The psychometric properties of the social functioning in dementia scale over different dementia severities are unknown.We interviewed 299 family carers of people with mild, moderate, or severe dementia from two UK research sites; examined acceptability (completion rates); conducted exploratory factor analysis; and tested each factor's internal consistency and construct validity.spending time with other people, correlating with overall social function  and activities of daily living ; communicating with other people correlating with activities of daily living ; and sensitivity to other people correlating with quality of life  and inversely with neuropsychiatric symptoms . The three factors' correlations with other domains were similar across all dementia severities.Of 299, 285 (95.3%) carers completed questionnaires. Factor analysis indicated three distinct factors with acceptable internal consistency: The social functioning in dementia scale carer version measures three social functioning domains and has satisfactory psychometric properties in all severities of dementia. It has been found to have high acceptability and reliability and moderate concurrent validity against a single item rating overall social functioning 1.11.test SF-DEM's acceptability for assessing social function in people with dementia of any severity2.examine the underlying factor structure of SF-DEM using EFA3.test the internal consistency and construct validity of the scale and its derived factors against measures of a person with dementia's quality of life, symptoms, and dementia severity. We hypothesized that social functioning would be correlated positively with better quality of life and that social functioning would be inversely correlated with dementia severity and neuropsychiatric symptoms.We aimed to2This cross-sectional study of family carers of people with dementia was nested in the CDEMQOL study (Alzheimer's Society project grant: 234 AS-PG-14-017), and the South Central–Hampshire A Research Ethics Committee gave ethical approval (15/SC/0605).2.1We recruited study participants from clinical services based in three UK National Health Service mental health trusts in Sussex and North London, which provide dementia assessment and management services; Join Dementia Research network 2.2We recruited family carers, aged over 18 years, of people with dementia of any subtype and severity , who defined themselves as having a caring role for the person with dementia, and having primary responsibility for the well-being and decisions for the person with dementia, although others may have been involved in the process. We excluded paid carers or those who were unable to speak English sufficiently to complete the assessment.2.3Trained researchers conducted a single semistructured interview with family carers at their homes or in a university or hospital site according to their preference, between June 2017 and January 2018.2.4SF-DEM 1.Bristol Activities of Daily Living Scale (B-ADL) 2.Neuropsychiatric Inventory (NPI) 3.DEMQOL-Proxy 4.Clinical Dementia Rating Scale (CDR) We recorded the age, sex, ethnicity, marital status and level of education of the person with dementia and carer and their relationship, the living situation of the person with dementia, and the dementia subtype. We also completed the following validated carer-rated measures of the person with dementia's symptoms and dementia severity.2.5We completed all statistical analysis using SPSS (version 23). Ten percent of the collected data were checked through double entry for any disparities. We first described the demographic and clinical characteristics of the study sample.a priori that individual items would be classed as acceptable if ≥90% completion. We also recorded the time taken to complete the scale.We assessed SF-DEM's acceptability and feasibility in mild, moderate, and severe dementia. As missing data suggest that participants were unable or unwilling to answer questions, we reported the frequency of missing data according to level of dementia severity; we decided Although SF-DEM was developed to measure the interaction of people with dementia with society in general and their own personal environments, we did not have specific hypotheses about the factor structure of the developed measure. We therefore used EFA to empirically establish the common sources of variance underlying the item responses We then created sum scores for each of the SF-DEM factors derived from the EFA and then evaluated the construct validity of each, using other carer-reported measures of a person with dementia's quality of life, symptoms, function, and dementia severity. We used Pearson to assess correlation of SF-DEM factor score totals with the summary question overall impression of current social functioning, B-ADL total score, NPI total, DEMQOL-Proxy total score, and CDR.As secondary analyses, we repeated the validity tests stratified for the level of dementia severity as assessed by CDR. For these analyses, we examined the correlation between SF-DEM factors and the scale against which it had highest correlation.3We recruited 300 study participants; one carer withdrew before completing SF-DEM, so we included data from 299 carers. 3.1We report the SF-DEM questions, participants' responses, and presence of missing data fully in 3.2In the EFA, the first three factors explained 13.9%, 23.2%, and 11.3% of the variance, respectively. The three-factor solution, which explained 48.4% of the variance, was preferred because of the “leveling off” of eigenvalues on the scree plot after three factors  and the The three-factor solution was rotated obliquely to yield standardized factor loadings presented in 3.3spending time with other people; α = 0.79 for communicating with other people; and α = 0.72 for sensitivity to other people. One item (“Seeing friends and family in their own home”) had lower item-total correlation and poorer factor loadings but removing it would reduce face validity with little improvement in the Cronbach's α score, so we chose for all items to remain in the scale.Internal consistency for each of the factors was acceptable: Cronbach's α = 0.73 for spending time with other people; scale 2 = communicating with other people; scale 3 = sensitivity to other people).We then calculated sum scores for the three scales and used the sum scores for subsequent analyses , and communicating with other people and sensitivity to other people was weakly positively correlated with overall rating of function . Higher communicating with other people and sensitivity to other people ratings were moderately associated with less neuropsychiatric symptoms , and higher sensitivity to other people was associated with better quality of life .function . Higher spending time with other people and communicating with other people decreased with dementia progression, and the scores were significantly different between the four levels of CDR severity as determined by a one-way ANOVA . There was no significant difference between sensitivity to other people mean scores according to dementia severity .communicating with other people score and overall social functioning rating was lower for moderate and severe dementia; correlation between sensitivity to other people score and overall social function rating was also lower in people with severe dementia. Correlation between spending time with other people score and ADL rating (B-ADL score) remained consistent for severe dementia, but coefficients were smaller for very mild to moderate categories  sources. The social functioning in dementia scale (SF-DEM) is the only scale specifically designed to measure social functioning in dementia. One previous study examined SF-DEM's psychometric properties in assessing social function in mild dementia, and no analysis has examined its factor structure.2.pending time with other people and communicating with other people was associated with dementia severity but sensitivity to other people was not.Interpretation: SF-DEM was acceptable, valid, and reliable in people with dementia of any severity. We found in exploratory factor analysis (EFA) that it measures three distinct factors related to social functioning. The score for s3.Future directions: Future longitudinal research should test SF-DEM's responsiveness and correlates of social function change over time. Such testing should examine whether sensitivity to others changes with advancing disease and consider whether SF-DEM changes are early markers of disease in preclinical populations.Social function is an important domain to consider in the diagnosis of, and interventions for, dementia, and we report for the first time that the carer-rated SF-DEM scale has acceptable validity in assessing social function of people with any dementia severity. This validated measure informs future research such as enabling intervention evaluation studies to measure social function as an important patient-reported outcome; and SF-DEM may also be a useful scale for clinical use in tracking social decline in the context of disease progression. The results of our factor analysis can guide future SF-DEM use as it indicates that three subdomains of social functioning should be used and scored separately. The correlation between two SF-DEM domains and dementia severity suggests that these domains decline with increasing severity, but future longitudinal research should test the responsiveness of SF-DEM in measuring change and examine correlates of social function change over time. In particular, future research should examine sensitivity to others over time as this study is, to our knowledge, the first to find a preserved domain throughout the range of dementia severity. Finally, considering the difference in scores between even very mild and mild dementia, such testing should include preclinical populations such as those with mild cognitive or behavioral impairment and consider correlation with the Mild Behavioral Impairment checklist


Importantly, we used a full testing approach in which subjects were given as much time as they required to complete each task. For each task, we then compared the performance of subjects who completed the task within the expected number of testing days with those subjects who needed more testing time. We found that the two groups did not differ in task performance, and therefore opportunistic testing would have been justified without risking biased results. If our findings generalise to other species, maximising sample sizes by only testing consistently motivated subjects will be a valid alternative whenever full testing is not feasible.Dropouts are a common issue in cognitive tests with non-human primates. One main reason for dropouts is that researchers often face a trade-off between obtaining a sufficiently large sample size and logistic restrictions, such as limited access to testing facilities. The commonly-used opportunistic testing approach deals with this trade-off by only testing those individuals who readily participate and complete the cognitive tasks within a given time frame. All other individuals are excluded from further testing and data analysis. However, it is unknown if this approach merely excludes subjects who are not consistently motivated to participate, or if these dropouts systematically differ in cognitive ability. If the latter holds, the selection bias resulting from opportunistic testing would systematically affect performance scores and thus comparisons between individuals and species. We assessed the potential effects of opportunistic testing on cognitive performance in common marmosets  Cognitive performance in animals can be assessed as pure proof of principle, i.e. to test whether a specific ability can be found in a given species. However, cognitive performance is increasingly assessed also for comparative purposes, both between and within species. At the inter-specific level, two or more species are tested with the same cognitive task(s) in order to explore species differences and similarities in cognitive abilities. These differences are usually hypothesised to emerge because the species have faced different selection pressures in their evolutionary past, such as challenges posed by their social or ecological environment. Examples of such studies suggest that enhanced inhibitory control is found in species with a fission-fusion social group structure  or a feeCognitive comparisons can also focus on the individual level, allowing researchers to investigate differences and similarities between individuals of the same species. Examples include studies exploring the effects of aging –8, sex dWhenever the goal is to compare cognitive performance across species and individuals, it is crucial to take into account that an individual’s performance in a test is also affected by factors that are not primarily cognitive in nature, such as emotion, motivation and health. We may thus risk measuring individual differences in such non-cognitive factors rather than cognitive ability per se and therefore the performance scores would be biased or even meaningless. The same is true if individuals are selected for inclusion in a cognitive study based on such non-cognitive factors.In the last decade, researchers have started to systematically re-address the risk that cognitive tests may inadvertently measure individual (and species) differences in non-cognitive factors rather than true differences in the cognitive abilities. A number of experimental studies in non-human primates has systematically assessed both external (testing-related) and internal (subject-related) factors to determine how they affect the subjects’ cognitive performance . ExampleExamples of external non-cognitive factors that have been addressed so far include aspects of the test design, setup and procedure such as how the test apparatus and test stimuli are presented to the subject . Other An open question is therefore whether opportunistic testing, here defined as using strict stop criteria to restrict total testing time, results in a selection bias because the subjects included in the study differ in cognitive ability from a random sample of the population. and squirrel monkeys (Saimiri sciureus). The subjects were tested with a cognitive test battery consisting of five commonly used paradigms: Detour Reaching, A-not-B, Quantity Discrimination, Reversal Learning, and a two-choice Memory task , as wel monkeys ). Import from six family groups and eight adult common squirrel monkeys (Saimiri sciureus) from two bachelor groups participated in this study. The initial study sample consisted of the fifteen marmosets  in the upper section of the table who were tested with the whole test battery of subtests 1–5 (hereafter tasks). Kitty  who became Kantor’s partner later in the study  was tested with task 5. The remaining eleven marmosets  were only tested with both memory tasks (5 and 6) when we assessed in a parallel study whether memory task 5 truly measured the ability to remember the location of a reward [As summarized in a reward . The squ3, each of which was equipped with several climbing structures such as natural branches, a sleeping box, an infrared lamp and a bark mulch floor. Whenever the weather conditions allowed it, each group had free access to an outdoor enclosure. The squirrel monkeys’ indoor enclosures measured 16.55 m3  and 24.77 m3 (larger group) and were equipped with climbing structures, an infrared lamp and a bark mulch floor. Each group had constant free access to a fully roofed outdoor enclosure, and in addition, the two groups took turns in accessing a larger outdoor area of 86.4 m3. The marmosets were fed a vitamin and calcium-enriched porridge in the morning, fresh fruit and vegetables at lunchtime, and gum and mealworms in the late afternoon. In addition, they received a daily protein snack in the afternoon such as pieces of cooked egg. The squirrel monkeys were fed a mixture of pellets and cottage cheese in the morning, a variety of vegetables and a small amount of fruit at lunchtime, and a protein snack such as cockroaches in the late afternoon. Water was available ad libitum from water dispensers (marmosets) or water bowls (squirrel monkeys). All subjects were tested between their regular feedings and never food deprived during the study. Testing time was restricted to 8.30–12.00 hours in the morning and 13.00–16.30 in the afternoon. Each subject was only tested once per day and never during the monkeys’ general resting time from 12.00–13.00 hours. The monkeys could freely enter and leave the test enclosure and were not handled by the experimenter at any time before, during or after the cognitive test sessions. After the completion of this study, the monkeys continued living at the Primate Station, eventually participating in other non-invasive studies.All monkeys were captive-born and mother-reared and housed in family (marmosets) and bachelor groups (squirrel monkeys) consisting of two to eight individuals in indoor-and outdoor enclosures. The marmosets’ indoor enclosures had both daylight and artificial light and were composed of one to three components (depending on group size) measuring at least 4 mAll subjects were tested individually in a dedicated test enclosure that was connected to their home enclosures through a semi-transparent plastic tube. Once the doors on each end of the tube were opened, the subject could voluntary and freely enter the tube and walk to the test enclosure without being handled by humans at any time. The actual test compartment for the marmosets consisted of a white rear wall, a white floor, two lateral grid walls and a clear Perspex window front. Depending on the cognitive task and respective apparatus, the window front contained one or two rectangular openings through which the subject could reach with its arms to access the apparatus. Each test apparatus was placed on a wooden board (varying size and features) that was mounted on a height-adjustable test table and flush with the test compartment’s window. The test compartment used for the squirrel monkeys also had a white plastic wall in the back and a white floor, and three grid walls. The measurements of the test apparatuses see  were adaThe general testing procedure was identical for both species. Before and after each test trial, the experimenter positioned an occluder (a wooden or cardboard sheet) between the test compartment’s window and the test apparatus. Its removal announced the next trial to the subject and the experimenter called the subject’s name and said ‘look’ and made sure that the subject was attentive to the test apparatus and procedure. In the first task (Detour-Reaching), the test apparatus remained within the subject’s reach and was accessible to the subject as soon as the experimenter had removed the occluder between the test compartment’s front and the test apparatus. In all other tasks, the apparatus was mounted on a sliding platform and remained out of the subject’s reach until the baiting process was completed (tasks 2–6) or the time delay had expired (tasks 5–6). The experimenter then pushed the test apparatus into the subject’s reach so that the subject could make its choice. After a correct choice, the experimenter waited for the subject to consume the reward, retracted the test apparatus, set-up the occluder, and immediately continued with the next trial. After a wrong choice, however, the experimenter quickly retracted the test apparatus out of the subject’s reach so that no second choice was possible. She then retrieved the reward, showed it to the subject and placed it in a holding area for re-use in the next test trial. In tasks 1 and 2, the next trial was started immediately after the subject had completed a given test trial. In task 1, this ensured the monkeys’ wellbeing by keeping the duration of each test session as short as possible because a single test trial took up to 2 minutes. In task 2, it was important to quickly continue with the next trial in order to get the subjects into the intended routine of repeatedly choosing the same cup. In tasks 3, 4, 5 and 6, the experimenter only continued with the next test trial once a 15-second (tests 3–4) or 10-second (test 5–6) time interval had expired. This brief delay was intended to make a wrong choice more costly and encourage subjects to concentrate and attempt to choose correctly. Again, since test sessions in tasks 5 and 6 took inherently longer than in the other tasks because each test trial already involved a time delay of up to 30 seconds, the slightly shorter additional inter-trial interval after a wrong choice kept total testing time to a minimum. It is unlikely that this slight difference in inter-trial intervals was perceived by or affected the subjects’ motivation differently because in each task a beep tone announced the end of the delay after a wrong choice. Since food preferences differed between the two species, marmosets received mealworms, crickets or locusts as rewards, whereas squirrel monkeys received mealworms or small pieces of cashew nut.If a subject lost motivation during a test session and expressed this by refusing to make a choice in 3 consecutive test trials or showed behavioural signs of emotional arousal or avoidance (as determined by the stop criteria in ), the exThe criteria to determine the expected amount of testing time in which each task would have had to be completed under an opportunistic testing regime were adopted from a similar study  with theFor those subjects who needed longer than expected to complete a task but were not excluded under our full testing regime, we determined the maximum total testing period for each task based on what appeared reasonable depending on the total number of test trials (one to five months). This extended time period allowed subjects ample opportunity to complete each task at its own pace including breaks from testing as required . We discontinued testing after this time period had expired because it was not justified to continue testing for a completely unlimited time period (i.e. after complete motivation loss).In this section, and in Measured cognitive ability. Inhibition of a predominant motor response (directly reaching for a food item that is placed behind a transparent barrier) in favour of applying an adequate and successful response (reaching around the barrier to be able to successfully access the food item).Test apparatus. A static transparent barrier (a quadratic Perspex panel) was vertically attached to the top of a wooden board. The task required the subject to reach around the barrier in order to grasp a food item that was placed behind it.Familiarisation phase. The subject was given the opportunity to explore the test apparatus in the test compartment without a food reward present for a duration of 10 minutes.Test phase. In each trial, the experimenter placed the food item on the board and behind the Perspex barrier in one of 3 positions: left, central or right. In the two lateral conditions that simply served as distractors, only half of the reward was positioned behind the left or right-side edge of the Perspex barrier while its other half remained exposed. To successfully retrieve the reward, the subject could directly reach for it and grasp it. In the actual test condition , however, the reward was placed in the middle of the Perspex barrier so that it was fully occluded by it. To successfully retrieve the reward, the subject had to inhibit directly reaching for the reward as this would have resulted in its hand colliding with the Perspex barrier. It had to reach around the barrier instead so that it could grasp the reward without reaching into the barrier (successful detour-reaching).If the subject successfully reached around the barrier at its first attempt, the trial was noted as correct. If it failed to do so and reached into the barrier instead, the trial was noted as incorrect. Yet, contrarily to an earlier study , the appThe Detour-Reaching task entailed 5 sessions of 12 trials. In each test session, the reward appeared 4 times in each of the 3 positions  in a counterbalanced and pseudo-randomised order, with the rule that the reward never appeared in the same location on more than two consecutive trials. This generally resulted in the completion of the full 5 test sessions and 60 trials in 5 consecutive days. However, subjects who did not complete a whole session or refused to participate at all on a given day were tested the following day. This resulted in the test sessions being spread over more than the expected 5 days. We stopped a test session on a given day if a subject was not motivated to complete all trials. However, we allowed these subjects to eventually complete all 5 sessions of the task in as many testing days as it needed.Outcome variables. (1) Successful detour-reaching: per cent correct trials of the 20 central trials at first attempt. (2) Learning from error: per cent correct trials at first attempt plus after initial failure (further detour-reaching attempts).Stop criterion in opportunistic testing. In opportunistic testing, the five sessions of the Detour-Reaching task would have been supposed to be completed within five consecutive days .Of the 23 subjects in our study sample who completed the Detour-Reaching task, nine of the 15 marmosets  and 3 of the 8 squirrel monkeys  needed longer than five testing days to complete the five test sessions. These 12 subjects would have been excluded from (further) testing owing to motivational issues, had we stopped testing after five consecutive days as in opportunistic testing.In our full testing, in which we gave the monkeys a total testing time period of 1 month to complete the Detour-Reaching task, all 12 individuals who needed more testing time than expected eventually also completed the task and no subject had to be excluded.Measured cognitive ability. Inhibition of a learned routine, i.e. to avoid choosing the same location that had consistently been rewarded and switching to a second location instead that was now being rewarded. In order to successfully choose the correct cup, the subject had to inhibit a learned association between cup B and the food reward.Test apparatus. Two opaque black plastic cups (A and B) were placed upside-down on a wooden sliding platform. The cups were attached to the board in a way that the experimenter could easily open and close them by flipping them back and forwards.Familiarisation. The subjects learned (1) how to open the cups, (2) that only one cup at the time would contain a reward, and (3) that only one choice was possible in each trial.Test. The test consisted of 6 trials conducted in a single session. During the first 5 trials, the experimenter placed a reward under cup B while the subject was watching, closed both cups simultaneously and quickly pushed the test apparatus within the subject’s reach. The subject could then reach out to choose one of the two cups, by lifting it or attempting to do so. In the 6th trial, the experimenter placed the cricket under cup A instead of B.Cup A was only baited once a subject had correctly chosen cup B in 5 consecutive trials. If the subject did not correctly choose cup B in the first 5 consecutive trials, the experimenter repeated baiting cup B until the subject succeeded. If a subject did not make 5 consecutive correct choices within a single test session of 14 trials, it was tested again the following day. If a subject stopped participating during the session, it was also tested the following day.Outcome variable. Correct choice in the last trial (cup A instead of B): yes/no.Stop criterion in opportunistic testing. In opportunistic testing, testing with subjects who failed to choose cup B in the first 5 consecutive trials would have been stopped and those subjects would have been excluded from further analysis.In our study, this would have resulted in losing 9 of the 22 subjects in the analysis who participated in the actual test. Eight of the 15 marmosets  and one of the 7 squirrel monkeys (S8) did not achieve 5 correct B-choices in the initial 5 consecutive B-trials and therefore needed longer to succeed in 5 consecutive B-trials.In our full testing approach in which we gave the monkeys one month (including the familiarisation phase) to complete the A-not-B task and as many B trials as needed, all 9 subjects who needed more than 5 B-trials to reach criterion eventually did so and completed the actual test. We only had to exclude one other squirrel monkey (S7) because he still refused to complete the familiarisation trials once all other subjects had completed the full task.Measured cognitive ability. Understanding of quantities by distinguishing between pairs of different quantities, i.e. two amounts of edible items.Test apparatus. Schmitt & Fischer [ Fischer  had repo Fischer , 27, thePre-test. The subjects were presented with a single and obvious numerical contrast (1 vs. 6 pellets), and the location of the larger quantity was counterbalanced and pseudo-randomised for all contrasts. This ensured all subjects understood they had to choose the larger of two pellet quantities in order to obtain the cricket. Before proceeding to the test phase, they had to reach the criterion of ≥ 80% correct trials within a single pre-test session of 10 trials.Test. The test consisted of 3 sessions of 10 trials each. In each session, the subjects were presented with 10 different numerical contrasts . As in the pre-test phase, the location of the larger quantity was counterbalanced and pseudo-randomised for all contrasts.Outcome variable. Per cent correct trials (choice of the larger amount) across all 3 test sessions.Stop criterion in opportunistic testing. Under an opportunistic testing regime, testing would have been stopped if a subject did not complete the 30 trials within 3 sessions that were conducted on 3 consecutive days and this subject would have been excluded from the analysis.Of the 15 marmosets who had entered the pre-test, 12 subjects participated in the actual Quantity Discrimination test. Nine subjects  completed all 3 test sessions within the expected time frame of 3 testing days . The remaining 3 male subjects needed up to twice as long to complete the task  and would have been excluded in opportunistic testing.Under our full testing regime, in which we gave the monkeys one month (including the pre-test) to complete the task, the 3 above mentioned male individuals also completed the task. The only individuals we had to exclude were 3 other males  who were still not motivated to fully participate in the pre-test after a total testing period of one month and once their conspecifics had completed the whole task.Measured cognitive ability. Cognitive flexibility, i.e. reversing a previously learned association to a new one if the previously learned association is no longer rewarded.Test apparatus. The apparatus consisted of a wooden board that contained two food wells and two rectangular wooden plates with two distinct black and white patterns printed on top.Familiarisation phase. Each subject was familiarized with the basic setup and procedure in order to ensure it understood that it needed to uncover the baited well to obtain the reward by pushing away the correct plate. In this phase, both wooden plates looked identical (no patterns). In each trial, the experimenter placed a cricket in full sight of the subject in one of the two food wells and covered both wells with the plates. Pushing away the plate from the baited well allowed the subject to retrieve the cricket. To pass to the test phase, the subjects had to reach the criterion of 10/12 correct trials.Test Phase 1 –Unhidden baiting–Learning of an association. The subjects were allowed to watch the baiting process to learn the association between one of the two patterns and the food reward. As in the familiarisation phase, the now patterned plates were initially each positioned behind one of the two food wells. The experimenter then placed a cricket in one food well and simultaneously covered both wells with the patterned plates. The reward was always under the same pattern and only choosing the correct one of the two patterns was rewarded. The criterion to move on to the next phase was again 10/12 correct trials.Test Phase 2 –Strengthening of the learned association. The experimenter baited the food well with the same pattern as in phase 1 but now behind the occluder so that the subject could not observe the baiting process. As in phase 1, she simultaneously placed both patterned plates on top of the two food wells so that the subject could not make use of any auditory cues. She then removed the occluder and the subject could make its choice. The criterion to move on to the next phase was again 10/12 correct trials.Test Phase 3 –Reversal of the learned association. The experimenter followed the same procedure as in phase 2 with the crucial difference that she now always placed the food reward under the opposite pattern (i.e. the one that had previously not been rewarded). The criterion was again 10/12 correct trials.Outcome variables. Number of sessions to reach criterion  in each of the 3 test phases: (1) learning the association between one pattern and the food reward, (2) strengthening of the learned association and (3) reversal of the learned association. In addition, we used a cognitive flexibility measure, the Transfer Index (TI) [dex (TI) , 30, whiStop criterion in opportunistic testing. Had we used an opportunistic testing regime, subjects who did not complete each test phase in a maximum of 7 sessions would have been excluded from further testing. Since none of the marmosets in our study completed phase 3 within 7 sessions, this approach would have resulted in eventually losing the whole study sample.TI could be determined.In our full testing approach, we did not use such a strict criterion but we discontinued testing with subjects who (1) needed more than 19 test sessions of 12 trials in the last of the 3 test phases  or (2) failed to complete phase 2 or 3 owing to motivational issues over a total testing period of 5 months. Of the 15 marmosets who participated in the Reversal Learning task, only one male subject (M13) lost motivation very early on and failed to complete phase 1. Of the 14 remaining subjects, two males (M14 and M15), did not complete phase 2, despite having reached criterion very quickly in phase 1. Finally, of the 12 subjects who had completed phase 2 and entered phase 3, five subjects could not be tested until completion of the reversal: three males  who completely refused to participate owing to motivational issues and two females (M2 and M11) who developed a strong side bias in phase 3 which they did not overcome. However, since all 12 subjects had completed the first session of phase 3, the Measured cognitive ability. Remembering in which of two locations a food reward has been hidden over increasing time delay periods.Memory task 1a). In the traditional two-choice Memory task version 1a, the test apparatus and procedure largely corresponded to the Hidden Reward Retrieval test in [ test in . Two whiMemory task 1b). In the two-choice Memory task version 1b, the test apparatus consisted of two black containers with loosely placed lids that were attached to the middle one of three wooden platforms held together by a wooden frame (for details see [ails see ).Pre-test. A no-delay condition was used as a pre-test. In this pre-test, the subjects could witness the baiting and choose immediately afterwards, without delay. Pre-test criterion was reaching ≥ 80% correct choices in a single 10-trial session. This ensured the subjects understood the task at hand before being exposed to time delay.Test. At the beginning of each trial, both containers were covered with a rectangular piece of bark mull in Memory task 1a or the containers were closed in Memory task 1b. Once the subject was attentive, the experimenter did the following: In Memory task 1a, she removed the cover of one of the containers, placed a piece of a locust in the container and put the cover back in place so that the bright yellow locust was no longer visible and both containers looked the same. In Memory task 1b, the experimenter lifted the lid of one of the two cups, placed a mealworm in the cup and closed it so that the mealworm was no longer visible to the subject. During the time delay period (5–30 seconds in Memory task 1a and 5–20 seconds in Memory task 1b), the test apparatus remained out of the subject’s reach. As soon as the time delay had expired, the experimenter pushed the test apparatus into the subject’s reach allowing it to choose one of the two containers. The subjects were tested with one session of 10 (Memory 1a) or 12 (Memory 1b) trials per day. A correct choice consisted of uncovering (Memory 1a) or opening (Memory 1b) the baited container.Outcome variable: Number of correct trials (per cent correct choices) across all delay conditions in Memory 1a  and Memory 1b , as well as in both task versions combined .Stop criterion in opportunistic testing.Memory 1a). In opportunistic testing, subjects would have been excluded if they did not complete all 6 test sessions in the expected 6 testing days . Since However, the majority of subjects completed the task within 12 testing days which we set as expected testing time instead.In our full testing approach, we gave each marmoset as many test sessions as it needed to complete the pre-test and the 6 test sessions during a 5-month testing period. Of the subjects who needed more than 12 testing days, five  eventually completed Memory task 1a. However, three other marmosets  had not completed the whole task once the 5-month testing period expired and had to be excluded from statistical analysis.Memory 1 b). In opportunistic testing, subjects would have been excluded if they failed to complete the 4 test sessions in the expected 4 testing days. This would have resulted in excluding 4 of the 8 marmosets  and one of the 8 squirrel monkeys (S3) who participated in Memory task 1b.We gave each subject a 5-month testing period (including the pre-test) to complete the memory task. Fifteen of the 19 monkeys (8 of the 11 marmosets of study sample 2 and 7 of the 8 squirrel monkeys) who had entered Memory task 1b eventually completed the full task within the given testing period. The remaining 3 marmosets  and one squirrel monkey (S6) had lost motivation early in the pre-test or after a very small number of test trials and the experimenter could not regain their motivation to fully participate. Using our full testing approach, we thus only had to exclude these 4 subjects from statistical analysis who were not sufficiently motivated to participate in the actual test.Since the two versions of Memory test 1 slightly differed in terms of used methodology which could potentially have affected the results, we first analysed and reported them separately. In a third analysis, we then also combined the subjects of marmoset sample 1 who had completed the first 4 test sessions of Memory task 1a and the subjects of marmoset sample 2 and the squirrel monkeys who had completed Memory task 1b.Using our full testing approach in which we had given each subject sufficient time to complete the (first) four delay conditions of Memory task 1, we obtained performance scores for one more male marmoset from sample 1 (M14) who had dropped out in Memory task 1a because he had not completed all 6 delay conditions. Therefore, we only had to exclude two subjects of study sample 1 (M15 and M16) who had not completed all trials of the 4 test sessions after expiration of the 5 months testing period.Three other marmosets  of study sample 2 and one squirrel monkey (S6) also had to be excluded because they had dropped out in the pre-test phase or after only a few test trials owing to motivational issues.Measured cognitive ability. Remembering in which of nine locations a food reward has been hidden over increasing time delay periods.Apparatus and procedure. This memory task differed from task 5 in that nine rather than two options were available to choose from. The apparatus and procedure were identical to the ones used in Memory task 1b with the only difference being that 9 rather than only 2 cups were used. The method has been described in detail in an earlier publication [lication .Outcome variable. Per cent correct choices across all delay conditions .Stop criterion in opportunistic testing. In opportunistic testing, subjects would have been excluded if they had not completed the 4 delay conditions in 4 sessions conducted on 4 consecutive days.In our full testing approach, we continued testing for a total testing period of 5 months in order to allow every subject to complete the test. Seven of initially 11 marmosets and 7 of initially 8 squirrel monkeys completed the whole Memory task 2 whereas one marmoset (M24) only completed the first test session (the 5-second delay condition). Three marmosets  and one squirrel monkey (S6) eventually had to be excluded because they had either not completed the pre-test or only a very small number of test trials before they refused to participate at all.For each of the six cognitive tasks, we analysed whether individuals who finished the task in the expected time (i.e. would have been included in opportunistic testing) differed in performance from individuals who needed longer (i.e. would have been excluded in opportunistic testing but were included in our full testing). We ran Generalised Linear Mixed Models (GLMMs), with the exception of the A-not-B task (task 2) for which we computed Fisher exact tests owing to the small number of trials in this task. The outcome variables for cognitive performance were the response variables, and subject was included as a random factor in all models. Testing time needed to complete a given test was included as a binary fixed factor: expected amount of time vs. longer. Individual test session, species (where applicable) and all two-way interactions were also included as fixed factors in the models. We calculated all models with biologically meaningful factor combinations and identified the best model using the Akaike criterion corrected for small sample sizes .F = 8.48, p = .000), species  = 1.26; p = .274), and their interaction  = 5.51, p = .001). Thus, individuals who finished the task in the expected time did not differ in performance from individuals who took longer to complete it  whereas 12 individuals took longer than expected . The best model to explain performance in the Detour-Reaching task included the fixed factors test session (e it see .1. The ie it see .p = 0.178). Likewise, there was a non-significant trend for squirrel monkeys to outperform marmosets   completed the test trials in time . Nine individuals  took longer than the expected amount of testing time  to reach criterion  before receiving the final (A) trial. Overall, 8 of the 15 marmosets and all 7 squirrel monkeys who completed the A-not-B task correctly chose cup A in the last trial and therefore passed the task whereas the remaining 8 marmosets failed it. Since a GLMM was not applicable owing to the relatively small number of test trials, two Fisher exact tests were conducted instead. The first Fisher exact test determined whether testing time  affected the subjects’ performance . The second test determined whether the species difference in performance was significant. Although there was a non-significant trend that subjects who needed more time to complete the A-not-B task were more likely to fail it, testing time had no significant effect on the monkeys’ performance  see .2.Mean score = 71% correct choices, SD = 8%; t(11) = 8.53, p = .000). The best model to predict cognitive performance was the one that only included one fixed effect, testing time, which did not affect the marmosets’ performance  = 0.42; p = 0.534)  whereas 3 individuals took slightly longer than expected . Overall, the 12 subjects who completed the Quantity Discrimination test performed well above chance (534) see .3.TI [For the Reversal Learning task, we first report how long (number of test sessions) the subjects needed to reach the learning criterion  in each of the 3 test phases. We also report whether the number of days the subjects needed on average to complete a 12-trial test session affected their performance . Finally, we answer the central question: whether the amount of testing time  affected the subjects’ cognitive flexibility (as measured by the TI ).M = 2.07, SD = 1.60), with the minimum being 1 session and the maximum 6 sessions  within an average of 2 test sessions   who completed phase 2 (strengthen the learned association), seven  did so in time whereas 5  needed longer than expected. The number of 12-trial sessions needed to complete phase 2 ranged from 4 to 13 sessions, with the average being 7 sessions (.11) see .M = 12.57, SD = 4.96)  who completed phase 3 , only 1 female subject (Vesta) did so in time whereas 6 subjects  needed longer. The marmosets completed phase 3 in 7 to 19 sessions, with the average being 13 sessions (.96) see .TI was calculated for each of these subjects. In order to determine whether testing time, i.e. the number of days a subject needed to complete a session in phase 2  or a subject’s sex had an effect on its cognitive flexibility (as measured by the TI), we ran GLMMs with the fixed effects time, sex, and the interaction of these two factors. The two models that only included testing time and sex, respectively, only differed marginally from each other (ΔAICc < 1). Neither time  = 0.62, p = 0.808) nor sex  = 0.91, p = .362) significantly affected a subject’s TI  = 0.71; p = 0.420. The two models that only differed marginally from the best model (ΔAICc < 1) each also included only one fixed effect, delay and sex respectively. Delay condition had a significant effect on a subject’s performance  = 3.44; p = .009) whereas a subject’s sex did not  = 0.17; p = 0.690)  who completed the 2-choice Memory task 1 a (study sample 1), 6 subjects did so in our set time (≤ 12 days) whereas 6 subjects took longer (> 12 days). The best model to predict whether time needed to complete the task had an effect on the monkeys’ performance only included the fixed effect time which did not have a significant effect on performance : 690) see .Of the 15 monkeys (8 marmosets and 7 squirrel monkeys) in study sample 2 who completed the two-choice memory task, eleven individuals  did so in time (≤ 4 days) whereas the other 5 individuals  needed longer (> 4 days).F = 0.38; p = .544) (see The best model only included the fixed factor time which did not have a significant effect on the monkeys’ performance (544) see .F = 5.33, p = .002). Although the next best model also included time, required testing time did  not significantly affect performance  = 3.00, p = 0.095) (see When combining the (first) four delay conditions of those marmosets (sample 1 and 2) and squirrel monkeys who had completed all essential trials of the two-choice memory task, we obtained performance scores for two more male marmosets from sample 1 (Kapi and Kantor) who had dropped out in Memory task 1a because they had not completed all 6 delay conditions. Of the 28 monkeys (7 squirrel monkeys and 21 marmosets) who completed the four delay conditions, ten (4 marmosets and 6 squirrel monkeys) did so within the expected time (4 days) whereas 18  took longer (> 4 days). The best model only included the fixed factor delay which had a highly significant effect on the monkey’s performance (095) see .5.F = 5.48; p = .003, indicating that memory performance decreased with increasing retention delay. While species had no effect on overall performance in any of the models, there was a significant interaction of delay and species in the third-best model (see Of the 14 monkeys (7 marmosets of study sample 2 and 7 squirrel monkeys) who completed the nine-choice memory task, nine individuals  did so in time (4 days) whereas the other 5 individuals  needed longer (> 4 days). Time to complete the 9-choice memory task (4 days versus > 4 days) did not affect the subjects’ performance in the 9-choice memory task (no significant effect in any of the models). The best model only included the fixed effect delay which did affect performance (i.e. the proportion of correct choices): odel see .6.The main question was whether the amount of testing time a subject required to complete a task affected its cognitive performance in that task. Our results show that this was not the case and that in each task, the performance of subjects who needed longer than expected to complete the task did not differ from those who completed the task in time.It is important to stress that even in our full testing approach, we lost subjects from the sample. This was because the monkeys were never forced to enter the testing enclosures but had to be at least sufficiently motivated to approach the testing area voluntarily to be included. While we had made every effort to allow each individual to complete each task at its own pace, it was not justified to continue testing for a completely unlimited time period. Therefore, we cannot rule out the possibility that the few subjects who dropped out in some tasks despite being given ample time to complete them, differ in cognitive ability from subjects who completed the tasks.Despite this possible exception, our findings suggest that opportunistic testing in primates (i.e. only testing subjects who readily participate) does not bias the results of cognitive tasks in several physical domains, at least in marmosets and squirrel monkeys. To what extent they generalise to tasks from the social domain or to paradigms that do not require subjects to choose between a set of presented options remains to be established.Species differences in cognitive performance were minor and mostly non-significant, even though we cannot exclude that this may be owing to the relatively low number of squirrel monkeys in our sample. The only significant effect was that in the detour-reaching task, marmosets increased their performance over time, whereas squirrel monkeys did not, which led to increased inhibition performance in marmosets in later trials see . One posOur finding that the selection bias that results from opportunistic testing does not affect cognitive performance in marmosets and squirrel monkeys in several physical cognition tasks adds to the list of non-cognitive factors that have been studied so far regarding their potential to affect cognitive performance . While rTo sum up, we showed that at least for two New World monkey species and the given set of cognitive tasks, opportunistic testing does not bias the test results. If our findings generalise to other non-human primate species and cognitive tasks, then maximising sample sizes by testing only consistently motivated subjects will be a valid alternative whenever a sufficiently large number of subjects is available but full testing is not feasible owing to time constraints.Future studies should extend the list of non-cognitive factors that affect cognitive performance, so that the validity and reliability of existing cognitive tests can be further improved and newly developed tests will be less prone to performance biases. Once this task is achieved, cognitive test batteries can then be used more reliably to compare the cognitive abilities of non-human primate individuals and species.This study was performed in accordance with the Swiss legislation and licensed by the Veterinary Office of the Canton of Zurich . Thus before, during and after this study, the monkeys were never constrained or subjected to any pain, suffering or injury and their general state of health was not impaired. All cognitive tasks were conducted non-invasively between the monkeys’ regular feeding times. The monkeys could freely enter and leave the test enclosure without being handled by humans at any time and were never isolated from their social groups. After the completion of this study, the monkeys continued living at the Primate Station, eventually participating in other non-invasive studies.S1 TableEach test apparatus was placed on a wooden board (varying size and features) that was mounted on a height-adjustable test table and flush with the test compartment’s window. Depending on the cognitive task, the Perspex window front used for the marmosets contained one or two openings with rounded edges at a distance of 10 cm above the test compartment’s floor: (1) one large opening (18 x 2.5 cm), (2) two small openings: 4 x 2.4 cm, 12 cm apart.(PDF)Click here for additional data file.S1 FigSuccessful detour-reaching in the inhibition trials at (a) first attempt and (b) after initial failure to reach around the transparent barrier. Marmosets (grey solid bars)/squirrel monkeys (yellow dotted bars).(PDF)Click here for additional data file.S2 FigFewer  sessions indicate better performance, hence the negative prefix on the y-axis. Phase 1—learning of the initial association between a pattern and a reward. Phase 2—strengthening of the learned association. Phase 3—reversal of the learned association.(PDF)Click here for additional data file.S3 FigThe dotted line represents the expected amount of time (number of testing days) after which testing would have been discontinued and subjects who needed longer would have been excluded had we used the classical opportunistic approach. (a) The marmosets of study sample 1 (represented by ID numbers M1-M16 and gender symbols) were tested with version a of Memory 1 , (b) the marmosets of study sample 2 (ID numbers M17-M27 and gender symbols) and the squirrel monkeys (represented by yellow triangles) were tested with version b . The two versions of Memory 1 slightly differed in details of experimental set-up (apparatus) and design Click here for additional data file.


CHRM2), related to heart rate recovery, was replicated in recent genome-wide association studies (GWASs). Additional 17 candidate causal genes were identified for heart rate increase and 26 for heart rate recovery in these GWASs. Nine of these genes were associated with both acute increase and recovery of the heart rate during exercise. These genes can be broadly categorized into four categories: (1) development of the nervous system ; (2) prolongation of neuronal life span (SYT10); (3) cardiac development (RNF220 and MCTP2); (4) cardiac rhythm (SCN10A and RGS6). Additional 10 genes were linked to long-term modification of the heart rate response to exercise, nine with heart rate increase and one with heart rate recovery. Follow-up will be essential to get functional insights in how candidate causal genes affect the heart rate response to exercise. Future work will be required to translate these findings to preventive and therapeutic applications.The acute heart rate response to exercise, i.e., heart rate increase during and heart rate recovery after exercise, has often been associated with all-cause and cardiovascular mortality. The long-term response of heart rate to exercise results in favourable changes in chronotropic function, including decreased resting and submaximal heart rate as well as increased heart rate recovery. Both the acute and long-term heart rate response to exercise have been shown to be heritable. Advances in genetic analysis enable researchers to investigate this hereditary component to gain insights in possible molecular mechanisms underlying interindividual differences in the heart rate response to exercise. In this review, we comprehensively searched candidate gene, linkage, and genome-wide association studies that investigated the heart rate response to exercise. A total of ten genes were associated with the acute heart rate response to exercise in candidate gene studies. Only one gene (The online version of this article (10.1007/s00018-019-03079-4) contains supplementary material, which is available to authorized users. The regulation of resting heart rate is complex; autonomic tone, central and peripheral reflexes, hormonal influences, and factors intrinsic to the heart are all important determinants , 2. DespThe acute heart rate response to exercise, heart rate increase during and heart rate recovery after exercise, offers unique insights into cardiac physiology compared to heart rate in rest and can therefore be exploited to obtain additional information on cardiac function . ImpaireBoth the acute and long-term responses of heart rate to exercise have been shown to have a large heritable component –17. DeveIn general, the regulation of the circulatory system during exercise involves several adaptations. These adaptations include dilatation of resistance vessels in the active muscles, a decrease in vagal outflow to the heart, followed by an increase of sympathetic outflow. If exercise is intense, the cholinergic fibers to the adrenal medulla are also activated, resulting in release of epinephrine into the circulation . Under nThe increase of heart rate during exercise is for a major part attributable to the decrease in vagal tone followed by an increase in sympathetic outflow and an increase in levels of circulating catecholamines . It has ACE gene was one of the first candidate genes thoroughly investigated for its possible relationship with the heart rate response to exercise . F. F, related to heart rate recovery, was replicated in recent GWASs. Additional 17 candidate causal genes were identified for heart rate increase and 26 for heart rate recovery in these GWASs. Nine of these genes were associated with both acute heart rate increase and recovery during exercise. These genes can be broadly categorized into four categories: (1) development of the nervous system ; (2) prolongation of neuronal life span (SYT10); (3) cardiac development (RNF220 and MCTP2), and (4) cardiac rhythm (SCN10A and RGS6). Of the total of 43 genes, nine showed overlap with resting heart rate and heart rate variability, six with atrial fibrillation and coronary artery disease, two with ECG traits, and nine with blood pressure. The current findings support the idea that the autonomic nervous system is a major player in the regulation of the acute heart rate response to exercise. Heart rate recovery is especially influenced by parasympathetic nervous system genes (ACHE and CHRM2), in line with the previous research [In the current review, we found a total of 10 genes associated with the acute heart rate response to exercise in candidate gene studies. Only one gene Below is the link to the electronic supplementary material.


Transcriptional responses to the appropriate temporal pattern of action potential firing are essential for long-term adaption of neuronal properties to the functional activity of neural circuits and environmental experience. However, standard transcriptome analysis methods can be too limited in identifying critical aspects that coordinate temporal coding of action potential firing with transcriptome response. A Pearson correlation analysis was applied to determine how pairs of genes in the mouse dorsal root ganglion (DRG) neurons are coordinately expressed in response to stimulation producing the same number of action potentials by two different temporal patterns. Analysis of 4728 distinct gene-pairs related to calcium signaling, 435,711 pairs of transcription factors, 820 pairs of voltage-gated ion channels, and 86,862 pairs of calcium signaling genes with transcription factors indicated that genes become coordinately activated by distinct action potential firing patterns and this depends on the duration of stimulation. Moreover, a measure of expression variance revealed that the control of transcripts abundances is sensitive to the pattern of stimulation. Thus, action potentials impact intracellular signaling and the transcriptome in dynamic manner that not only alter gene expression levels significantly (as previously reported) but also affects the control of their expression fluctuations and profoundly remodel the transcriptional networks.  Expression of the 20,000–25,000 protein-coding genes in mammalian neurons is regulated by a complex, inter-coordinated network that is constantly in a dynamic state of modulation influenced by neural impulse activity. The most common method of analyzing gene transcription in cells is by measuring the abundance of tens of thousands of specific gene transcripts simultaneously in cells by microarray or RNA sequencing. The values obtained from multiple biological replicate experiments are represented by a single measure of central tendency of gene expression level for each gene product, typically the arithmetic mean. Statistical comparisons are made to identify mRNA transcripts that are significantly increased or decreased in abundance following an experimental treatment relative to a control. Although this approach is valid and insightful, it is limited in determining how transcriptional networks may be modulated in response to an external stimulus, especially so when the stimulus applied is temporally dynamic and fails to drive the cell into a steady state. Standard analysis also ignores changes in the control of transcripts abundances and in the interplay of functional pathways caused by the experimental treatment.This is the situation in neurons responding to action potential input of different temporal patterns within the physiological range of activation. Under normal conditions, transcriptional networks are being dynamically activated (or inhibited) by action potential firing. However, this activity may fail to produce a measurable increase or decrease in abundance of a given mRNA transcript, even though the transcript is part of a transcriptional network that is being modulated dynamically. This may occur because, in contrast to receptor–ligand-mediated signaling in most non-neuronal cells, neuronal response to sensory input, and information processing within neural networks, is a temporal code of transient membrane depolarizations. In neurons, the action potential firing frequency and burst patterns, together with synchrony and phase relationships among populations of interconnected neurons, is the fundamental mechanism of operation . NeuronsAnalysis of only the expression level of individual genes fails to exploit the richer information available from the microarray and sequencing data which quantifies the expression levels of all genes simultaneously, beyond what could have been obtained by measuring each gene separately. By analogy, measuring volume of traffic of different types of vehicles on roads would be insufficient to elucidate the complex structure and operation of a transportation network; how the traffic is coordinately changing is an essential feature. A convoy is not typically recognized by identifying vehicles exceeding the speed limit, but rather by the highly coordinated changes in speed among all the vehicles in the convoy. Likewise, the critical aspect of gene network function is the coordinated activity of elements in the network. Various forms of (weighted and unweighted) correlation network analyses were developed and widely used in the genomics field e.g., ,3,4,5) t,4,5 t3,42, N2O, N2O3, N2O4, and N2O5), each combination being dominant in particular conditions, how to conceive that tens of far more complex entities (genes) can form unique, universal and rigid pathways? Therefore, we consider that the genes responsible for a particular biological process  can be networked in various ways, depending on the nature of the biological system and can remodel in response to external stimuli. As such, the correlations between expressions of genes are not fixed forever, but dynamic and subject to change to optimize the functional pathways they are involved in. Distinct from the common paradigm, our correlation analysis determines how genes coordinate their expressions not in in different conditions or time-points but in biological replicas of the same condition/time point. The biological replicas (here four dishes of DRG neurons subjected to the same firing pattern and duration) can be considered as the same system subjected to slightly different environment that do not alter significantly the abundances of their transcripts. However, when two atoms, nitrogen and oxygen, can combine in several ways  neurons in cell culture. These neurons were chosen because they are not spontaneously active and, critically, they fire single action potentials in response to a brief electrical pulse, they also do not form dendrites or synapses on themselves. These properties of cultured DRG neurons allow precise experimental control of the firing pattern ,14. www.ncbi.nlm.nih.gov/geo) accession number GSE84872. The results of that analysis showed that hundreds of genes were regulated by the specific pattern of action potential firing. The analyses presented in this study refer to the transcript abundance variability and expression coordination, the other two independent measures in addition to the average expression level that can be associated to every neuronal gene in each stimulation paradigm. These analyses were applied to determine whether and how the control of the transcript abundances changes and functional pathways remodel after electrical stimulation. Similar types of statistical variation and covariance methods have been used by us to ascertain the reconfiguration of gene networks in cancer [A standard analysis of data from these experiments has been published ; expressn cancer ,17, infen cancer  and neurn cancer ,20,21, thttps://www.kegg.jp) and Gene Ontology (www.geneontology.org). We decided to analyze TRF genes because they modulate the expression of all other genes. CAS genes were analyzed owing to the important role of calcium signaling in controlling almost all major cellular processes. This is especially relevant in neurons, where Ca2+ signaling is a critical component linking action potential firing with intracellular signaling pathways and gene expression. Finally, we analyzed the voltage-dependent ion-channels because they are the most sensitive to the electrical activity of the neurons.93 genes related to the calcium signaling (CAS), 934 transcription factors (TRF) and 41 voltage-dependent ion channels were selected using Kyoto Encyclopedia of Genes and Genomes  one day following plating for 5 days. Culture conditions have been optimized to enrich for medium-large type DRGs. Genetic markers of small type (nociceptive) neurons, Tyrosine hydroxylase and P2rX3 are expressed at low levels. Cultures were subsequently used for experiments 3–4 weeks after plating by which time axons extend from the side compartments into the central compartment under the central barriers and could be electrically stimulated. These cultures contain no detectable Schwann cells or astrocytes. The Schwann cell specific isoform of the cell adhesion molecule L1 is undetectable by RT-PCR [gfap is not detected by microarray [All experiments were conducted in accordance with animal study protocols approved by the NICHD Animal Care and Use Committee . For dissociated DRG cell culture, multi-compartment Campenot chambers made of Teflon, were affixed by vacuum grease to collagen coated 35 mm cell culture dishes as previously described . Neuronsy RT-PCR , and thecroarray  or RNA sDRG cultures with axon outgrowth under the Teflon barrier 23 weeks post plating were selected for electrical stimulation. A complete media change to media lacking NGF and sera was carried out and stimulating lids were placed onto each dish. Cultured cells were then placed back into the incubator and left undisturbed overnight. The following day, cultures were stimulated through platinum electrodes in contact with media on opposite sides of the Teflon barrier and the bulk media. Stimulation parameters and responses to stimulation have been reported previously . For thi12 = −0.985) which at its turn is positively correlated with G3 (ρ23 = 0.986). Thus, the present study offers not overlapping but complementary perspectives to the previously published results [Microarray experiment was carried out in four replicas of each of the five stimulation conditions on the Agilent-026655 Whole Mouse Genome Microarray 4 × 44K v2 two-color microarray platform GPL10333 as previously described . All con results  that reli probed by spot k , one gets the average expression level ikμ and the standard deviation ikσ. For the purpose to account for the multiple testing of the same gene and to give more credit to the transcripts probed by larger number of spots, we replaced the traditional coefficient of variation CV = σ/μ by the Relative Expression Variability (REV). REV is defined for each gene in each condition as the mid chi-square interval estimate of the pooled CVs for i -14R degrees of freedom and 5% level of significance: An important descriptive statistic beyond measures of central tendency is the variance in individual gene expression observed within experimental replicates. The dispersion around mean results from both technical noise  and biological variability. In addition to the stochastic nature of the chemical reactions involved in transcription ,24, the iR = 1,2, …, 28, the redundancy correction took values from 0.960 (iR = 28) to 1.566 (iR = 1). Expression of some genes is more variable than others, as different genes are regulated by distinct mechanisms in an intricate network of regulatory pathways. Immediate early genes, for example, would be expected to exhibit higher variance because these genes respond rapidly to a wide variety of stimuli and physiological changes [With  changes . HoweverExpression variability is an indicator of the cell priorities in controlling the transcript abundances, with genes critical for the cell survival allowed the least expression variation (lowest REV) across biological replicas .2) expression levels of gene pairs in biological replicas. We apply this analysis only on results from biological replicas to avoid gene pairing being compromised by exposure to different conditions (here different stimulation paradigms). The analysis identifies the genes whose expressions are significantly (p < 0.05) positively or negatively coordinated, and of genes whose expressions are significantly (p < 0.05) uncorrelated. The statistical significance of the Pearson correlation coefficient for the number of paired (log2) expression levels was determined using a two-tail t-test for the degrees of freedom df = 4*R (number of spots probing redundantly each of the correlated transcripts) − 2. For most gene pairs, df = 4 − 2 = 2. If there are two spots probing each gene, then df = 8 − 2 = 6, for 3 spots df = 12 − 2 = 10 and so on. When both genes are probed by a single spot, then for the four pairs of expression levels in the biological replicas the p < 0.05 significant correlation applies for |ρ| > 0.95. If both genes are probed by 2 spots each, then p < 0.05 significant correlation is for |ρ| > 0.71, and so on, larger the numbers of probing spots, less Pearson coefficient is necessary for the p < 0.05 significant correlation. However, not many genes had exactly the same number of probing spots, in which case we had to consider the pooled values with the redundancy groups of spots. https://www.youtube.com/watch?v=Kc3M5x7125A presents a tutorial on of how to determine the Pearson correlation coefficient and its significance in Excel.Expression variability further allows calculation of Pearson product-momentum correlation coefficient  between As analyzed by Marbach et al. , the PeaThe selected genes formed 4278 distinct CAS–CAS gene pairs, 435,711 TRF–TRF pairs, 86,862 CAS–TRF pairs and 820 pairs of voltage-gated ion channels that were further analyzed for expression correlation and interplay. We have developed a Python version that performs the coordination analysis for the entire transcriptome and then sort the pairs according to their GO category in less than 10 min instead of days in Excel .i and j. Pair-wise relevance (PWR) analysis ,18 was ui is a CAS gene and j is a TRF). Because both the correlation analysis and the PWR analysis are symmetrical to the switch of the genes within the pair, neither one can determine what gene in the pair is the regulator and what gene is the regulated.PWR analysis was also used to determine the interplay between the calcium signaling and the transcription factors (in Equation (2), The REV values are reported in Itpkc, the most stably expressed CAS gene in the unstimulated neurons, associated with NGF-driven neurite outgrowth [P2rx4 which is the most stably expressed at 18/1 2 h but becomes very variably expressed if the stimulation is extended for 5 h . The case of P2rx4 indicates that the neuronal homeostatic mechanisms are highly dynamic and changes not only with the pattern but also with the duration of the stimulation. utgrowth , becomesp < 0.05) significant coordinations of the gene pairs were categorized as positive, negative or independent across all five conditions. The level of expression coordination was determined for all 97,629,351 distinct pairs of transcripts under each experimental condition. A summary of the 4728 CAS–CAS pairs, 435,711 TRF–TRF pairs and 86,862 CAS–TRF pairs are shown in Voltage-gated ion channels are transmembrane ion transporting proteins that are activated in response to changes in the membrane potential. We analyzed whether the action potential firing has consequences on the expression level and coordination of the genes encoding such channels. Scn3a (Nav1.3) but significantly down-regulated Scn10a (Nav1.8) and Scn11a (Nav1.9), and altered calcium currents as previously shown [Scn3a as unaltered in all conditions but 18/1 5 h, where it was significantly up-regulated by 2.06x. We found also that Scn10a was not affected by any stimulation, while Scn11a was up-regulated only in 18/1 5 h (by 2.10x). The differences between the new results and the 2003 results illustrate again the sensitivity of the transcriptome to the environmental conditions (here different patterns of stimulation). However, the coordination analysis revealed previously unknown coupling between these sodium channels and the potassium channels. Interestingly, the positive correlations of Scn3a with Kcna2, Kcnb1, Kcnc1, and Kcnd3 in 18/1 2 h neurons are not present in unstimulated neurons and are canceled in all other stimulation paradigms, indicating how responsive are the gene networks to the neuronal electrical activity. However, Scn3a establishes a new negative correlation at 90/5 5 h with the pseudogene Kcne1l , known to slow the activation of the Kcnq1 channel [It is interesting to compare the results from ly shown . Under t channel .The similarity in responses in coordinated expression between pairs of calcium related genes and pairs of transcription factor genes, suggests interaction between these two types of genes in a transcriptional network responding to patterned action potential firing. Adcy8-Calm2) or in the pattern 18/1 for the same 2 h duration  or in the unstimulated neurons . Camk1d-Hoxb3) or in the pattern 90/5 for the same 2 h duration . Together these results indicate that not only the pattern in which the same number of action potentials are delivered but also the duration of delivery profoundly alters the coordinated activity between transcription factor genes. Interestingly, in addition to considerably increasing the percentage of significantly coordinately expressed gene pairs, the electrical stimulation also reversed the type of coordination existing in the non-stimulated condition. Coordination reversal may occur not only in stimulated versus unstimulated neurons but also when changing the pattern of stimulation for the same duration or the duration for the same pattern. For instance, In Changes in gene expression in DRG neurons after action potential stimulation were analyzed in terms of the variability and coordinated activity between pairs of calcium signaling related genes and transcription factor genes. The analysis provides an important and different perspective on the activity-dependent transcriptional response in neurons than is obtained by simple identification of genes that are up or down regulated significantly following stimulation. Not only the expression levels of the neuronal genes are affected by stimulation but also the organizational principles of the  transcriptome  and the p < 0.05 level. However, the specific pairs of transcription factors and calcium signaling genes that are coordinately activated and with high prominence that are revealed here will provide a valuable database for researchers in studies related to a wide range of functions.The specific functional consequences of the two (18/1 and 90/5) patterns of action potential firing are not known; indeed, it is uncertain whether any persistent functional consequences would be produced by action potential firing in these two patterns. Determining the possible functional consequences of firing neurons in these two patterns for 2 or 5 h was not the objective of the present series of experiments. Instead, these experiments test whether the transcriptional network is altered by differences in the pattern of action potential firing detected by analyses of expression variability and expression coordination between pairs of genes, even if the levels of expression of these genes may not change sufficiently at the By hypothesis, we expected increased correlated activity between pairs of genes after action potential stimulation, and this was observed. However, we had no a priori expectation that the majority of gene pairs would be positively correlated; that is, both genes increasing or both decreasing in expression level in tandem after stimulation. A surprising result was that so few genes were negatively correlated (one gene increased while the other decreased in expression). Since a gene product in a network may have either a facilitatory or inhibitory influence, negatively correlated gene pairs could either promote or inhibit a pathway or functional response, depending on whether the inverse correlation uncouples two genes having the same direction of influence or disinhibits opposing influences. The finding of so few negatively correlated gene pairs, and an increase in the number of negatively correlated gene pairs with more prolonged impulse trains and prolonged stimulation, indicates that calcium-regulated genes and transcription factor genes in these neurons tend to influence the transcriptional network in the same direction in response to action potential firing. Camta2 and the calcium signaling gene Capn5, both of which are upregulated in cells in which cholesterol metabolism is altered [Tceb1-Capn5 is the only pairing carried over into any of the other conditions, 18/1 2 h and 90/5 2 h and the numbers are lower in both conditions. Pair-wise relevance analysis illustrated in  altered . Three oTceb1, however the calcium signaling genes in each pairing are unique to each time point. This likely shows a change in transcriptional priorities and calcium signaling in the simulated cells from cellular metabolic activity signature seen in the unstimulated condition to a more focused genetic signature related to the decoding of external signals. In support of this hypothesis it has recently been demonstrated that the activity of transcription factor Tceb1 is regulated by experience dependent neuronal plasticity driven by specific external stimuli in Caenorhabditis elegans [Tceb1 is a transcriptional regulator of transcription elongation, therefore it has the capacity to be a common regulator of many genes, including calcium signaling genes, which are controlled by neuronal activity firing patterns. Multiple steps in the transcriptional process are dependent upon calcium signaling, including transcriptional elongation and termination [Tceb1 and a novel pairing of Camta2-Cib1 is seen. As is observed at 2 h, the calcium signaling genes have varied roles in intracellular signaling and are all linked to neuronal function. The pairings in the 18/1 pattern at 2 h and 5 h stimulation time are dominated by the transcription factor  elegans . Tceb1 imination . After 5Tceb1-Cacng7 pairing, as we do with the 18/1 2 h time point, however a unique pairing of Camta2-Cagna7, not seen with the 18/1 stimulus is observed, indicating perhaps a regulated gene network specific to the 90/5 stimulus pattern. However, after 5 h of 90/5 stimulus we observe not only unique gene parings but also a new set of transcription factors and calcium genes. Creb3 transcription factor is a regulator of cytosolic calcium concentration [Aplysia [Cib2, a gene required for calcium dependent mechanotransduction and growth of auditory hair cells [The gene pairings with the 90/5 stimulus pattern show a different set of predominating transcription factors and novel gene pairings of transcription factors and calcium genes from the 18/1 stimulus and unstimulated cells, particularly after 5 h of stimulus. After 2 h of stimulus we again see the ntration  and memo[Aplysia  and is pir cells . TherefoThe gene pairs in the PWR analysis are computationally related, but may not influence each other directly if they operate in different pathways. However, with or without direct interaction, all functional pathways should work in a coordinated manner to optimize the cellular processes. In this respect, the covariance analysis provides a unique screen for possible biological interactions which could not be found by the traditional method of comparing expression levels of genes.The emphasis here has been on presumed activity-dependent effects on transcription on calcium signaling related genes and transcription factor genes. Nonetheless, action potential firing activates multiple intracellular signaling networks, notably via intracellular calcium signaling and subsequent activation of kinases and phosphatases, that impinges on the full spectrum of biological processes determining the abundance of specific gene transcripts, from gene transcription to mRNA degradation. The level of expression of any gene transcript measured by microarray in these experiments reflects the net result of all the multiple cellular processes influencing mRNA abundance. Although this analysis focused on calcium signaling related genes and transcription factors, in principle, the findings should apply to other gene pairs and to other cellular processes impacted by action potential firing that influence multiple aspects of gene expression.It is well-known that the correlation metric determines how genes are clustered. However, we do not construct gene regulatory networks according to their responses across conditions (including knockouts, as algorithms such as ENET , GENIE3,p-value should be corrected for multiple testing to establish whether a gene is significantly regulated when comparing two conditions. The correction, using either Benjamini and Hochberg [Decades-long debate did not yet decide whether the Hochberg , BenjamiHochberg , BonferrHochberg , HochberHochberg , Holm [4Hochberg , or HommHochberg  method, The study has some limitations owing to the inherent technical noise of the microarray platform. However, the strikingly different patterns of coordinated gene expression seen in the five different stimulus conditions are consistent with expected differences in calcium signaling produced by the two action potential firing patterns and with the apparent homeostatic changes over stimulus time. Overall, these results indicate that the transcriptome is being altered in a coordinated manner, despite signal-to-noise considerations.


A receptor δ, α4 and β2 subunit mRNA expression in the hypothalamus, which are predominantly involved in promoting tonic neuronal inhibition. Allopregnanolone, an allosteric modulator of GABAA receptors, which increase the expression of these subunits in the amygdala and hypothalamus, is shown to reduce the hypertension and sympathetic nervous system contribution in BPH/2 mice. Thus far, evidence suggests that BPH/2 mice have aberrant GABAergic inhibition, which drives neuronal overactivity within amygdalo-hypothalamic brain regions. This overactivity is responsible for the greater sympathetic contribution to the hypertension in BPH/2 mice, thus making this an ideal model of neurogenic hypertension.It has been 45 years since Gunther Schlager used a cross breeding program in mice to develop inbred strains with high, normal, and low blood pressure . Thus, it is timely to gather together the studies that have characterized and explored the mechanisms associated with the hypertension to take stock of exactly what is known and what remains to be determined. Growing evidence supports the notion that the mechanism of hypertension in BPH/2 mice is predominantly neurogenic with some of the early studies showing aberrant brain noradrenaline levels in BPH/2 compared with BPN/3. Analysis of the adrenal gland using microarray suggested an association with the activity of the sympathetic nervous system. Indeed, in support of this, there is a larger depressor response to ganglion blockade, which reduced blood pressure in BPH/2 mice to the same level as BPN/3 mice. Greater renal tyrosine hydroxylase staining and greater renal noradrenaline levels in BPH/2 mice suggest sympathetic hyperinnervation of the kidney. Renal denervation markedly reduced the blood pressure in BPH/2 but not BPN/3 mice, confirming the importance of renal sympathetic nervous activity contributing to the hypertension. Further, there is an important contribution to the hypertension from miR-181a and renal renin in this strain. BPH/2 mice also display greater neuronal activity of amygdalo-hypothalamic cardiovascular regulatory regions. Lesions of the medial nucleus of the amygdala reduced the hypertension in BPH/2 mice and abolished the strain difference in the effect of ganglion blockade, suggesting a sympathetic mechanism. Further studies suggest that aberrant GABAergic inhibition may play a role since BPH/2 mice have low GABA Experimental models of hypertension have been developed over the last half century and have made a major contribution to our understanding of the mechanisms underlying the development and treatment of hypertension. Of particular note here is the spontaneously hypertensive rat (SHR), the stroke-prone SHR from Japan, and other inbred strains such as the Milan  and LyonGenetically hypertensive mice are one of three lines of mice, namely BPL/1 (Blood Pressure Low), BPH/2, and BPN/3 , which were concurrently selectively bred based on their BP . It is wHypertensive BPH/2 mice display higher BP than control lines from as young as 6 weeks of age, based on tail-cuff measurements . SBP in The introduction of radio-telemetry measurement for mice was developed in 2000  nearly aDavern and colleagues measured the cardiovascular response to a range of acute stressors in BPH/2 mice compared with BPN/3 mice . These iThe earliest indication that BPH/2 mice may have a neurogenic form of hypertension came from findings showing an inverse relationship between whole brain noradrenaline levels and BP in the three lines of mice  . Howevervia influences on the SNS.The amygdala integrates sensory  and cognitive information to initiate behavioral responses to specific stimuli during aversive psychological and physiological stress as well as reproductive stimuli. In addition, a major pathway for sympathetic activation involves projections from the amygdala to the anterior hypothalamus, the dorsomedial hypothalamus (DMH), and the ventromedial hypothalamus (VMH) . These pThe MeAm was also one of the brain regions with greater c-Fos containing neurons in BPH/2 mice compared with BPN/3 mice following exposure to 1 h of dirty cage switch stress . This inThe idea that the amygdala contributes to hypertension is not new and was shown to be important to the development of hypertension in SHR, based on the antihypertensive effect of electrolytic lesions of the amygdala . FukumorMicroinjections into the MeAm with ibotenic acid, which ablates cell bodies but not fibers of passage, resulted in a hypotensive effect that abolished 64% of the hypertension in BPH/2 but had no effect in BPN/3 . MeAm levia multiple routes  and for a range of durations (30 min to 2 weeks) revealed that the sympathetic vasomotor inhibitory effect of rilmenidine is minimal and similar in hypertensive BPH/2 and normotensive BPN/3 mice  subunits in the hypothalamus of BPH/2 compared to BPN/3 . A major study by Marques and colleagues used microarray and quantitative real-time polymerase chain reaction (qPCR) of the hypothalamus of young and adult BPH/2 and BPN/3 mice . This woto BPN/3 ,b. This 2 strain .AR is a heteromeric pentamer made up of five of 19 currently known subunits arranged in a combination of two α, two β, and a third  subunit all arranged around a chloride conducting central pore. The combination of subunits gives rise to the vast number of structurally unique GABAAR, which contributes to its diverse functionality. The amygdalo-hypothalamic pathway contributes to hypertension in both BPH/2 mice and SHR and may involve a common mechanism leading to increased sympathetic vasomotor tone. Combining the microarray finding of altered GABAAR subunit expression in the hypothalamus with the lower levels of GABAAR in the hypothalamus and amygdala of SHR  reduced BP by 40% in BPH/2 after 14 days with no effect in BPN/3 mice  has been explored in a variety of ways in BPH/2 mice including genetic investigations, analysis of expression or activity of RAS components, and pharmacological assessment of the RAS contribution to BP. Yet, delineating the contribution of the RAS to the hypertension in BPH/2 mice has proven quite complex. Angiotensin II (AngII) is an important effector hormone, being part of the RAS, which is well known to contribute to BP regulation by influencing dipsogenic and sodium appetitive responses, vasopressin release, and importantly sympathetic vasomotor tone . In 20011R) antagonist], Palma-Rigo and colleagues found little difference between the hypertension induced by the drug in both strains, which was in the order of 23–25 mmHg  both acutely and chronically in BPH/2 and BPN/3 mice. Acute administration of candesartan actually produced a lesser depressor response in BPH/2 mice compared with BPN/3 mice during the dark (active) period, whereas the response was comparable between strains in the light (inactive) period (1R contribution to BP is not evident in BPH/2 mice. Reactive oxygen species (ROS) such as superoxide can be second messengers for AngII-mediated signaling in the brain  period  in BPH/2 mice. Nonetheless, it seems that in contrast to the peripheral RAS, overactivity of the central RAS does not contribute to the hypertension in BPH/2 mice.The suggestion that augmented central AngII stimulation of ATH/2 mice . This wa) period . Subcutahe brain ; therefohe brain . Taken t) period , 2018. E) period . Furtherc manner . It woulDespite being hypertensive from a relatively young age, BPH/2 mice do not have greater absolute heart weight nor left ventricle weight compared with normotensive BPN/3 mice . HoweverTo date, the impact of hypertension on the structure of the vasculature has only been assessed in cerebral arterioles. Baumbach and colleagues reported that BPH/2 mice exhibit cerebral arteriole hypertrophy but no reduction in external diameter (remodeling) such as that seen in other models of hypertension . This isHypertensive BPH/2 mice are reported to release double the amount of hydrogen peroxide from the aorta compared with BPN/3, which may indicate a greater level of oxidative stress . MeasureHypertensive BPH/2 mice have similar kidney-to-body weight ratio as BPN/3  but haveAn important phenotypic difference observed in BPH/2 mice is that their body weight is lower compared with BPN/3 mice . HoweverThe lower fat mass may also contribute to why hypertensive BPH/2 mice are less capable of thermoregulation than BPN/3 mice, as indicated by a greater rate of rise of body temperature, higher end temperature, and shorter time until death following exposure to 43°C heat . These dAn additional subline of mice produced by Schlager and colleagues is the BPH/5 strain. Compared with C57Bl6 mice, this strain has moderately elevated BP, which is increased further during pregnancy . As suchA more recent cross of BPH/2 with the apolipoprotein E-deficient mouse (APoE) was developed to examine the interaction between hypertension associated with high SNS activity with an atherosclerotic-prone mouse . These mWatson and colleagues recently examined the effect of streptozotocin-induced diabetes in the BPH/2 and BPN/3 strains . While dAR dysfunction within the CNS and most likely within the amygdala and hypothalamus (The development of the strains of hypertensive mice by Gunther Schlager and colleagues in the 1970s has led to a comprehensive examination of mechanisms of hypertension. This led to the discovery that the hypertension is principally related to chronic activation of the SNS and as such is one of the few true models of neurogenic hypertension . Our ownthalamus . Neurostthalamus . Excitinthalamus . Howeverthalamus .To date, the evidence for the involvement of limbic regions in human essential hypertension is limited to indirect associations. For instance, using blood-oxygen-level-dependent functional magnetic resonance imaging, greater amygdala activity is associated with greater BP reactivity to stress, which in turn is associated with greater risk of developing hypertension . ImportaAll authors contributed to the drafting, editing and finalizing of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.