Appetitive trace conditioning (TC) was examined over 6 months in younger-adult (2–8 months) and middle-aged (12–18 months) male Wistar RccHan rats, to test for early age-related impairment in working memory. Novel object recognition (NOR) was included as a comparison task, to provide a positive control in the event that the expected impairment in TC was not demonstrated. The results showed that TC improved at both ages at the 2 s but not at the 10 s trace interval. There was, however, evidence for reduced improvement from one day to the next in the middle-aged cohort tested with the 2 s trace conditioned stimulus. Moreover, within the 10 s trace, responding progressively distributed later in the trace interval, in the younger-adult but not the middle-aged cohort. Middle-aged rats showed NOR discriminative impairment at a 24 h but not at a 10 min retention interval. Object exploration was overall reduced in middle-aged rats and further reduced longitudinally. At the end of the study, assessing neurochemistry by HPLC-ED showed reduced 5-HIAA/5-HT in the dorsal striatum of the middle-aged rats and some correlations between striatal 5-HIAA/5-HT and activity parameters. Overall the results suggest that, taken in isolation, age-related impairments may be overcome by experience. This recovery in performance was seen despite the drop in activity levels in older animals, which might be expected to contribute to cognitive decline. Trace conditioning (TC) procedures are used to test animals’ ability to associate events across a time interval, in this case between a conditioned stimulus  offset and an unconditioned stimulus  onset . Even quTC is reliably demonstrated in both appetitive (e.g. food US) and aversive (e.g. footshock US) procedures. However, aversive procedures have more typically been used in studies of its neural substrates . Aversiv1 receptor agonists have been found to restore working memory deficits in aged monkeys  pathways . Moreove monkeys .1 receptor modulation of appetitive TC  was selected because early behavioural changes are key to the detection of biomarkers for the onset of age-related cognitive decline, prior to the onset of irreversible neuropathological changes. Thus we set out to examine TC as a prospective behavioural screening tool, to assess the efficacy of treatments to restore cognitive function. Measures at different timepoints (every 6 weeks) enabled us to track age-related decline in the ability to condition a noise CS with food US over a trace interval. Importantly, a matched cohort of younger adult rats was conditioned under identical conditions but from 2 to 8 months of age, to provide some control for the effects of repeated training which would potentially offset age-related decline. Delay conditioning can (in principle) provide a suitable control, against which assess changes in TC. However, delay conditioning is itself susceptible to age-related decline . In the Novel object recognition (NOR) was included as a comparison task, to provide a positive control in the event the expected impairment in TC was not demonstrated. The exploratory behaviours measured in NOR tasks are spontaneous, relying on rodents’ preference for (relatively) novel objects or locations, no further motivation is required . NOR tes1 receptor modulation on NOR  for later assay by high-pressure liquid chromatography with electrochemical detection (HPLC-ED), in order to examine how age-related behavioural changes related to neuropharmacological substrates. The monoamine neurotransmitters and brain regions examined were selected because of their likely relevance to the cognitive changes anticipated and guided by our earlier experimental studies of TC  as well n on NOR . The hyp22.1On arrival in the laboratory, male Wistar RccHan rats (Harlan) were caged in weight-matched groups of 4 in large double-decker individually ventilated cages (IVCs) which also provide animals with the opportunity to retreat from social interaction. They were kept on a 12:12 h light/dark cycle with free access to water, as well as unrestricted access to food during a 2 week acclimatization period. After 4 days’ acclimatization, all rats were handled daily for 1 week on arrival and at least twice weekly thereafter for the duration on the study. Weights were recorded every 2–3 days, more frequently in the case of any sudden weight loss , or if daily husbandry checks indicated any signs of malaise.ab libitum. Housing density was reduced to 2 rats per cage if the rats became too large to be comfortably accommodated at the original stocking density of 4/cage. The rats were closely monitored in house until they reached the age of 12 months (at 466–701 g), at which point the experimental phase of the study commenced. Two rats developed non-specific signs  prior to any conditioning, one later became prone to nose bleeds. In total 3 rats showing non-specific symptoms were humanely killed.The 24 rats to be used as the middle-aged group arrived at 16 weeks of age (at 324–452 g) from the Harlan Netherlands barrier. Following a total 2 week acclimatization period, this first cohort (now at 335–472 g) was food restricted to limit the development of adiposity which plays a major role in the development of inflammation and insulin resistance over the course of aging, such that the older rats did not rapidly exceed 500–600 g (as per veterinary instructions). Weight control was achieved by housing the rats in weight-matched groups (with subsequent adjustments to their housing as necessary) and tailoring the daily food ration. In addition to the sucrose pellets provided during conditioning, all rats received approximately 5 g per 100 g bodyweight food ration up to 20 g per day, this ration was adjusted as necessary to allow for healthy weight gain and to stabilise weights once these exceeded 400 g. The home cage ration was available until it was consumed, as the duration of food deprivation at the point of testing was not critical. Water was available The middle-aged cohort was confirmed pathogen free. However, rodent parvovirus infection was later identified at Harlan Netherlands and they were therefore unable to supply matched rats to meet the experimental schedule. Harlan (now trading as Envigo) UK were able to supply Wistar RccHan rats, of matched genetic background and health status, as required 2 weeks prior to the scheduled start of the experimental phase. The 24 rats to be used in the younger-adult group arrived at 6 weeks of age from the Harlan UK barrier (at 151–180 g). The younger adult rats were acclimatized and handled, as for the middle-aged group, until they reached the age of 2 months (at 221–275 g), at which point the experimental phase of the study commenced.All rats were introduced to sucrose reward pellets in their home cage over two days following the introduction of food restriction for experimental purposes. Regulated procedures were carried out in accordance with the principles of laboratory animal care, specifically the United Kingdom (UK) Animals Scientific Procedures Act 1986, Project Licence number PPL 40/3716, with the approval of the University of Nottingham Animal Welfare and Ethical Review Body (AWERB) committee and following the ARRIVE guidelines.2.2Experimental testing was conducted within a set of 4 fully automated ventilated conditioning chambers, adapted for appetitive conditioning. The food magazine  was constantly illuminated for the duration of the pre-conditioning and conditioning sessions . Thus the magazine light helped the rats to find the food  but was not predictive like the CS. Access to the magazine was via a magazine flap. Nose-pokes were recorded by the breaking of the photo beam within the food magazine. The US was two 45 mg sucrose pellets  dispensed into the magazine. The CS was a mixed frequency noise, presented via a loudspeaker in the roof of the chamber, set at 80 dB and of 5 s duration. An experimental background stimulus was provided by three wall mounted stimulus lights and the house light flashing on (0.5 s) and off (0.5 s), continuously for the duration of the conditioning session. This ‘envelope stimulus’ provided an additional measure of contextual conditioning, as per earlier studies of the neural substrates of trace conditioning conducted in the same laboratory using the same behavioural procedure .2.3Allocation to TC groups was counterbalanced by box. The two cohorts of rats were also run in a counterbalanced sequence over two replications at each assessment timepoint. On each conditioning day there were 30 pairings of noise CS and food presented at a 2 s or 10 s trace interval. Conditioning was conducted on two consecutive days at each of the five timepoints.2.3.1There were two days of shaping to accustom rats to eating from the magazine. On the first day the rats were shaped in pairs, on the second and subsequent days of pre-conditioning they were placed individually in the conditioning chambers. On each of the first two days, rats had access to a pre-load of 15 reward pellets with an additional 15 rewards over 15 min to familiarise rats with the food deliveries. The tray flap door was propped open on days 1 and 2 but was closed on subsequent days so that the rats were required to nose-poke the door open to collect food. Then followed two days of baseline sessions, during which there were 30 unsignalled rewards over 60 min, delivered on a variable interval around 2 min, to habituate rats to the sounds produced by food delivery.2.3.2Depending on the experimental group, the sucrose US was delivered 2 s or 10 s after CS offset (in the two different trace groups). Thirty signalled reward deliveries were presented on a variable interval, with the constraint that the inter-trial-interval (ITI) was always at least 1.5 times longer than the inter-stimulus-interval (ISI) length. Throughout acquisition, the background stimulus (flashing lights) was presented continuously. This continuous presentation also encompassed the 2 s or 10 s ISI, as applicable, which added to the overall duration of a 60 min session so that conditioning sessions were of either 61 or 65 min total duration.The dependent variables to assess TC were the number of nose-pokes during the 5 s CS and during the 2 s or 10 s trace interval between CS and US (the ISI). This responding was compared with that seen in the 5 s after the delivery of the US in acquisition; and in the remainder of the session . Latency to enter the food magazine was not recorded and there was no way to measure latency to consume the rewards with the automated set-up in use. However, no food was left (by either cohort) at the end of the conditioning sessions.Because it was continuously presented, contextual conditioning to the flashing lights background could not be distinguished from (age-related changes in) ITI responding over the course of acquisition. We therefore examined responding to the experimental background, over and above any contextual conditioning to the cues provided by the experimental chambers, with a final set of extinction tests conducted 2 weeks after the fifth conditioning session. Following the test format of earlier studies, these used 30 5 s presentations of the flashing light stimulus over 30 min . The numThere were five timepoints of TC testing: starting from when the rats were aged 54 (the middle-aged cohort) or 10 weeks (the younger-adult cohort); at 6 weekly intervals ; up to when the rats were aged 78 (the middle-aged cohort) or 34 weeks (the younger-adult cohort).2.4Testing was conducted in a 38 × 40 cm opaque plastic rectangular arena with 54 cm high walls. An overhead camera (JVC Everio GZ-EX515) was used to record the rats’ exploratory behaviour. The object stimuli were provided by duplicate copies of bottles and flasks made of glass, metal or plastic, and of varied shape, colour and size, and of weight too heavy to be displaced by the rat. We used the same pair of objects to test all the rats at timepoint 1. A different pair was used for the re-test at timepoint 5, matched for discriminability on the basis of previous data . The tes2.5NOR procedures were based on established protocols . Rats weThere were two timepoints of NOR testing: (1) two weeks after the first conditioning session when the rats were aged 56 (the middle-aged cohort) or 12 weeks (the younger-adult cohort); and (2) the week after the fifth and final conditioning session when the rats were aged 79 (the middle-aged cohort) or 35 weeks (the younger-adult cohort).2.6Tissue samples were taken, using established micro-punch procedures . FollowiThe brains were removed rapidly and were placed dorsal side up in an ice-chilled rat brain matrix . Using ice-chilled razor blades, three 2 mm coronal brain sections were cut. The posterior side of the slices corresponded to approximately +3, +1 and −1 mm (anterior/posterior) from bregma according to the atlas of g for 20 min at 4 °C; Eppendorf centrifuge 5417R). The supernatant was then filtered through a 0.45 µM syringe tip filter (ThermoFisher Scientific). Neurotransmitter and metabolite levels in the samples were detected using HPLC-ED, separation was achieved using an autosampler (PerkinElmer Series 200), column  and mobile phase, which was delivered at a flow rate of 45 µl/min by an isocratic pump (Dionex P680). Analytes were detected with a Decade SDC electrochemical detector fitted with a Sencell (Antec), using an oxidation potential of 0.75 V vs the Ag/AgCl reference at 32 °C. The mobile phase consisted of 20 mM potassium dihydrogen orthophosphate, 20 mM sodium acetate, 0.1 Mm EDTA, 0.15 mM octanesulfonic acid and 10% v/v methanol adjusted to pH 3.89 by adding concentrated orthophosphoric acid. Both samples and standard were prepared for injection at 10 µl. The external standard consisted of 5-HT, DA and their metabolites, in concentrations of 10−7, 4 × 10−8, 2 × 10−8 and 10−8 M for calibration.The tissue samples were later thawed, sonicated (Soniprep150: MSE Scientific Instruments) for 15 s in 250 µl (mPFC & NAc) or 500 µl  0.1 M perchloric acid containing 0.4% sodium metabisulphite and 0.2% disodium ethylenediaminetetraacetic acid (EDTA), and centrifuged , ratios to index 5-HT and DA utilisation were computed for each brain region from the raw levels of metabolite and neurotransmitter (i.e. 5-HIAA/5-HT and DOPAC/DA). HVA levels were not reliably determined in NAc and mPFC but [DOPAC + HVA]/DA could be examined in dorsal striatum.2.7The dependent variable used to examine age-related differences in TC was the number of responses (nose-pokes into the food magazine) made during the CS presentations. To separate out effects on motor responding or motivation for food reward, similar analyses were conducted on the ITI and US response measures. Difference scores (CS minus the average ITI responding/5s) provided an adjusted measure of CS responding. Additionally, the responding of the animals during the 10 s trace interval between CS offset and US delivery was also examined for any age-related change in the distribution of responding within the trace interval. Thus, the 10 s ISI was broken down into two second bins of time and analysed using repeated measures ANOVAs by bins and days. Responding to the light background was examined only upon completion of the conditioning procedures at timepoint 5. Responding to 5 s light presentations was compared to that seen 5 s prior to the presentations (pre-stimulus) and 5 s after offset (post-stimulus), as well as in the ITI.t-test.The behavioural data were analysed in mixed design with the between subjects factors of trace (at 2 levels: 2 s and 10 s) and cohort (at two levels: younger-adult and middle-aged). To assess effects over the course of acquisition, the repeated measures (within subjects) factors were timepoint  and days (at 2 levels: 2 days’ conditioning per timepoint). Additionally, within day acquisition was examined by blocks ; and the distribution of responding within the 10 s trace interval was examined by bins (at 5 levels: 5 bins of 2 s within the 10 s trace). Responding to the light background was examined using the same factorial design applied to test within day acquisition to the noise CS, in this case in relation to six blocks of five unreinforced stimulus presentations (since there was only one day of extinction testing). Trend analyses of interactions involving timepoint, blocks or bins obviate the need for multiple comparisons . Selectet-test.The dependent variable used to examine NOR was exploration time for novel vs familiar objects over the 3 min test presentation. Exploration times were compared in a mixed design with the between subjects factor of retention interval (at 2 levels: 10 min and 24 h) and cohort (at two levels: younger-adult and middle-aged); the within-subjects factors were familiarity (at 2 levels: novel versus familiar) to assess NOR and timepoint (at 2 levels: 1 and 5) to assess effects longitudinally over the course of aging (within the timeframe of the study). Further analyses of the discrimination ratios used a single between subjects factor of group . Plannedt-test. Additionally, associations between neurochemical and behavioural parameters were explored by correlational analyses. These analyses were conducted using summary scores that reflected TC and NOR performance (for both cohorts) at the final time point. For TC, we used CS, ISI, US and ITI responding at the final (fifth) timepoint (averaged over the two days). For NOR, we used sample exploration, and exploration of the novel versus familiar object at timepoint 5 (when the second NOR test was conducted).Differences in neurotransmitter turnover between the cohorts at the final timepoint were determined by Student’s 33.1The middle-aged group (M = 138.729 ± 16.703) responded overall less than the younger-adult cohort , F = 13.778, p = 0.001. There were no significant effects by trace condition-to be, Fs < 1.3.2There was a significant effect of cohort × timepoint, F = 13.396, p < 0.001, as well as cohort × timepoint × trace, F = 5.561, p < 0.001. Timepoint was also significant by trace, F = 2.589, p = 0.039, as well as by day, F = 2.691, p = 0.033. The difference between the cohorts is of most importance because differences in ITI responding potentially confound the measure of TC provided by CS responding. The younger-adult cohort responded more in the ITI at timepoint 1, in the 2 s, t(20) = 3.642, p = 0.002, but not in the 10 s trace conditioned group, t(22) = 0.037, p = 0.971 A.Fig. 1M3.3In order to ascertain whether aged rats were less motivated by the sucrose rewards, responding in the 5 s following US delivery was also examined .As might be expected, ANOVA showed a number of effects involving timepoint, including by cohort, F = 4.710, p = 0.001. This arose because whilst older rats maintained consistent levels of responding , the younger-adult rats showed some overall reduction in US responding as training progressed . 3.4This was assessed by the use of a difference measure, to adjust for ITI responding (described above). There was a significant effect of cohort × timepoint, F = 3.495, p = 0.009. As shown in There was also a timepoint × trace interaction, F = 13.673, p < 0.001, significant also in the linear trend, F = 19.066, p < 0.001. This arose because overall conditioning improved in both cohorts as the study progressed, at the 2 s but not at the 10 s trace interval. The three-way interactions with cohort were not significant. The four-way interaction, F = 3.654, p = 0.007, significant also in the linear trend, F = 11.422, p = 0.002, appeared to relate to older rats’ reduced improvement from one day to the next, at the early timepoint measures of CS responding at the 2 s trace interval (which is further examined below). ANOVA of the raw scores showed the same statistical outcomes and the raw scores are shown in Initial acquisition was further examined by ANOVA of the timepoint 1 data . As would be expected based on the above analysis of CS responding averaged over the day, there were interactions between cohort and days, F = 4.525, p = 0.039 and cohort × trace × days, F = 12.601, p = 0.001. There was also a main effect of cohort, F = 15.678, p < 0.001, because the older rats responded overall less. Blocks was significant by trace, F = 4.236, p = 0.001, also in the linear trend, F = 8.085, p = 0.007, reflecting more rapid acquisition to the CS conditioned at 2 s versus 10 s. Of the interactions involving cohort and blocks none was significant, except for the four-way in the linear trend F = 6.956, p = 0.012, which was further explored by the selected comparisons shown in 3.5There was an effect of cohort × bin, F = 2.628, p = 0.040, significant also in the linear trend, F = 4.450, p = 0.046, as well as a three-way interaction of cohort × bin × timepoint, F = 1.735, p = 0.039, significant also in the linear trend, F = 4.308, p = 0.05. Therefore the interactions arise because a linear relationship best describes the change in responding across the 2 s time bins of the 10 s trace and this is different by cohort and timepoint. A further three-way interaction cohort × timepoint × day, F = 2.730, p = 0.034, did not relate to any systematic shift across the timepoints and will not be further discussed.3.6There were no effects of cohort on any of the corresponding measures of responding to the experimental background provided by the light stimulus , maximum F = 1.234, p = 0.273. ANOVA of responding to successive presentations of the light over the course of extinction (by the 6 blocks of 5 presentations) confirmed this conclusion: there were no effects by cohort, maximum F = 1.234, p = 0.273. As would be expected, overall response levels within these sessions were relatively low: middle-aged cohort M = 19.708 (±4.391); younger-adult cohort M = 23.958 (±4.066).3.7There was a main effect of cohort because older rats explored overall less at the test stage, F = 30.053, p < 0.001. There was also an effect of timepoint, F = 15.120, p < 0.001, because rats explored overall less at timepoint 5. An interaction between timepoint and delay, F = 8.311, p = 0.006, arose because test exploration showed a greater drop at the 24 h retention interval.The preference for the novel object was confirmed by the main effect of familiarity, F = 78.396, p < 0.001, which – as would be expected – depended on the retention delay, F = 11.729, p = 0.001, and the minute of the test session, F = 11.129, p < 0.001. Importantly, there was an interaction between familiarity and cohort, F = 4.129, p = 0.049, because the younger-adult cohort showed a greater difference in the exploration of the novel compared to the familiar object. However, none of the other interactions between familiarity, cohort and timepoint was significant, maximum F = 1.604, p = 0.207.As might be expected given the age-related effects on test exploration, there was also a clear effect of cohort on sample exploration, F = 34.171, p < 0.001, because the younger-adult group explored more than the older group. There were also effects of timepoint, F = 106.676, p < 0.001, as well as a timepoint by cohort interaction, F = 16.079, p < 0.001, because sample exploration reduced from timepoint 1 to timepoint 5, particularly in the younger-adult cohort which showed higher levels of sample exploration to begin with. improved NOR performance at the second NOR test conducted at timepoint 5. As expected, there was an effect of delay, F = 10.633, p = 0.002 because NOR performance was better at the 10 min than at the 24 h retention interval. None of the interactions involving age was significant, maximum F = 2.449, p = 0.125. However, when the between subjects factors of cohort and delay were collapsed  = 7.010, p = 0.011. However, this arose because of overall ollapsed , there w3.8In relation to the summary TC measures, there was a positive correlation between the 5-HIAA/5-HT ratio in the NAc and responding in the ITI at the final timepoint, r(45) = 3.23, p = 0.031 C. There 4The ages examined in the present study are approximately equivalent to human ages 45–60 years (in the middle aged cohort) versus 20–35 years (in the younger-adult cohort). Thus, the rats in the older cohort should have been pre-senescent, at least at the start of the study. Additionally, age-irrespective, rats were expected to show positive transfer from earlier training sessions which would counteract age-related decline. On these grounds, behavioural changes indicative of cognitive impairment might be expected to be modest. Nonetheless, statistically significant effects involving timepoint and cohort confirmed that there were two age-related differences in performance in the appetitive TC procedure: (1) reduced improvement in conditioning from one day to the next in the middle-aged cohort, at the first timepoint measures of responding to the 2 s trace CS ; and (2)Testing conditioning over successive days and at different timepoints over an extended period requires retention to show improvement . TherefoAnalyses of responding within 10 s trace ISI bins showed that whilst the younger-adult rats eventually distributed their responding towards the latter part of the ISI, there was no such tendency in the middle-aged cohort; neither was there any evidence that the middle-aged cohort expected the US deliveries any earlier – responding remained evenly distributed across the 2 s bins of the ISI. In the absence of motivational differences between the age-groups (as suggested by the similar levels of US responding), this pattern of findings may reflect some age-related deficit in timing ability. In human eyeblink conditioning, it has been reported that aged participants showed impaired TC but preserved ability to time blink CRs in relation to airpuff US deliveries, but the trace interval is much shorter in eyeblink procedures 500 ms; .4.1Aversive studies have demonstrated TC impairments in aged animals, e.g. using eyeblink conditioning in rabbits  and miceTC, including the fear conditioning variants employed in studies of aging , is know4.2It was predicted that the middle-aged cohort of rats should show impaired NOR, particularly at the 24 h retention delay . It was Thus, in line with the further acquisition seen in the TC procedure, NOR discrimination was equivalent when the cohorts reached 8 and 18 months of age. The only behavioural parameter which changed over time and was different by age group was sample exploration . Specifi4.3First, the results showed an overall reduced 5-HIAA/5-HT ratio in the dorsal striatum of the middle-aged cohort. This finding is consistent with the most striking age-related behavioural change which was in activity as measured by NOR sample exploration . Second,Thus, changes in 5-HT function were linked to activity-related parameters in both TC and NOR procedures. Reduced striatal 5-HT turnover might account for overall decreased object exploration but was nonetheless unrelated to NOR cognitive performance. It is well-established that 5-HT modulates motor function and behavioural arousal  and both4.4Due to circumstances beyond our control, there was a confound in the design in that we had to order the younger cohort of Wistar Han rats from a different barrier. This switch in supply could have compromised comparison between the groups if there were systematic differences in behavioural phenotype between the barriers. It must also be acknowledged that the design did not include further groups of untrained aged rats, for inclusion in the neurochemical analyses, to examine the effects of age independent of training. However, animals do not age in a vacuum and whatever the experience of these animals it would doubtless beg further questions as to the role of environmental enrichment and other aspects of the husbandry regime in healthy brain ageing. Thus there is a more general limitation to be considered in that the time rats take to age inevitably conflates their experiences to that point: environmental enrichment and social caging (for example) would be expected to mitigate against the effects of aging. The younger rats were delivered at 6 weeks of age, and  acclimatized in our animal unit for two weeks before the behavioural testing was started. The older rats were delivered at 16 weeks of age and further aged in house until they reached 12 months. Thus the older cohort experienced an additional 10 weeks under the suppliers’ conditions and some 8 months in our animal unit. In house aging was preferable in that we could achieve some weight control during the aging period (following the standard 2-week acclimatization) and the regular handling to weigh them (every 2–3 days) reduced the rats’ reactivity. The provision of cardboard tubing, chew sticks and nesting materials is standard in our animal unit. We have no way to show what the effect of husbandry on in house aging may have been but our presumption is that our handling and enrichment regime should have (if anything) reduced the cognitive deficits otherwise associated with aging, possibly for non-specific reasons related to reduced reactivity in novel situations and/or the health benefits of the food restriction. In house aging guaranteed the availability of the older group of rats at the time scheduled to complete the study and – although the environment was not enriched beyond our standard level – we could at least be confident that any age-related impairment was not simply attributable to environmental deprivation rather than older age per se.Possibly related to the use of environmental enrichment during the in house aging , the age-related impairments shown in the present study were not particularly marked. The adaptation of the appetitive TC procedure for the longitudinal aspect of the design may also have been suboptimal. For example, there were no interim extinctions sessions with the noise CS. These would not have eliminated the effects of earlier conditioning sessions but they would be expected to reduce the positive transfer of learning from one timepoint to the next. However, any age-related differences in the course of interim extinctions would have confounded the assessment of acquisition at the subsequent timepoints. In any case, based on previous work with the same conditioning procedure there were no grounds to assume that conditioning would be at asymptote after in total 60 conditioning trials at timepoint 1 . Indeed,There was no evidence at all for any age-related differences in contextual conditioning, as measured by responding to the experimental light background. However, conditioning to the light background was directly assessed only by a single extinction test 2 weeks after the end of an extended training . Possibly, stronger evidence of conditioning to the light background would have been obtained had this been tested at an earlier timepoint. A further consideration arises in that age-related differences in the sensitivity to distracting stimuli could have confounded interpretation of the rats’ responding to the flashing light background stimulus or the target CS. Distractors have been found to interfere with trace conditioning . However4.5Counter to expectation, age-related decline was not demonstrated longitudinally as rats progressed through to middle age. On the contrary, the results showed further acquisition at the 2 s trace interval at the later conditioning timepoints. There was, however, evidence for reduced improvement from one day to the next in the middle-aged cohort, at the first timepoint measures of responding to the 2 s trace conditioned stimulus. Neither cohort showed much responding to the CS when this was conditioned at the 10 s trace interval, but there was evidence for impaired timing within the 10 s trace in the middle-aged cohort. This difference between the cohorts increased over the course of the study not because of any further impairment with further aging but because timed responding developed only over the extended period of training in the younger-adult cohort.Notwithstanding the challenges posed by designs of the kind used in the present study, the results underscore the importance of longitudinal studies. Between groups comparison showed NOR discriminative impairment in middle-aged rats tested at the first timepoint . Object exploration at the sample stage of the procedure was overall reduced in middle-aged rats and further reduced longitudinally. However, despite further reduced sampling of the objects, the middle-aged rats showed recovery when re-tested on NOR at the final timepoint. The difference in 2 s TC between the middle-aged and younger-adult cohorts was similarly transient. Thus taken in isolation, cross-sectional studies may overestimate age-related impairments.Age-related decline in middle-age might be expected to be modest and potentially difficult to demonstrate over the course of an extended acquisition with the appetitive TC procedure in use. Nonetheless early behavioural changes are key to the detection of biomarkers for the onset of age-related cognitive decline, prior to the onset of irreversible neuropathological changes, offering prospective screening tools to assess the efficacy of interventions to restore behaviour, for example with DA D1  or muscaTC is likely supported by a wider network of distributed mechanisms  and, of All of the authors have made a substantial contribution to the experimental design, data analyses and/or write up of the study. The submitted version of the manuscript has been seen and approved by all authors and none of the data have been previously published (except in abstract form for conferences). This work was supported by the BBSRC (ref. BB/K004980/1). The authors have no competing financial interests or other conflicts of interest and nothing to disclose. The BBSRC had no further role in the design of the study, collection and analysis of data or the decision to publish the data.


Vaccinium, commonly known as the kind of blueberry, inhibited the early steps of influenza virus (IFV) infection to host cells, and that the activity was correlated with the total polyphenol content. Particularly potent inhibitory activity was observed for Vaccinium oldhamii. In this study, we identified the active components in Vaccinium oldhamii involved in the inhibition of IFV infection. We sequentially fractionated the Vaccinium oldhamii extract using a synthetic adsorbent resin column. High inhibitory activity was observed for the fractions eluted with 30%, 40%, and 50% ethanol, and three peaks  considered to represent polyphenols were identified in the fractions by HPLC analysis. Among these peaks, high inhibitory activity was detected for peak A and B, but not for peak C. These peaks were analyzed by LC/MS, which revealed that peak A contained procyanidin B2 and ferulic acid derivatives, whereas peak B contained two ferulic acid O-hexosides, and peak C contained quercetin-3-O-rhamnoside and quercetin-O-pentoside-O-rhamnoside. It is already known that these polyphenols have anti-IFV activity, but we speculate that ferulic acid derivatives are the major contributors to the inhibition of the early steps of IFV replication, such as either adsorption or entry, observed for Vaccinium oldhamii.We previously reported that extracts from plants of the Ericaceae genus  The blueberry is popular around the world as a functional fruit. It is reported to promote the recovery of sight in the dark  and possRibes nigrum). The blackcurrant extract demonstrated antiviral activities against herpes simplex virus (HSV) type 1 + gave product ions at m/z 427, 429, and 291. Further, the UV/Vis spectrum of peak #1 showed a maximum absorption at 279 nm. These results indicated that peak #1 was an epicatechin dimer, such as procyanidin B1 or B2. We next compared the mass and MS/MS spectra as well as the UV/Vis spectra of a commercially available procyanidin B2 standard and peak #1, and confirmed peak #1 to be procyanidin B2.Peak #1 at 7.10 min showed as an intense peak at − at m/z 355, and product ions at m/z 295 ([M − H − 60]−), 235 ([M − H − 120]−), 217 ([M − H − 120 − 18]−), 193 ([M − H − 162]−), and 175 ([M − H − 162 − 18]−). The UV/Vis spectra were also almost the same and gave a maximum absorption at around 329 nm. The positive-ion mass and MS/MS spectra did not give informative peaks. It was reported that ferulic acid derivatives in black carrot showed [M − H]− at m/z 355, product ions at m/z 295, 217, 193, 175, and 134, and λ max at 330 nm [Peak #2 at 7.45 min and peak #3 at 7.99 min showed almost the same mass and MS/MS spectra in the negative-ion mode. These peaks gave [M − H](M − H − 2−), and − at m/z 355. However, the MS/MS spectra were different from those of peaks #2 and #3. [M − H]− at m/z 355 gave an intense product ion at m/z 193 ([M − H − 162]−), with no other intense peaks observed. This pattern was previously reported for ferulic acid hexoside in dried plums by Fang [Peaks #4 (11.05 min) and #5 (11.46 min) of fraction B also showed [M − H] by Fang  and in h by Fang . These dm/z 447 and m/z 579, respectively. In the positive-ion mode, peaks #6 and #7 gave [M + H]+ at m/z 449 and m/z 581, respectively, and the dominant product ions were observed at m/z 303 ([M + H − 146]+) and m/z 449 ([M + H − 146]+), respectively. As for the MS/MS spectrum of peak #7, a similar fragmentation pattern to that of peak #6 was observed. That is, product ions other than the one at m/z 449 were observed at m/z 431 ([M + H − 132 − 18]+), 413 ([M + H − 132 – 18 × 2]+), 345 and 303 ([M + H − 132 − 146]+). Further, the UV/Vis spectra of peaks #6 and #7 showed maximum absorption around 349 nm. These data suggested that peaks #6 and #7 were quercetin derivatives, such as quercetin-3-O-rhamnoside and quercetin-O-pentoside-O-rhamnoside, respectively [In the negative-ion mass spectra of peaks #6 (12.47 min) and #7 (12.55 min), intense peaks were observed at ectively .Vaccinium oldhamii, showed potent inhibitory activity against IFV infection, with procyanidin B2 and ferulic acid derivatives identified as the major active components in these fractions. Moreover, quercetin derivatives were identified from the fraction showing weak anti-IFV activity.Berry fruits such as blackcurrant ,9,10, arVaccinium oldhamii.Ferulic acid and its derivatives have been reported to bind to neuraminidase and inhibit the initial stage of IFV infection, with its derivatives in particular showing a more potent effect . The twoProcyanidin B2 was previously reported to inhibit IFV replication in cells , but no The inhibitory activity of fraction C was weak, with the major component in the fraction found to be quercetin glycoside. The anti-IFV activity of quercetin rhamnoside was reported previously, but this activity was due to the suppression of IFV replication in cells, not by the inhibition of virus infection to host cells . In ordeVaccinium oldhamii, but it was clarified that the major contributors to the inhibition of the early stage of IFV infection were ferulic acid derivatives. However, we should also consider the possibility of a synergistic effect among various components. For example, the anti-IFV activity of catechin was enhanced when multiple catechins were mixed [Vaccinium oldhamii on IFV infection might not be due only to the effect of the ferulic acid derivatives but also a synergistic effect among the various polyphenols contained in the fruit. Further study is required to clarify the network of components in the plant that might act synergistically. Also, in recent years, research has been actively conducted on processing methods for suppressing the decrease in active components, such as polyphenols [From these results, some polyphenols having anti-IFV activity were found to be contained in re mixed . Therefoyphenols ,29, so i


Objective: We investigated the independent and joint associations of changes in estimated cardiorespiratory fitness (eCRF) and symptoms of anxiety and depression with brain volumes in individuals from the general population.Method: 751 participants  from the Nord-Trøndelag Health Study (HUNT) MRI cohort were included. eCRF obtained from a non-exercise algorithm and symptoms of anxiety and depression were assessed twice; at HUNT2 (1995–97) and HUNT3 (2006–08). Brain MRI was performed shortly after HUNT3. Brain parenchymal fraction (BPF), bilateral hippocampal and total cortical volume were extracted from brain MRI obtained at 1.5T, using FreeSurfer and Statistical Parametric Mapping.Results: Multiple regression revealed that participants whose eCRF increased had larger BPF  and larger hippocampal volume  compared to participants whose eCRF remained low. Participants whose eCRF remained high had larger BPF  and larger cortical volume . Participants whose anxiety symptoms worsened had smaller BPF  and cortical volume  than participants whose anxiety symptoms remained low. Each ml/kg/min increase in eCRF was associated with larger cortical volume among individuals with worsening of anxiety symptoms , and larger BPF among individuals whose depressive symptoms improved .Conclusion: Promoting exercise intended to improve eCRF may be an important public health initiative aimed at maintaining brain health among middle-aged individuals with and without changing psychological symptoms. It is well established that the volume of the human brain decreases in normal aging. However, accelerated hippocampal and cortical atrophy may be an indicator of the development of mild cognitive impairment or dementia in older adults  has been proposed as a factor which may attenuate age-related brain atrophy , which is an estimate of structural brain reserves , a large population-based health survey from the Nord-Trøndelag County in Norway  study, a sub-study in HUNT3, participants were required to have participated in HUNT1,−2, and−3, be between 50 and 65 years of age at inclusion, live within 45 min traveling distance from the location of the MRI examination, and not have standard MRI contraindications such as body weight above 150 kg. Of 1,494 invited participants, 1,088 (73%) agreed to participate in the study, and 1,006  had successful MRI examinations and were defined as MRI participants. For details on inclusion and characteristics of participants, non-participants, and non-invited see Honningsvag et al. .This study was approved by the HUNT study board of directors and the Helse Midt-Norge Regional Ethics and Health Research Committee (REK midt). All participants were legally competent adults, and gave their written informed consent.Brain MRI was performed using a 1.5 T GE Signa HDx 1.5 T MRI scanner, equipped with an eight-channel head coil  and software version pre-14.0M4. All participants underwent the same scan protocol, for details see Haberg et al. . The folhttp://enigma.ini.usc.edu/protocols/imaging-protocols/). The sum of left and right hemisphere volume measurements was used.MRI data was analyzed with FreeSurfer V5.3.0  toolbox, full description in Hansen et al.  was estimated based on a combination of T1 and T2 images using an automated version of the reverse brain mask method  specific medians of the eCRF distribution. The participants were then stratified into four eCRF change groups: “remained low” (“low” at both HUNT2 and HUNT3), “decreased” , “increased” , and “remained high” (“high” at both HUNT2 and HUNT3). For greater statistical power, the continuous change variable delta eCRF (ΔeCRF) was calculated by subtracting eCRF at HUNT2 from eCRF at HUNT3 for each participant. Whereas the categorical change variable provides a relative measure of eCRF change dependent on the age- and sex-specific median, the continuous change variable provides data on absolute change in ml/kg/min from HUNT2 to HUNT3.Symptoms of anxiety and depression were assessed using the Hospital Anxiety and Depression Scale (HADS). The HADS is a self-assessment scale developed in 1983 by Zigmond and Snaith . The Norn = 3, HUNT3 n = 114) or HADS-D  scale were excluded from the analyses. For participants who had answered 5 or 6 questions , the total score was extrapolated by multiplying the sum by 7/5 or 7/6, respectively. A total score of 8 or above on the HADS-A or HADS-D subscale was used as an indication of clinically relevant anxiety or depression symptoms, respectively  and 7 items that cover depressive symptoms (HADS-D), giving a total of 14 items. Each subscale item has a 4-point Likert scale ranging from 0 (no symptom) to 3 (highest symptom level), with a maximum score of 21 on each scale indicating the highest symptom load. Participants who had responded to <5 questions on either the HADS-A  and “high” (≥8) HADS-categories at HUNT2 and HUNT3. Next, four HADS change groups were created: “remained low” (“low” at both HUNT2 and HUNT3), “improved” , “worsened” , and “remained high” (“high” at both HUNT2 and HUNT3). For greater statistical power, we also calculated delta HADS-A and HADS-D (ΔHADS-A and ΔHADS-D) by subtracting HADS-scores at HUNT2 from HADS-scores at HUNT3. Whereas the categorical HADS change variables provide a relative measure of HADS change as they depend on the cut-off for clinically relevant symptoms of anxiety and depression, the continuous change variables provide data on absolute change in HADS score from HUNT2 to HUNT3.Forty-five participants were excluded from further analyses due to brain pathology, and 50 participants were excluded due to failed or incorrect FreeSurfer processing. Further, 160 participants were excluded due to missing data on eCRF or HADS. The final sample comprised 751 participants (390 women) with a mean age of 58.9 years (range 50–67) at the time of the HUNT MRI examination, and with a mean time of 11.8 ± 0.2 years between HUNT2 and HUNT3.r) and Cramér's V (ϕc) were computed to estimate correlation between changes in eCRF, HADS-A, and HADS-D. We used multiple regression models to assess standardized regression coefficients (β) and 95% confidence intervals (CI) for the associations of changes in eCRF and HADS, both as categorical and continuous variables, with BPF, total hippocampal volume, and total cortical volume. The “remained low” eCRF and “remained low” HADS groups served as reference categories. The role of age and sex in the eCRF prediction models is to refine the estimate of CRF, not to remove the effect of age and sex in the relationships between eCRF and brain volumes. Thus, all models were adjusted for age, sex, education , and smoking . Models with hippocampal and cortical volumes were also adjusted for ICV. In addition, models with ΔeCRF and ΔHADS were adjusted for eCRF at HUNT2 and HADS score at HUNT2, respectively.Descriptive statistics were used to assess study sample characteristics for the four categorical change in eCRF groups. Pearson's correlation .To assess the joint associations of changes in eCRF and HADS, we investigated the association of ΔeCRF with brain volumes, stratified by categorical change in HADS-A and HADS-D. This allowed us to assess the influence of changes in eCRF on brain volumes among different strata of HADS change. Interaction between categorical measures of eCRF and HADS could not be assessed due to low statistical power. Thus, we investigated interaction on an additive scale between ΔeCRF and ΔHADS on brain volumes. In additional analyses, we assessed the independent associations of changes in eCRF and HADS with brain volumes. Here, ΔeCRF, ΔHADS-A, and ΔHADS-D were included in one model, and categorical measures of change in eCRF, HADS-A, and HADS-D were included in another model. Finally, in order to better capture areas in the cortex that may be associated with changes in eCRF and HADS, we performed analyses with cortical thickness and area. Here, General Linear Models (GLMs) were fitted at each vertex across the cortical surface. Individual surface maps were smoothed with a full-width-half-maximum Gaussian kernel of 25 mm, averaged across participants . A r = 0.03, p = 0.410, ΔeCRF and ΔHADS-D, r = 0.004, p = 0.903, categorical eCRF change and HADS-A change, ϕc = 0.06, p = 0.497, categorical eCRF change and HADS-D change, ϕc = 0.09, p = 0.024. ΔHADS-A and ΔHADS-D were moderately correlated, r = 0.51, p < 0.001, as were categorical measures of change in HADS-A and HADS-D, ϕc = 0.61, p < 0.001. Results from the multiple regression models assessing the associations of changes in eCRF and HADS with BPF, hippocampal and cortical volume are shown in Study sample characteristics stratified by change in eCRF are shown in Participants in the “increased” eCRF group and in the “remained high” eCRF group had larger BPF; β = 0.09  and β = 0.15 , respectively, compared to participants in the “remained low” eCRF group. In addition, each ml/kg/min increase in ΔeCRF corresponded to a larger BPF, β = 0.16 .Participants in the “worsened” HADS-A group had smaller BPF, β = −0.09 , compared to participants in the “remained low” HADS-A group. Further, each unit increase in ΔHADS-A was associated with a smaller BPF, β = −0.07 . ΔHADS-D was associated with smaller BPF, although this result did not reach statistical significance, β = −0.07  .Participants in the “increased” eCRF group had a larger hippocampal volume, β = 0.09 , compared to participants in the “remained low” eCRF group. Furthermore, each ml/kg/min increase in ΔeCRF was associated with a larger hippocampal volume, β = 0.09 .Neither change in HADS-A nor HADS-D was significantly associated with hippocampal volume .Participants in the “remained high” eCRF group had larger cortical volume, β = 0.05 , compared to those in the “remained low” eCRF group. In addition, each ml/kg/min increase in ΔeCRF corresponded to a larger cortical volume, β = 0.05 .Participants in the “worsened” HADS-A group had smaller cortical volume compared to those in the “remained low” HADS-A group, β = −0.05 . Further, each unit increase in ΔHADS-A was associated with smaller cortical volume, β = −0.04 . Change in HADS-D was not significantly associated with cortical volume .In analyses stratified by categorical HADS change, each ml/kg/min increase in ΔeCRF was associated with larger BPF , hippocampal  and cortical volume  among participants in the “remained low” HADS-A group. In the “worsened” HADS-A group, each ml/kg/min increase in ΔeCRF was associated with larger cortical volume, β = 0.13 . In the “remained low” HADS-D group, each ml/kg/min increase in ΔeCRF was associated with larger BPF , hippocampal , and cortical volume . In addition, each ml/kg/min increase in ΔeCRF was associated with larger BPF in the “improved” HADS-D group, β = 0.28  .ps ≥ 0.188). Finally, no localized associations were observed for cortical thickness or area.Analyses assessing independent changes in eCRF, HADS-A, and HADS-D did not alter the associations, although the association between ΔHADS-A and BPF and ΔHADS-A and cortical volume was attenuated and was no longer statistically significant after adding ΔHADS-D, HADS-D at HUNT2, ΔeCRF, and eCRF at HUNT2 to the model . No inteIn our study of 751 adults from the general population, we found that increasing or maintaining a high eCRF during middle-age was associated with larger BPF, hippocampal and total cortical volume. Worsening of anxiety symptoms was associated with smaller BPF and total cortical volume. In the stratified analyses, increasing eCRF was associated with larger brain volumes among individuals whose anxiety and depressive symptoms remained low, among individuals with worsening anxiety symptoms, and among those with improving depressive symptoms.The increased brain volumes observed among individuals whose eCRF increased or remained high is in accordance with previous studies showing larger whole brain (Zhu et al., Earlier studies investigating gray matter volume in anxiety disorders have found smaller volumes in the hippocampus, midbrain, thalamus, insula, and superior temporal gyrus among patients with general anxiety disorder (Moon et al., Surprisingly, we did not observe a statistically significant association between changes in depressive symptoms and brain volumes, although we observed a similar reduction in BPF associated with ΔHADS-D as observed for ΔHADS-A see . These nTo our knowledge, no previous study has investigated the joint associations of long-term changes in eCRF and symptoms of anxiety and depression on brain volumes. The results from stratified analyses showed that increased eCRF was associated with larger cortical volume among participants whose symptoms of anxiety worsened and with larger BPF among participants whose symptoms of depression had improved. With few exceptions, eCRF was positively associated with larger brain volumes across all strata of change in HADS-A and HADS-D, even though a number of these associations did not reach statistical significance. Given that anxiety and depressive symptoms are common among older adults (Reynolds et al., The present study has several strengths. First, the study included a large, validated, population-based sample with a relatively narrow age range and an acceptable participation rate of 71.1% for the age group 60–69 at HUNT3 (Krokstad et al., The main limitation of our study is the lack of longitudinal brain MRI data, making causal inferences about the observed associations between changes in eCRF, symptoms of anxiety and depression, and change in brain volumes impossible. However, there is evidence indicating that both anxiety (Mah et al., In conclusion, our study demonstrated that increasing or maintaining high eCRF during midlife was associated with larger BPF, and hippocampal and cortical volume in a sample of middle-aged adults drawn from the general population. Worsening of anxiety symptoms was associated with smaller BPF and cortical volume. Importantly, increased eCRF appeared to be especially beneficial for cortical volume among individuals with worsening anxiety symptoms. In sum, our findings underline the importance of physical activity promotion, and anxiety prevention, as a means of promoting healthy brain aging in the general population. Health care professionals in primary clinical practice should promote physical activity and emphasize treating anxiety symptoms in order to maintain brain health among middle-aged individuals.https://www.ntnu.edu/hunt/data.The data analyzed in this study was obtained from the HUNT Research Center. Instructions and requirements for data access are available on the following website EZ, CP, ØS, GS, AH, and LE all contributed to the conceptualization and design of the study, drafting and revising of the manuscript prior to submission and during revision.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.


The aim of this study was to assess the incidence of fractures in infancy, overall and by type of fracture, its association with accidents, metabolic bone disease risk factors, and abuse diagnosis.The design was a population-based register study in Sweden. Participants: Children born 1997–2014, 0–1 years of age diagnosed with fracture-diagnosis according to International Classification of Diseases (ICD10) were retrieved from the National Patient Register and linked to the Swedish Medical Birth Register and the Death Cause Register. Main outcome measures were fractures of the skull, long bone, clavicle and ribs, categorized by age (younger or older than 6 months), and accident or not.n = 4663). Major fracture localisations were long bone (44·9%), skull (31·7%), and clavicle (18·6%), while rib fractures were few (1·4%). Fall accidents were reported among 71·4%. One-third occurred during the first 6 months. Metabolic bone disease risk factors, such as maternal obesity, preterm birth, vitamin D deficiency, rickets, and calcium metabolic disturbances, had increased odds of fractures of long bones and ribs in early infancy (0–6 months): birth 32–36 weeks and long bone fracture [AOR 2·13 (95%CI 1·67–2·93)] and rib fracture [AOR 4·24 (95%CI 1·40–12·8)]. Diagnosis of vitamin D deficiency/rickets/disorders of calcium metabolism had increased odds of long bone fracture [AOR 49·5 (95%CI 18·3–134)] and rib fracture [AOR 617 (95%CI 162–2506)]. Fractures without a reported accident had higher odds of metabolic risk factors than those with reported accidents. Abuse diagnosis was registered in 105 infants, with overrepresentation of preterm births, multiple births and small-for-gestational age.The incidence of fractures during infancy was 251 per 100 000 infants (Metabolic bone disease risk factors are strongly associated with fractures of long bone and ribs in early infancy. Fracture cases with abuse diagnosis had a metabolic bone risk factor profile. Incidence of fractures during infancy 0–1 year) has been addressed as part of fractures during childhood. Compared to later childhood and adolescence, a lower incidence of fractures is found for infants, 80–326 per 100 000. One reason for this variation might be whether birth-related fractures are included  year has. SpecifiThe most common cause of fractures during childhood is fall accidents, when Landin’s modified description of trauma level categories of light, moderate, and severe, is applied to define the understanding of clinically evident fractures , 5, 6. Ith and 16th week and may range from a silent condition to multiple fractures [Bone strength and stiffness is lower during infancy, being lowest around 4–5 months, compared to adolescents and adults . During ractures , irresperactures . Ricketsractures –16. Suggractures , twinninractures , and smaractures . Potentiractures  and ethnractures  could coEarly bone health is affected by several genetic disorders, such as Osteogenesis imperfecta (OI) –24 and EThere are several knowledge-gaps regarding fractures during infancy on a population level. To our knowledge, neither incidence nor etiologies, as perceived cause of trauma, abuse, and risk factors of metabolic bone disease, have been addressed in population studies. We hypothesised: 1) if the suggested risk factors for bone fragility are valid, they should be overrepresented in infants having fractures; and 2) the infant age distribution should be pathophysiologically compatible with the suggested bone fragility factors. The aim of this national population register study was to assess the incidence of fractures in infancy, overall and by type of fracture, age, sex, and prematurity, and its association with accidental injury, genetic disorders, abuse diagnosis and metabolic bone disease risk factors.N = 1 855 267) who had been registered in the National Patient Register (NPR) (n = 395 812) [n = 182 974) [n = 731 901) [This was a nationwide population register study which included infants born in Sweden from 1997 to 2014 with follow-up to one year of age. A flow chart of the data is presented in 395 812) . From th182 974) . Thus, a182 974)  and Caus182 974)  was link731 901) . MaternaOutcome measures were fracture of the skull, long bone, clavicle and the ribs with or without transport accidents, or fall accidents. These were categorized by age younger or older than 6 months according to the understanding of bone development during infancy , 9, and Metabolic risk factors of possible importance for fracture risk during infancy following exposure factors were assessed: (1) maternal overweight/obesity defined as BMI at start of pregnancy categorized by overweight (25–29·9) and obesity by class 1 (30–30·9), 2 (35–39·9), and 3 (40+); (2) mother born in Africa, Asia or Latin America; (3) maternal smoking in pregnancy week 30–32 1–9/10- cigarettes; (4) sex; (5) multiple births; (6) preterm birth 32–36/<32; (7) small-for-gestational age (SGA) (<2·5 or <10 percentiles) and SGA Term and Preterm; and (8) infant diagnosis of vitamin D deficiency, Rickets and Disorders of calcium metabolism.The incidence proportion, or cases per 100 000 infants with 95% confidence intervals (CI), was calculated. Mean, median, and reported accidents were described with descriptive statistics. Mantel-Haenszels chi-square test and Fisher’s exact test were applied to assess differences. Deaths were presented by reported cause of death. Fractures associated with OI and EDS/Hypermobility Syndrome were assessed separately in relation to the study population. In the further analysis of background factors for fractures, OI and EDS were excluded. The distribution of metabolic risk factors, maternal and infant, were displayed for the major fracture groups for the first and second half of the first year. To assess metabolic risk factors for long bone and rib fractures during the first half year of life, crude and adjusted odds ratios, with 95% confidence intervals, were analysed. The statistical software package IBM SPSS 25·0  was used for data analyses.This study was approved by regional ethics committee in Uppsala (2014-11-19 No 383) and was conducted on de-identified data.In total, 4 663 fractures were reported in the first year of life in Sweden during 1997–2014, giving an incidence of 2513 per 100 000 infants .n = 2 093), 19 had fracture of the clavicle, 24 a fracture of the ribs, and 15 a fracture of the skull. However, for those having a rib fracture (n = 66), 24 had also had a long bone fracture (32·4%), 13 had a skull fracture (17·6%), and 8 had a clavicle fracture (10·8%). One-third of all occurred within the first six months of life. Mean age at diagnosis for any fracture was 7 months, skull fracture was 5·7 months, shaft fracture of long bone fracture 5·1 months, and for rib fracture, 3·3 months (p = 0·02).Almost half of the fractures, 44·9%, were in the long bones, and 31·73% were skull fractures, while only 1·4% had rib fractures . Few of 3 months . Pretermn = 3 500), 26·6% (n = 932) had a slight trauma , and 23·7% (n = 831) had a moderate trauma , while the others had unspecified fall accidents.Out of all fractures, 71·4% had a reported fall accident, 4·2% a transport accident, and 2·8% a pinch accident. This pattern was consistent by type of fracture, with the exception of rib fractures, where only 36·4% had a report of an accident . Out of The cause of death of 54 infants with fractures were: transport accidents (24), diseases (8), malformations (5), perinatal causes (4), falls (3), specified accident events, undetermined intent (5), abuse/homicide (4), and complication of surgery (1).p<0·0000001). There were 511 infants whose mothers had either EDS or EDS/hypermobility syndrome diagnosis, and six of those had fractures (1·2%) compared with the study population (p 0·036).There were 84 infants with a diagnosis of osteogenesis imperfecta (OI), an incidence of 4·5 per 100 000 infants. Of those with OI, 29 had fractures , out of which 15 had a reported accident, hence, 14 were not reported as being related to an accident. Compared with the study population, infants with OI had an increased risk of contracting a fracture : 21 (20%) preterm-born (p<0·0000001), 10 (9·5%) multiple births (p<0·0006), and 25 (23·8%) small-for-gestational age <10th percentile (p<0·000007). Two infants had a diagnosis of vitamin D deficiency/rickets/calcium metabolic disturbance, and one infant had a diagnosis of osteogenesis imperfecta. Three of the infants with an abuse diagnosis died, and all three had subdural haemorrhage. Other diagnoses were skull fracture (2), clavicle fracture (1), rib fracture (2), and retinal haemorrhage (1). One of these other diagnoses had a transport accident, whereas none had a fall accident.Out of all fracture cases, 105 (2·3%) also had an abuse diagnosis. The distribution by localization of fracture was: skull (33), clavicle (17), ribs (28), shaft long bone fracture (25), and non-shaft long bone fracture (32). Other concomitant diagnoses were subdural haemorrhage (15), retinal haemorrhage (9), and superficial injury (5). A transport accident was reported for 8 infants, 31 had a reported fall accident, and there was one reported pinch accident. Perinatal risk factors in this group were , strengthening its population representativeness. Further, the Swedish health registers are considered to have a high validity , 38. RegThe overall incidence found in this national study was in the lower range of what has been reported from a relatively small hospital-based study  , and a sIn our study, 1/3 of the infants with fractures did not have an ICD-code for accident, while a Swedish hospital-based study had very few cases in which the trauma mechanism could not be determined, namely 4% during 1993–1994, and 0% in 2005–2006 [Our results indicate that, in addition to the different genotypes for osteogenesis imperfecta, the parental phenotype of Ehlers-Danlos/hypermobility syndrome should also be investigated. The clinical observations made by Holick et al. , indicatA main finding of this study was the very high odds of fractures of the long bones and ribs during the infant’s first six months when having a diagnosis of vitamin D deficiency, rickets, or disorders of calcium metabolism. This supports the hypothesis that metabolic bone disease might be one cause for being a fracture-prone infant , 11–13. This study also adds knowledge of the importance of also mapping the maternal conditions, such as obesity  and mateSeveral of the risk factors for fractures in infants found in this study, not only extreme prematurity but also moderate preterm and twins, and, further, small-for gestational age, show that it is important that guidelines on the diagnosis of the etiology for infant fractures are not limited to the sampling of well-known biochemical markers for metabolic bone disease and x-rays on the child. Parameters such as parathyroid hormone (PTH), alkaline phosphatase (ALP), ionized calcium, phosphorus, magnesium, and vitamin D may be normal or only slightly affected in a prematurely born infant who has osteopenia with or without signs of rickets at several weeks old . Also, cEpidemiological risk factor analysis cannot assert a causal relationship and should be interpreted in relation to bio-mechanical properties of infant bone. There is a scarcity of bio-medical information about infant bone. Ambrose et al.  performeIn this study, 105 infants had a fracture diagnosis and abuse diagnosis , 57 had long bone fractures, and 28 had rib fractures. Within our study design we could not ascertain whether those fracture were clinically overt with symptoms or whether they were occult/silent. Occult fractures are usually detected when a child is investigated for suspected child abuse, which is initiated when an infant has an acute symptomatic fracture, a subdural hematoma or hygroma, or acute deterioration of consciousness associated with some kind of intracranial finding considered to be of traumatic origin, usually a subdural hematoma or hygroma [It might be speculated that the occult fractures detected by skeletal survey for suspected infant abuse in fact reflect metabolic bone disease. The high predictive value for infant abuse, as previously stated –32, mighWe suggest that all infants, not only premature ones, having unexplained or occult fractures with no obvious cause in early infancy should be investigated regarding bone fragility as a possible explanation. Our results suggest that in such an investigation the mandatory sampling of biochemical markers for osteopenia and skeletal x-ray investigation need to be complemented with consideration given to all conditions that may affect fetal bone mineralization and that DEXA, or preferable US giving no radiation, should be recommended.The incidence of infant fracture is comparable to that previously reported; 2/3 had reported fall accidents. Bone fragility of osteogenesis imperfecta and vitamin D/disorders of calcium metabolism are well known. This study also indicates that bone fragility is associated with Ehlers-Danlos/hypermobility syndrome. Metabolic bone disease risk factors are strongly associated with fractures of the long bone and ribs before 6 months of age, especially without reported accident. The group fracture and abuse diagnosis had a metabolic bone risk factor profile of being preterm born, multiple birth and small-for-gestational age.


PICALM, BIN1, CD2AP, and RIN3—suggested to be involved in blood–brain barrier amyloid-β transcytosis pathways—associate with Alzheimer’s disease, all dementia, suggested vascular dementia, and stroke, and whether such associations are independent of the strong ε4 APOE risk allele. In a prospective cohort study of 74,754 individuals from the general population we genotyped PICALM (rs10792832), BIN1 (rs6733839), CD2AP (rs10948363), and RIN3 (rs10498633), and generated a weighted and a simple allele score. Multifactorially adjusted hazard ratios for the fourth quartile versus the first quartile of the weighted allele score were 1.42  for Alzheimer’s disease, and 1.33 (1.19–1.48) for all dementia. For suggested vascular dementia and stroke the corresponding estimates were 1.71 (1.18–2.49) and 1.12 (1.04–1.22), respectively. Hazard ratios were similar after APOE adjustment. Genetic variants in PICALM, BIN1, CD2AP, and RIN3 are associated with increased risk of Alzheimer’s disease, all dementia, and suggested vascular dementia independent of the strong APOE ε4 allele. These findings may suggest that clathrin-mediated endocytosis in clearance of amyloid-β across the blood–brain barrier is important for the integrity of both brain tissue and cerebral vessels.To test whether genetic variants in The online version of this article (10.1007/s10654-019-00498-2) contains supplementary material, which is available to authorized users. Alzheimer’s disease and other forms of dementia are devastating neurodegenerative diseases currently affecting more than 47 million people globally, and expected to triple in 2050 . There aPICALM (phosphatidylinositol-binding clathrin assembly protein) [BIN1 (bridging integrator 1) [CD2AP (CD2-associated protein) [RIN3 (Ras and Rab interactor 3) [PICALM consistently associate with dementia in GWAS [APOE (apolipoprotein E gene) [Interestingly, a number of genome-wide association studies (GWAS) risk genes , PICALM protein) , 18, BINrator 1) –21, CD2Aprotein) , 23, andactor 3) , encode actor 3) –32. Part in GWAS , 33–35,  E gene) , an asso E gene)  and sinc E gene) , 35, 37. E gene) , 38. Rec E gene) .PICALM, BIN1, CD2AP, and RIN3 previously identified as top hits for Alzheimer’s disease [Our aim was dual. First, we wanted to establish the exact risk increases at the individual level for Alzheimer’s disease and all dementia for four potential blood–brain barrier pathway genes. Second, we wanted to test the impact on specific cerebral vascular endpoints –suggested vascular dementia and stroke—since the blood–brain barrier most likely is central for dementia related vascular events. We genotyped four variants in  disease , in two The Copenhagen General Population Study (CGPS) is a prospective study of the Danish general population initiated in 2003 and still recruiting –42. IndiThe Copenhagen City Heart Study (CCHS) is a prospective study of the Danish general population initiated in 1976–1978 with follow-up examinations in 1981–1983, 1991–1994 and 2001–2003 –42. IndiCombining the two studies yielded a total of 74,754 individuals for Alzheimer’s disease and all dementia analyses, of whom 2514 developed dementia during a median follow-up of 10 years (range 0–23 years). For the analyses of suggested vascular dementia and stroke we included 72,612 individuals, of whom 5016 developed stroke and 248 developed suggested vascular dementia during a median follow-up of 10 years (range 0–25 years). No individuals were lost to follow-up. Follow-up began at the time of blood sampling (2003 and onward for CGPS and 1991–1994 or 2001–2003 for CCHS). Follow-up ended at occurrence of event , death , emigration , or on March 22nd, 2017 (last update of the registry), whichever came first. Years at risk were calculated for each participant as the time difference between baseline and the end of follow-up. Written informed consent was obtained from all individuals. Individuals in both studies were white and of Danish descent.In CGPS and CCHS, information on births, deaths, emigrations and immigrations was collected from the national Danish Civil Registration System. Information on diagnoses of dementia and stroke, and age at diagnosis was drawn from the national Danish Patient Registry and the national Danish Causes of Death Registry. The national Danish Patient Registry has information on all patient contacts with all clinical hospital departments in Denmark since 1977, including emergency wards and outpatient clinics from 1994. The national Danish Causes of Death Registry contains data on the causes of all deaths in Denmark, as reported by hospitals and general practitioners. The national Danish registries are regarded among the best of its kind , 44, howStandard hospital assays measured electrolytes, glucose, liver-, kidney-, and inflammatory parameters as well as total cholesterol, HDL (high-density lipoprotein) cholesterol and triglycerides . LDL (low-density lipoprotein) cholesterol was calculated using the Friedewald equation  when plaPICALM rs10792832, BIN1 rs6733839, CD2AP rs10948363, RIN3 rs10498633 and for p.Cys130Arg  defining the ε4 allele and p.Arg176Cys  defining the ε2 allele of the APOE gene [Taqman-based  or KASP technology based assays  were used to genotype for Body mass index was measured weight in kilograms divided by measured height in meters squared. Hypertension was use of anti-hypertensive medication, a systolic blood pressure of 140 mm Hg or greater, and/or a diastolic blood pressure of 90 mm Hg or greater. Diabetes mellitus was self-reported disease, use of insulin or oral hypoglycemic agents, and/or non-fasting plasma glucose levels of more than 11 mmol/L (> 198 mg/dL). Estimated glomerular filtration rate was calculated according to CKD-EPIcrea . SmokingIGAP  . Probability values < 0.001 are given as powers of 10. Kruskal–Wallis one-way analysis of variance or Pearson’s Combining all genotypes, we generated two different genetic risk scores for dementia and stroke. The first genetic score, named “weighted allele score”, was calculated for each individual using a weighted sum of alleles for increasing risk of Alzheimer’s disease and all dementia, subsequently categorized into quartiles of approximately equal size to maximize statistical power. The weights correspond to the regression coefficients for the dementia risk increasing alleles in each individual adjusted for age and gender  (SupplemAPOE genotype (ε2/ε3/ε4 APOE genotype).Cumulative incidences of Alzheimer’s disease and all dementia were plotted against age and weighted/simple allele score group, using the method of Fine-Gray , to acco2 statistics and was considered as low (I2 ≤ 25%), moderate (25% < I2 < 49%), or high (I2 ≥ 50%). Results are presented for both fixed- and random-effects models.Meta-analyses were conducted using the user-written metan command from Stata/S.E. v13.0 to estimate fixed and random effects odds ratios by regression coefficients and standard errors for each of the four genetic variants. Between-study heterogeneity was assessed by Cochran’s Q test and IBaseline characteristics of the 74,754 individuals enrolled in the study are shown by quartiles of weighted allele score for Alzheimer’s disease in Table P for trend ≤ 2 × 10−5) . After stratification in age groups , results were similar although attenuated in the ≥ 80 years age group, most likely due to lack of power . There was no interaction between the genetic scores and age at baseline . Further no interaction was present between the genetic scores and APOE genotype .Cumulative incidences of Alzheimer’s disease and all dementia increased stepwise as a function of increasing weighted/simple allele score groups  and 1.14 (1.11–1.17) with I2 of 20% (P for heterogeneity = 0.29)  for both overall fixed- and random-effects, with I2 of 0% (P for heterogeneity = 0.97)  for both overall fixed- and random-effects, with I2 of 0% (P for heterogeneity = 0.92)  for both overall fixed- and random-effects, with I2 of 0% (P for heterogeneity = 0.70)  for suggested vascular dementia and 1.12 (1.04–1.22) for stroke. When prevalent stroke was excluded, risk of suggested vascular dementia remained (fourth versus first quartile 2.01 (1.32–3.07), data not shown). For the simple allele score multifactorially adjusted hazard ratios for 5–8 alleles versus 0–3 alleles were 1.65 (1.20–2.26) for suggested vascular dementia and 1.03 (0.96–1.10) for stroke , plasma levels of electrolytes , renal function , liver function , glucose level or high-sensitivity C-reactive protein .PICALM, BIN1, CD2AP and RIN3, four genes suggested to be involved in blood–brain barrier amyloid-β transcytosis, are associated with increased risk of Alzheimer’s disease, all dementia, and suggested vascular dementia in the general population independent of the strong APOE genotype. There was no conclusive association with risk of stroke. These findings may suggest that compromising blood–brain barrier function has implications for both brain tissue—manifested by increased Alzheimer’s disease risk—as well as for cerebral vasculature—manifested by increased risk of suggested vascular dementia.The principal findings of this study are that genetic variation in PICALM has consistently been associated with Alzheimer’s disease in several GWA studies with risk estimates in the range of 1.14–1.27 [BIN1 in the range of 1.15–1.36 [CD2AP in the range of 1.10–1.12 [RIN3 with an odds ratio for the risk increasing allele of 1.10 [To our knowledge this is the first study to simultaneously assess risk of dementia and stroke as a function of genetic variation in all four, potential blood–brain barrier transcytosis genes. .14–1.27 , 33, 34..15–1.36 , 37, CD2.10–1.12 , 31, 37, of 1.10 . The prePICALM in Alzheimer’s disease pathology, emphasizing clathrin-mediated endocytosis as a potential important mechanism in amyloid-β clearance across the blood–brain barrier [Picalm/−+ mice had a reduction in amyloid-β40 and amyloid-β42 efflux across the blood–brain barrier of 41% and 61%, respectively, compared to littermate controls. Using in vitro endothelial monolayer to mimic the blood–brain barrier, they found that amyloid-β binding to LRP1 enhanced the binding of PICALM, which initiated PICALM/clathrin-dependent endocytosis of the amyloid-β-LRP1 complex and subsequent transcytosis involving GTPases Rab5 and Rab11 controlling, respectively, early endosome formation and vesicle exocytosis [The biological mechanisms of our findings remain to be determined and the evidence from population studies linking the four selected variants to blood–brain barrier amyloid-β transcytosis pathways is limited. Recently new light was shed on the function of  barrier . Zhao etocytosis –60. In pocytosis , and RINocytosis , 27. Takocytosis .Vascular dementia is a disorder in the blood supply of the brain, caused by preceding hemorrhage or ischemia located in either larger vessels or in the microvasculature . AccumulAn important limitation to this study is that endpoints are based on ICD registry codes from hospitals and death certificates diagnosed in routine clinical practice and thus only captures individuals in contact with hospitals. This is in contrast to research studies where all individuals living in one area are examined and diagnosed with use of standardized instruments, trained staff and standardized diagnostic methods . ConsequAPOE genotype. Due to the extensive phenotyping of biochemical and other quantities in the present cohorts, we could also evaluate blood pressure, heart rate, electrolytes, and measures of kidney function, liver function, glucose metabolism and inflammation. We found no significant variation across weighted/simple allele score groups, suggesting that this molecular pathway may be safe to target therapeutically. The sufficient quality of the Danish registry-based all dementia and Alzheimer’s disease diagnoses was further supported by the well-known association with the apolipoprotein E ε4 allele in the present cohorts [Strengths of this study are the prospective design and the large, well-characterized, ethnically homogeneous cohort of the general population with no losses to follow-up. Furthermore, no differences of participant characteristics across weighted/simple allele score groups were observed and therefore obvious confounding of the gene scores could be excluded. Furthermore, there was no interaction between the genetic scores and age at baseline, age at diagnosis, or  cohorts  and by t cohorts . Diagnos cohorts .APOE ε4 allele. These findings may suggest that clathrin-mediated endocytosis in clearance of amyloid-β across the blood–brain barrier is important for the integrity of both brain tissue and cerebral vessels.In conclusion, by combining common genetic variation in four genes with possible function in the blood–brain barrier, we found an increased risk of Alzheimer’s disease, all dementia and suggested vascular dementia with increasing weighted/simple allele score groups independent of the Supplementary material 1 (DOCX 631 kb)Below is the link to the electronic supplementary material.


The original article has been corrected.The article “Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy”, written by “Aaron B. Simmons, Colin A. Bretz, Haibo Wang, Eric Kunz, Kassem Hajj, Carson Kennedy, Zhihong Yang, Thipparat Suwanmanee, Tal Kafri and M. Elizabeth Hartnett”, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 05 May 2018 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 20 June 2018 to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (


Arabidopsis thaliana were exposed by foliar spray to 30 mg L−1 of CuZn nanoparticles (NPs). The NPs were synthesized by a microwave-assisted polyol process and characterized by dynamic light scattering (DLS), X-ray diffraction (XRD), and transmission electron microscopy (TEM). CuZn NPs effects in Arabidopsis leaves were evaluated by chlorophyll fluorescence imaging analysis that revealed spatiotemporal heterogeneity of the quantum efficiency of PSII photochemistry (ΦPSΙΙ) and the redox state of the plastoquinone (PQ) pool (qp), measured 30 min, 90 min, 180 min, and 240 min after spraying. Photosystem II (PSII) function in young leaves was observed to be negatively influenced, especially 30 min after spraying, at which point increased H2O2 generation was correlated to the lower oxidized state of the PQ pool. Recovery of young leaves photosynthetic efficiency appeared only after 240 min of NPs spray when also the level of ROS accumulation was similar to control leaves. On the contrary, a beneficial effect on PSII function in mature leaves after 30 min of the CuZn NPs spray was observed, with increased ΦPSΙΙ, an increased electron transport rate (ETR), decreased singlet oxygen (1O2) formation, and H2O2 production at the same level of control leaves.An explanation for this differential response is suggested. Young and mature leaves of  Both zinc (Zn) and copper (Cu) are essential elements for plant growth . Zn defiNanoparticles (NPs) in agriculture are used to reduce the amount of sprayed chemical products by the smart delivery of active ingredients, diminish nutrient losses in fertilization, and increase yields through optimized water and nutrient management ,13,14,15Since plant production is driven by photosynthesis, it is possible to estimate the fate of plant growth and development by evaluating photosynthetic function . The pro−1 and 30 mg L−1 of CuZn NPs sprayed on tomato plants by determining their effects on the light reactions of photosynthesis [−1 of NPs, the application of 30 mg L−1 of CuZn NPs resulted in a reduced plastoquinone (PQ) pool that gave rise to H2O2 generation [−1 of CuZn NPs will result in differential effects on them, with young leaves retaining a more oxidized PQ pool and less H2O2 accumulation. Arabidopsis thaliana was selected as the plant material to test our hypothesis, since it has been widely used as a model system in understanding the physiological mechanisms of higher plants [The synthesis of hydrophilic CuZn NPs is challenging and has been hardly reported before ,37. We hynthesis . The evaynthesis . While nneration . A reducneration ,41—or, ineration ,43,44. Yneration , and thineration ,46,47. Br plants ,48.3)2·4H2O (2.0 mmol) and Cu(NO3)2·3H2O (2.0 mmol) were mixed and dissolved in 20 mL of triethylene glycol (TrEG). After centrifugation at 2800 g, the supernatant liquids were discarded, and the black-brown precipitate was washed three times with ethanol. NPs were re-dispersed in water via sonication assistance to form stable aqueous suspensions at concentrations <200 mg L−1. Based on our previous results, a modified synthesis of CuZn NPs was followed using a commercial microwave accelerated reaction system, the Model MARS 6-240/50-CEM . Equal aPrimary particle size and morphology was determined by a conventional transmission electron microscope (TEM)  . The crystal structure was investigated through X-ray diffraction (XRD) performed on a Philips PW 1820 diffractometer  at a scanning rate of 0.050/3 s, in the 2θ range from 10° to 90°, with monochromatized Cu Kα radiation (λ = 1.5406 nm) .The hydrodynamic size of CuZn NPs was determined by dynamic light scattering (DLS) measurements, which were carried out at 25 °C utilizing a Nano ZS Zetasizer  apparatus . Arabidopsis thaliana ecotype Columbia (Col-0) seeds obtained from the Nottingham Arabidopsis Stock Centre (NASC) were sown on a soil and peat mixture in a controlled-environment growth chamber under 22 ± 1/18 ± 1 °C day/night temperature with a 16-hour day at 130 ± 20 μmol photons m−2 s−1 light intensity and 50 ± 5/60 ± 5% day/night humidity. Four and six-week-old Arabidopsis plants were spayed with 30 mg L−1 of CuZn NPs. Rosette leaf 8 from four-week-old  and six-week-old (mature to senescing leaf) were selected for photosynthetic measurements 30 min, 90 min, 180 min, and 240 min after the foliar spray with 30 mg L−1 of CuZn NPs, while control plants were sprayed with distilled water.−1 of CuZn NPs. In each leaf, eight to 11 areas of interest (AOI) that covered the whole leaf area were selected for analysis. Four to five leaves from different plants were measured at each treatment at the actinic light intensity of 140 μmol photons m−2 s−1. By using the Imaging Win software , we measured the effective quantum yield of photochemistry in PSII (ΦPSII), the quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ), the quantum yield of non-regulated energy loss in PSII (ΦNO), the photochemical quenching (qp) that is a measure of the redox state of the PQ pool, the non-photochemical quenching (NPQ), and the relative PSII electron transport rate (ETR).Chlorophyll fluorescence analysis was conducted with an Imaging-PAM Chlorophyll Fluorometer  as described in detail previously . ChloropPSΙΙ) and the redox state of PQ pool (qp) are also shown as color-coded images, after 5 min of illumination with 140 μmol photons m–2 s–1.Representative results of the effective quantum yield of photochemical energy conversion in PSII  in the dark, as described before [The presence of ROS was detected in young and mature leaves of d before . After 3p < 0.05 with the StatView software  [Four to five leaves from different plants were analyzed for each treatment, and the differences between chlorophyll fluorescence parameters were separated by paired t-test at a level of CA, USA) . 47Zn29/γ-Cu9Zn15 based on the X-ray diffractions. TEM images of CuZn NPs  a. The obCuZn NPs  revealed−1 aqueous suspension of CuZn NPs after 24 h of incubation was found to be 1.8 mg L−1 for Cu and 2.2 mg L−1 for Zn, respectively. CuZn NPs are hydrophilic, and thus readily disperse in water. The hydrodynamic diameter provided by DLS number measurements b was 35 PSII, ΦNPQ and ΦNO, which sum to one. The quantum yield of photochemical energy conversion (ΦPSΙΙ) at 30 min, 90 min, and 180 min after the foliar spray with 30 mg L−1 of CuZn NPs presented a significant decrease in young leaves, while in mature leaves, it increased significantly compared to controls  at 30 min, 90 min, 180 min, and 240 min after the CuZn NPs spray increased significantly, compared to control, in young leaves , which is a loss process due to PSII inactivity, increased significantly in young leaves 30 min after the CuZn NPs spray compared to control, while it remained unchanged in mature leaves, where it decreased significantly later on  , but retained control values 240 min after spraying with the NPs  . In youn the NPs . ΦNO in l values .The non-photochemical quenching (NPQ) increased significantly at 30 min and 90 min after the CuZn NPs spray in young leaves, compared to the control, while it decreased 180 min after spraying, and increased again significantly 240 min after spraying a. NPQ in−1 of CuZn NPs, while at the same time it increased significantly in mature leaves compared to controls (The relative electron transport rate at PSII (ETR) decreased significantly in young leaves 30 min, 90 min, and 180 min after the foliar spray with 30 mg Lcontrols b. ETR recontrols b. The ETcontrols b.qp), which is a measure of the fraction of open PSII reaction centers, decreased significantly at 30 min and 180 min after the CuZn NPs spray in young leaves, compared to control; in contrast, it was at control values 90 min and 240 min after spraying , the mature leaves were in a more oxidized state than control .PSΙΙ) in control young leaves showed a spatial heterogeneity, with higher values in the midrib of the leaves than in the lamina  leaf area   e.PSΙΙ compared to control young leaves  of control young leaves showed a spatial heterogeneity, with higher values in the proximal (base) midrib of leaves  leaf area  of control mature leaves showed leaf homogeneity rather than leaf heterogeneity  was observed in young leaves   via the triplet state of chlorophyll (3chl*) [1O2 formation in young leaves compared to mature leaves. NPQ is one of the most important photoprotective mechanisms in plants [2O2 production at 90 min after the NPs spray to control levels  a that coΙΙ) (NPQ b. ΦNO co (3chl*) ,54,55,56n plants ,41,57,58l levels c. An eff2 occurs b, which 2 occurs b possibl2 occurs c, resultaf level c.Arabidopsis thaliana young and mature leaves with 30 mg L−1 of CuZn NPs revealed a spatiotemporal heterogeneity of ΦPSΙΙ and qp measured (at 140 μmol photons m−2 s–1) 30 min, 90 min, 180 min, and 240 min after spraying , an increased electron transport rate (ETR), an increased fraction of open PSII reaction centers (qp), decreased 1O2 formation, and no notable changes in H2O2 generation.In contrast to previous reports that young leaves acclimatize better to environmental changes and can maintain a better ROS homeostasis ,45,69, m−1 of CuZn NPs resulted in an excess supply of them, causing negative effects on PSII function and increased ROS production. Increased H2O2 generation in young leaves after 30 min of spraying with 30 mg L−1 of CuZn NPs  resulting from a significant decrease of the PsbS protein, which is the key regulator of the energy dissipation process [Previously, both the positive and harmful impacts of NPs on terrestrial and aquatic plants have been established, which are mainly due to the concentration, size, and specific surface area of NPs, the exposure methodology, and the plant species that was examined ,84,85,86 process . Neverth process ,84.The present study demonstrates that considering the leaf developmental stage is important for understanding the mechanisms underlying leaf growth responses to environmental stresses ,42,43,88