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Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is an autosomal dominant neurodegenerative disease caused by mutations in colony-stimulating factor 1 receptor (CSF1R). We sought to identify the role of microglial CSF1R haploinsufficiency in mediating pathogenesis. Using an inducible Cx3cr1 CreERT2/+-Csf1r +/fl system, we found that postdevelopmental, microglia-specific Csf1r haploinsufficiency resulted in reduced expression of homeostatic microglial markers. This was associated with loss of presynaptic surrogates and the extracellular matrix (ECM) structure perineuronal nets. Similar phenotypes were observed in constitutive global Csf1r haploinsufficient mice and could be reversed/prevented by microglia elimination in adulthood. As microglial elimination is unlikely to be clinically feasible for extended durations, we treated adult CSF1R+/- mice at different disease stages with a microglia-modulating dose of the CSF1R inhibitor PLX5622, which prevented microglial dyshomeostasis along with synaptic- and ECM-related deficits. These data highlight microglial dyshomeostasis as a driver of pathogenesis and show that CSF1R inhibition can mitigate these phenotypes.

Microglia dyshomeostasis induces pathology in CSF1R+/− mice, which can be reduced with pharmacological CSF1R inhibition., Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is an autosomal dominant neurodegenerative disease caused by mutations in colony-stimulating factor 1 receptor (CSF1R). We sought to identify the role of microglial CSF1R haploinsufficiency in mediating pathogenesis. Using an inducible Cx3cr1CreERT2/+-Csf1r+/fl system, we found that postdevelopmental, microglia-specific Csf1r haploinsufficiency resulted in reduced expression of homeostatic microglial markers. This was associated with loss of presynaptic surrogates and the extracellular matrix (ECM) structure perineuronal nets. Similar phenotypes were observed in constitutive global Csf1r haploinsufficient mice and could be reversed/prevented by microglia elimination in adulthood. As microglial elimination is unlikely to be clinically feasible for extended durations, we treated adult CSF1R+/− mice at different disease stages with a microglia-modulating dose of the CSF1R inhibitor PLX5622, which prevented microglial dyshomeostasis along with synaptic- and ECM-related deficits. These data highlight microglial dyshomeostasis as a driver of pathogenesis and show that CSF1R inhibition can mitigate these phenotypes.

The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.

Microglia, the resident immune cell of the brain, can be eliminated via pharmacological inhibition of the colony‐stimulating factor 1 receptor (CSF1R). Withdrawal of CSF1R inhibition then stimulates microglial repopulation, effectively replacing the microglial compartment. In the aged brain, microglia take on a “primed” phenotype and studies indicate that this coincides with age‐related cognitive decline. Here, we investigated the effects of replacing the aged microglial compartment with new microglia using CSF1R inhibitor‐induced microglial repopulation. With 28 days of repopulation, replacement of resident microglia in aged mice (24 months) improved spatial memory and restored physical microglial tissue characteristics (cell densities and morphologies) to those found in young adult animals (4 months). However, inflammation‐related gene expression was not broadly altered with repopulation nor the response to immune challenges. Instead, microglial repopulation resulted in a reversal of age‐related changes in neuronal gene expression, including expression of genes associated with actin cytoskeleton remodeling and synaptogenesis. Age‐related changes in hippocampal neuronal complexity were reversed with both microglial elimination and repopulation, while microglial elimination increased both neurogenesis and dendritic spine densities. These changes were accompanied by a full rescue of age‐induced deficits in long‐term potentiation with microglial repopulation. Thus, several key aspects of the aged brain can be reversed by acute noninvasive replacement of microglia.

Many high-priority and high-interest species are challenging to study due to the difficulty in accessing animals and/or obtaining sufficient sample sizes. The recent explosion in technology, particularly social media and live webcams available on the Internet, provides new opportunities for behavioral scientists to collect data not just on our own species, as well as new resources for teaching and outreach. We discuss here the possibility of exploiting online media as a new source of behavioral data, which we termed ‘video mining’. This article proposes epidemiological and ethological field techniques to gather and screen online media as a data source on diverse taxa. This novel method provides access to a rich source of untapped knowledge, particularly to study the behavior of understudied species or sporadic behaviors, but also for teaching or monitoring animals in challenging settings.

Iron deficiency is common throughout the world and has been linked to cognitive impairments. Using neonatal piglets to model human infants, we assessed the impact of iron deficiency on spatial learning and memory. Artificially reared piglets were fed 1 of 3 liquid diets with varying concentrations of iron: control (CON), mildly deficient (MID), or severely deficient (SID; 100, 25.0, or 10.0 mg iron/kg milk solids, respectively) for 4 wk. Relative to CON, SID and MID piglets had reduced hemoglobin (P < 0.05) as well as magenta skin color (P < 0.001), which correlated with hematocrit (R 2 = 0.76; P < 0.001). SID and MID hemoglobin differed at wk 3 and 4 (P < 0.05). In a hippocampal-dependent, spatial, T-maze task, SID piglets were unable to acquire the task (post hoc contrast:first vs. last day of acquisition), while MID piglets demonstrated deficits in reversal learning (P = 0.032). Iron concentrations in the liver (P < 0.001), serum (P = 0.003), and hippocampus (P = 0.004), but not prefrontal cortex, were lower in MID and SID compared with CON piglets. The level of the transferrin receptor mRNA (TFR) was greater in the prefrontal cortex of CON piglets than in MID and SID piglets (P = 0.001) but not the hippocampus. Gene expression of several neurotrophic factors and proinflammatory cytokines, as well as whole-brain and hippocampal volume, were not affected by dietary treatment. In conclusion, neonatal iron deficiency leads to cognitive impairment, which may be due in part to a reduced iron concentration in the hippocampus. J. Nutr. 142: 2050‐2056, 2012.

This study assessed the motivation of gestating sows housed in standard, barren gestation stalls (used for breeding/implantation and/or gestation) for access to environmental enrichment. Enrichment consisted of a cotton rope or rubber mat in comparison to positive (additional food when fed at commercial levels) and negative (empty trough) controls. Although environmental enrichment may improve animal welfare, sows' valuation of enrichments is largely unknown. This study used an operant panel and obtained behavioral measures to quantify motivation. As indicated by a higher price paid and lower latencies to press the panel and enter the treatment stall (all comparisons, p.05), sows demonstrated higher motivation for food compared with all treatments. Sows housed in gestation stalls did not demonstrate high motivation via operant responding for a cotton rope or a rubber mat; nor did they demonstrate any differences in behavioral measures (all comparisons, p.10). Although sows' motivation for a mat did not differ from that for an empty trough, previous work has demonstrated the welfare benefits associated with comfort flooring.

Pigs are a valuable animal model for studying neurodevelopment in humans due to similarities in brain structure and growth. The development and validation of behavioral tests to assess learning and memory in neonatal piglets are needed. The present study evaluated the capability of 2-week old piglets to acquire a novel place and direction learning spatial T-maze task. Validity of the task was assessed by the administration of scopolamine, an anti-cholinergic drug that acts on the hippocampus and other related structures, to impair spatial memory. During acquisition, piglets were trained to locate a milk reward in a constant place in space, as well as direction (east or west), in a plus-shaped maze using extra-maze visual cues. Following acquisition, reward location was reversed and piglets were re-tested to assess learning and working memory. The performance of control piglets in the maze improved over time (P < 0.0001), reaching performance criterion (80 % correct) on day 5 of acquisition. Correct choices decreased in the reversal phase (P < 0.0001), but improved over time. In a separate study, piglets were injected daily with either phosphate-buffered saline (PBS; control) or scopolamine prior to testing. Piglets administered scopolamine showed impaired performance in the maze compared to controls (P = 0.03), failing to reach performance criterion after 6 days of acquisition testing. Collectively, these data demonstrate that neonatal piglets can be tested in a spatial T-maze task to assess hippocampal-dependent learning and memory.

Gestating sow motivation for enriched environments is unknown, but is essential knowledge for developing housing that addresses animal welfare concerns. This study investigated whether the motivation of gestating sows for access to an enriched group pen (containing a rubber mat, straw, compost, and cotton ropes), and their behavior while in the pen, would differ due to social status. Motivation was measured using an operant panel and behavioral measures were obtained to test our hypotheses. Dominant and subordinate sows were similarly (58.38 ± 21.54 presses vs. 96.88 ± 48.30 presses [raw data], respectively; P = 0.72) and moderately (comparable operant responding to sows that had consumed 25–50% of their ad libitum diet) motivated for access to an enriched group pen. However, sow social status impacted aggression ( P = 0.007), enrichment use ( P = 0.001), and inactivity ( P = 0.002); where dominant sows were more aggressive (0.61 ± 0.29 vs. 0.06 ± 0.03 [raw data]; P = 0.008) and spent more time using enrichments (54.58 ± 6.02 vs. 29.57 ± 5.56 [raw data]; P = 0.001) upon entrance to the pen, while subordinate sows were more frequently displaced from enrichments ( P P = 0.003). Sow social status affected the pattern of enrichment use over the course of the day ( P = 0.02), where subordinate sows increased their use of enrichments the following morning during non-peak times ( P = 0.001). These findings demonstrate that regardless of social status, sows were able to access enrichments and valued an enriched group pen. Sow social status has the potential to greatly alter the effectiveness of enrichments in group settings and will become an increasingly important consideration as scientists and producers explore welfare friendly alternatives to barren sow housing.

Comfortable flooring may impact many aspects of an animal's welfare, including lying behavior, an animal's ability to change postures, and incidence of lameness and lesions. Therefore, we hypothesized that the addition of rubber mats to stalls of group pens would improve sow health, comfort and welfare during breeding. In this study, Landrace York- Yorkshire sows (128) were mixed post-weaning and housed in pens of four. The pens contained a slatted group area and four feeding stalls. Rubber mats (measuring 1.83 m 0.61 m 1.27 cm) were added to the feeding stalls of half of the pens and rotated to the opposite pens for each replication. The behavioral time budgets of the sows were recorded throughout the experiment; lesion and lameness scores were collected prior to mixing and at the end of the experiment. Data were analyzed as repeated measures mixed models with post hoc Tukey tests. The pattern of behaviors performed in the stall versus group area was different for mat and concrete treatments (REML: F 2,56 = 12.98; P < 0.001). Tukey tests revealed that only resting behaviors were affected. We then examined resting behavior in detail. Time spent in different resting postures (sternal versus lateral lying) differed between treatments (REML: F1,30 = 4.92; P = 0.03), where sows on mats spent more time lying laterally (P < 0.05) and performed more of their lying behavior in the stalls (GLM: F1,22 = 14.88; P = 0.001) compared to sows on concrete. Additionally, sows stood up and laid down more frequently (GLM: F1,30 = 13.53; P = 0.001) than sows on concrete. Warm temperatures reduced resting behavior in the stalls (REML: F2,56 = 8.75; P < 0.001). Sows in matted pens had a lower total lesion score at the end of the experiment in comparison to sows in concrete pens (REML: F1,30 = 5.03; P = 0.03). Lameness scores did not differ between treatments. These results imply that the provision of alternative comfort flooring may provide welfare benefits to breeding sows, though environmental temperature needs to be considered when providing rubber mats.

In addition to amyloid-β plaque and tau neurofibrillary tangle deposition, neuroinflammation is considered a key feature of Alzheimer's disease pathology. Inflammation in Alzheimer's disease is characterized by the presence of reactive astrocytes and activated microglia surrounding amyloid plaques, implicating their role in disease pathogenesis. Microglia in the healthy adult mouse depend on colony-stimulating factor 1 receptor (CSF1R) signalling for survival, and pharmacological inhibition of this receptor results in rapid elimination of nearly all of the microglia in the central nervous system. In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of ∼80% of microglia. Chronic microglial elimination does not alter amyloid-β levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.

Raising beef cattle on concrete floors can negatively impact their welfare by increasing joint swelling and body lesions, as well as abnormalities in resting behavior and postural changes. We hypothesized that the addition of rubber mats to concrete pens would improve beef cattle welfare by improving performance, health, hygiene, and resting behavior. Forty-eight crossbred Angus steers were housed in pens of 4 and randomly assigned to a single flooring treatment: (1) fully slatted concrete (CON), (2) fully slatted rubber mat (SLAT), or (3) solid rubber mat (SOLID; 60% of pen floor) from 36 to 48 wk of age. Weight, ADG, lesions, gait score, joint swelling, and animal and pen cleanliness were collected every 2 wk. Behavioral time budgets and frequency of postural changes (an indicator of floor traction and comfort) were collected at 0, 6, and 12 wk. No differences in weight gain or ADG were observed. Steers on SOLID flooring (0.80 ± 0.08) showed increased lesions compared to SLAT (0.38 ± 0.08) and CON (0.37 ± 0.08; both, = 0.05); however, there was no difference between SLAT and CON. SLAT steers (1.69 ± 0.04) showed a reduced gait score compared to SOLID (1.95 ± 0.04) and CON (1.98 ± 0.04; both, < 0.05), but SOLID and CON did not differ. Steers on SLAT flooring had less joint swelling (both knees and hocks) compared to SOLID and CON (all comparisons, < 0.05), but SOLID and CON did not differ. Steers on SOLID (3.64 ± 0.05) flooring were dirtier than those on SLAT (2.27 ± 0.05) and CON (2.19 ± 0.05; both, < 0.001), whereas SLAT and CON were similar. Additionally, SOLID and SLAT pens were less clean than CON pens ( < 0.001 and = 0.094, respectively), and SOLID was less clean than SLAT ( < 0.001). Time budget behavior was affected by treatment ( = 0.043), where SOLID differed from CON and SLAT (both, < 0.05). Steers on SOLID flooring preferred to rest on the rubber mat vs. slatted concrete ( = 0.001). Steers on SLAT flooring changed their posture more frequently than those on SOLID and CON flooring (both, < 0.05), but SOLID and CON did not differ. Compared to CON steers, SOLID steers showed an increase in lesions and a reduction in cleanliness, whereas SLAT steers showed a decrease in gait score and joint swelling and an increase in postural changes. Combined, these data suggest that the addition of slatted rubber mats to concrete pens may improve beef cattle welfare.

BACKGROUND: Microglia are dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival in the adult brain, with administration of the dual CSF1R/c-kit inhibitor PLX3397 leading to the near-complete elimination of all microglia brainwide. Here, we determined the dose-dependent effects of a specific CSF1R inhibitor (PLX5622) on microglia in both wild-type and the 3xTg-AD mouse model of Alzheimer's disease. METHODS: Wild-type mice were treated with PLX5622 for up to 21 days, and the effects on microglial numbers were assessed. 3xTg-AD mice were treated with PLX5622 for 6 or 12 weeks and effects on microglial numbers and pathology subsequently assessed. RESULTS: High doses of CSF1R inhibitor eliminate most microglia from the brain, but a 75 % lower-dose results in sustained elimination of ~30 % of microglia in both wild-type and 3xTg-AD mice. No behavioral or cognitive deficits were found in mice either depleted of microglia or treated with lower CSF1R inhibitor concentrations. Aged 3xTg-AD mice treated for 6 or 12 weeks with lower levels of PLX5622 resulted in improved learning and memory. Aβ levels and plaque loads were not altered, but microglia in treated mice no longer associated with plaques, revealing a role for the CSF1R in the microglial reaction to plaques, as well as in mediating cognitive deficits. CONCLUSIONS: We find that inhibition of CSF1R alone is sufficient to eliminate microglia and that sustained microglial elimination is concentration-dependent. Inhibition of the CSF1R at lower levels in 3xTg-AD mice prevents microglial association with plaques and improves cognition.

Copyright: © 2015 Elmore et al. Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ∼99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the original resident microglia without any apparent adverse effects in healthy adult mice.

Environmental insults during sensitive periods can affect hippocampal development and function, but little is known about peripheral infection, especially in humans and other animals whose brain is gyrencephalic and experiences major perinatal growth. Using a piglet model, the present study showed that inoculation on postnatal day 7 with the porcine reproductive and respiratory syndrome virus (PRRSV) caused microglial activation within the hippocampus with 82% and 43% of isolated microglia being MHC II+13 and 20 d after inoculation, respectively. In control piglets, +. PRRSV piglets were febrile (p< 0.0001), anorectic (p< 0.0001), and weighed less at the end of the study (p= 0.002) compared with control piglets. Increased inflammatory gene expression (e.g., IL-1β, IL-6, TNF-α, and IFN-γ) was seen across multiple brain regions, including the hippocampus, whereas reductions in CD200, NGF, and MBP were evident. In a test of spatial learning, PRRSV piglets took longer to acquire the task, had a longer latency to choice, and had a higher total distance moved. Overall, these data demonstrate that viral respiratory infection is associated with a marked increase in activated microglia in the hippocampus, neuroinflammation, and impaired performance in a spatial cognitive task. As respiratory infections are common in human neonates and infants, approaches to regulate microglial cell activity are likely to be important.

and Implications Lameness and leg injuries are common in the swine industry and are a serious welfare concern. The impact of rubber mats on measures of sow health, behavior and welfare were evaluated during 10 days at breeding. Sows preferred to rest in stalls with mats, showed a reduction in lesions and an increase in postural changes. The provision of rubber mats should be considered to improve sow welfare.

Environmental insults during sensitive developmental periods can affect neural cells and circuits, slow cognitive development, and increase the risk for behavioral disorders. However, the ramifications of postnatal infection on brain and cognitive development are poorly understood. Using a neonatal piglet model, we showed that infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a model of viral pneumonia, caused drastic microglial cell activation within the hippocampus with 82% and 43% of microglia being MHC-II positive at 13 and 20 days post-inoculation, respectively. In control piglets, less than 5% of microglia in the hippocampus were MHC-II positive. Increased inflammatory gene expression (e.g., IL-1B, IL-6, TNFa, and IFNY) was seen across multiple brain regions including the hippocampus. In a hippocampal-dependent spatial t-maze task, PRRSV piglets took longer to acquire the task, had a longer latency to choice, and a higher total distance moved. PRRSV also affected neurogenesis in the piglet dentate gyrus. In non-infected controls there were more BrdU-positive cells in males than females. PRRSV caused a reduction in surviving cells in males, but there were no changes in females. In addition, there was a ∼30% reduction in the number of new cells that developed into mature neurons in both males and females with PRRSV. These data suggest that early-life neuroinflammation has a profound effect on brain and cognitive development.