cytosolic calcium either back to the stores (Endoplasmic Reticulum (ER) and mitochondria) or the extracellular space  and sphingosine-1 phosphate (S1P) to the NAADP receptor and sphingolipid calcium release-mediating protein.The influx mechanisms are regulated by voltage-, receptor-  and store-operated channels. As mentioned earlier, the internal stores of calcium includes ER or a similar organelle named sacroplasmic reticulum (SR) in muscle cells, of which the calcium release is mainly controlled by the inositol-1,4,5 triphosphate receptor (InsP3R) and the ryanodine receptor (RYR). Calcium is released from the ER by the binding of InsP3 and cyclic ADP ribose (cADPR) to the InsP3R and RYR, respectively. InsP3 is released in response to binding of hormones, growth factors and the neurotransmitteracetylcholine to their cell surface receptors by the cleavage of phosphatidyl inositol 4,5-bisphosphate, a phospholipid present in the cell membrane. In addition, CaDevelopment of neuroblastoma tumourgenesis and malignancy arises due to unbalanced cell survival and apoptotic pathways. Nerve Growth Factor (NGF), Insulin-like Growth Factor (IGF), Epidermal Growth Factor (EGF), Platelet-Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF) are major growth factor signalling pathways in Neuroblastoma . Cell suAnaplastic Lymphoma Kinase (ALK) is also associated with cell survival signalling of neuroblastoma. It belongs to the family of insulin receptors with the trans-membrane receptor tyrosine kinase  and exer2+]i also exert its effect on intermediate kinases that are involved in cell survival signalling. NGF signalling is a predominant signalling pathway in neuroblastoma. Trktyrosine kinase receptor family is associated with the NGF signalling and includes TrkA for NGF, TrkB for brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and TrkC for neurotrophin-3 (NT-3)   50]. An. An2+]i fication ,52. In Peceptors ,54,55. Achannels . Thus, t by P13K  which is2+]i are considered as key regulators for intracellular signalling initiated by extracellular ligands like growth factors and hormones as they determine the fate of the signalling pathway  which activates AKT thereby phosphorylating the serine residue 136 of BAD and hence promote cell survival . In. In2+]i ay P3 [An increase in [Ca [Ca2+]i . These c]i [2+]i  85,86].,86.2+]i 1,4,5)P3   results either in differentiation or induction of apoptosis  across the mitochondrial membrane by creating a negative mitochondrial membrane potential i i irrespective of 3T3-p75 cells. Additionally, NGF helps in [Ca2+]i signalling via voltage-gated channels in mouse nociceptive neurons. A suggestion by Bonnington and McNaughton is that out of the three pathways activated by NGF induced TrKA receptor activation, PI3K pathway leads to the sensitization of TRPV1. This sensitization increased Ca2+-influx via voltage-gated channels. Two other pathways activated by TrKA receptors are the PLC gamma and ShC induced Ras pathway i ,120. In  pathway . Studieschannels . Others channels ,124.2+]i in inducing intrinsic apoptotic pathway. In addition, studies have confirmed the role of [Ca2+]i in inducing necroptosis in human neuroblastoma cells , a G-protein coupled receptor is a key mediator protein in maintaining the cellular responses and cell fate in response to external Ca.05–5 mM ,127. Chaiescence . In addiiescence . Loss oriescence ,128, as iescence ,130.Recent studies with neuroblastoma brought more insights into calcium and calcium sensing receptors. The expression of CaSR and smaller levels of parathyroid hormone-related protein were observed in differentiated, favourable neuroblastic tumours ,128. CaSin vitro experiments confirmed that when cells were exposed to Ca2+, cell vacuolization was observed along with an increase in number of dead cells. Thus, it was clear that CaSR was over expressed in neuroblastoma cells and when exposed to Ca2+ cells underwent apoptosis via ERK1/2signalling cascade i with the induction of necrosis or apoptosis. Studies report that arsenic trioxide (As2O3) and cisplatin (CDDP) [2+]i-homeostasis. Cisplatin is considered as the most effective chemotherapeutic drug for the treatment of neuroblastoma. Cisplatin exerts its anti-cancer therapeutic role by cytotoxicity (interacting with the purine bases in DNA at the nucleophilic N7-sites forming a interstrand and intrastrand of DNA-DNA and DNA-protein crosslinks) [2+]i was elevated by Ca2+ entry from the extracellular space [2+]i was CDDP-concentration dependent, leading to apoptosis by the activation of calpain mediated by ionsitol tri-phosphate (IP3) [2+]i through a different activation of IP3, the efficacy of cisplatin could be increased resulting in a higher rate of apoptosis. Cisplatin increased [Ca2+]i by Ca2+-entry and As2O3 by Ca2+-release from the internal stores [2+]i is allied with a cellular apoptosis with the activation of calpain and not caspase-8, confirming that cisplatin induced calcium influx via the IP3 receptors lead to cellular apoptosis with the activation of calpain i in neuroblastoma development, proliferation and apoptosis can be better understood when the complexity behind the Ca2+-concentration regulating the cellular process is considered. In neuroblastoma, [Ca2+]i is a key mediator and might not be restricted to growth factor signalling and the kinase activation. Apart from these, studies relating to [Ca2+]i on MYCN expression is questionable as neuroblastoma is often associated with MYCN over expression. Since [Ca2+]i rises with chemotherapeutic treatment, elevating [Ca2+]i agonist could improve the current treatment protocol. However, the studies should be extended to other biological and environmental factors like Vitamin D deficiency and calcium absorption as the disease prevalence is higher in regions with less sunlight. In addition, further insights into calcium deficiency in pregnancy and the scope of [Ca2+]i in treatment of other types of cancers are to be discussed.The role of [Ca2+]i regulates different cellular functions and has a key role in the development and prevalence of neuroblastoma. In neuroblastoma, these [Ca2+]i form a network with the intermediate kinase of major signalling pathways, either directly or indirectly, and its concentration determines the fate of the cells. Hence, studies could be more focused on [Ca2+]i, which could lead to the development of new Ca2+ based drugs or could also help in improving the efficacy of chemotherapeutic drugs that are currently used in treatment of neuroblastoma.[Ca