Long-duration animal tracking in difficult lighting conditions.

High-throughput analysis of animal behavior requires software to analyze videos. Such software typically depends on the experiments' being performed in good lighting conditions, but this ideal is difficult or impossible to achieve for certain classes of experiments. Here, we describe techniques that allow long-duration positional tracking in difficult lighting conditions with strong shadows or recurring "on"/"off" changes in lighting. The latter condition will likely become increasingly common, e.g., for Drosophila due to the advent of red-shifted channel rhodopsins. The techniques enabled tracking with good accuracy in three types of experiments with difficult lighting conditions in our lab. Our technique handling shadows relies on single-animal tracking and on shadows' and flies' being accurately distinguishable by distance to the center of the arena (or a similar geometric rule); the other techniques should be broadly applicable. We implemented the techniques as extensions of the widely-used tracking software Ctrax; however, they are relatively simple, not specific to Drosophila, and could be added to other trackers as well.

Evaluating intra-articular drug delivery for the treatment of osteoarthritis in a rat model.

Osteoarthritis (OA) is a degenerative joint disease that can result in joint pain, loss of joint function, and deleterious effects on activity levels and lifestyle habits. Current therapies for OA are largely aimed at symptomatic relief and may have limited effects on the underlying cascade of joint degradation. Local drug delivery strategies may provide for the development of more successful OA treatment outcomes that have potential to reduce local joint inflammation, reduce joint destruction, offer pain relief, and restore patient activity levels and joint function. As increasing interest turns toward intra-articular drug delivery routes, parallel interest has emerged in evaluating drug biodistribution, safety, and efficacy in preclinical models. Rodent models provide major advantages for the development of drug delivery strategies, chiefly because of lower cost, successful replication of human OA-like characteristics, rapid disease development, and small joint volumes that enable use of lower total drug amounts during protocol development. These models, however, also offer the potential to investigate the therapeutic effects of local drug therapy on animal behavior, including pain sensitivity thresholds and locomotion characteristics. Herein, we describe a translational paradigm for the evaluation of an intra-articular drug delivery strategy in a rat OA model. This model, a rat interleukin-1beta overexpression model, offers the ability to evaluate anti-interleukin-1 therapeutics for drug biodistribution, activity, and safety as well as the therapeutic relief of disease symptoms. Once the action against interleukin-1 is confirmed in vivo, the newly developed anti-inflammatory drug can be evaluated for evidence of disease-modifying effects in more complex preclinical models.

How does cognition evolve? Phylogenetic comparative psychology.

Now more than ever animal studies have the potential to test hypotheses regarding how cognition evolves. Comparative psychologists have developed new techniques to probe the cognitive mechanisms underlying animal behavior, and they have become increasingly skillful at adapting methodologies to test multiple species. Meanwhile, evolutionary biologists have generated quantitative approaches to investigate the phylogenetic distribution and function of phenotypic traits, including cognition. In particular, phylogenetic methods can quantitatively (1) test whether specific cognitive abilities are correlated with life history (e.g., lifespan), morphology (e.g., brain size), or socio-ecological variables (e.g., social system), (2) measure how strongly phylogenetic relatedness predicts the distribution of cognitive skills across species, and (3) estimate the ancestral state of a given cognitive trait using measures of cognitive performance from extant species. Phylogenetic methods can also be used to guide the selection of species comparisons that offer the strongest tests of a priori predictions of cognitive evolutionary hypotheses (i.e., phylogenetic targeting). Here, we explain how an integration of comparative psychology and evolutionary biology will answer a host of questions regarding the phylogenetic distribution and history of cognitive traits, as well as the evolutionary processes that drove their evolution.

Citizen Science as a New Tool in Dog Cognition Research.

Family dogs and dog owners offer a potentially powerful way to conduct citizen science to answer questions about animal behavior that are difficult to answer with more conventional approaches. Here we evaluate the quality of the first data on dog cognition collected by citizen scientists using the Dognition.com website. We conducted analyses to understand if data generated by over 500 citizen scientists replicates internally and in comparison to previously published findings. Half of participants participated for free while the other half paid for access. The website provided each participant a temperament questionnaire and instructions on how to conduct a series of ten cognitive tests. Participation required internet access, a dog and some common household items. Participants could record their responses on any PC, tablet or smartphone from anywhere in the world and data were retained on servers. Results from citizen scientists and their dogs replicated a number of previously described phenomena from conventional lab-based research. There was little evidence that citizen scientists manipulated their results. To illustrate the potential uses of relatively large samples of citizen science data, we then used factor analysis to examine individual differences across the cognitive tasks. The data were best explained by multiple factors in support of the hypothesis that nonhumans, including dogs, can evolve multiple cognitive domains that vary independently. This analysis suggests that in the future, citizen scientists will generate useful datasets that test hypotheses and answer questions as a complement to conventional laboratory techniques used to study dog psychology.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Considering the role of social dynamics and positional behavior in gestural communication research.

While the hominin fossil record cannot inform us on either the presence or extent of social and cognitive abilities that may have paved the way for the emergence of language, studying non-vocal communication among our closest living relatives, the African apes, may provide valuable information about how language originated. Although much has been learned from gestural signaling in non-human primates, we have not yet established how and why gestural repertoires vary across species, what factors influence this variation, and how knowledge of these differences can contribute to an understanding of gestural signaling's contribution to language evolution. In this paper, we review arguments surrounding the theory that language evolved from gestural signaling and suggest some important factors to consider when conducting comparative studies of gestural communication among African apes. Specifically, we propose that social dynamics and positional behavior are critical components that shape the frequency and nature of gestural signaling across species and we argue that an understanding of these factors could shed light on how gestural communication may have been the basis of human language. We outline predictions for the influence of these factors on the frequencies and types of gestures used across the African apes and highlight the importance of including these factors in future gestural communication research with primates.

The role of GRK6 in animal models of Parkinson's disease and L-DOPA treatment.

G protein-coupled Receptor Kinase 6 (GRK6) belongs to a family of kinases that phosphorylate GPCRs. GRK6 levels were found to be altered in Parkinson's Disease (PD) and D(2) dopamine receptors are supersensitive in mice lacking GRK6 (GRK6-KO mice). To understand how GRK6 modulates the behavioral manifestations of dopamine deficiency and responses to L-DOPA, we used three approaches to model PD in GRK6-KO mice: 1) the cataleptic response to haloperidol; 2) introducing GRK6 mutation to an acute model of absolute dopamine deficiency, DDD mice; 3) hemiparkinsonian 6-OHDA model. Furthermore, dopamine-related striatal signaling was analyzed by assessing the phosphorylation of AKT/GSK3β and ERK1/2. GRK6 deficiency reduced cataleptic behavior, potentiated the acute effect of L-DOPA in DDD mice, reduced rotational behavior in hemi-parkinsonian mice, and reduced abnormal involuntary movements induced by chronic L-DOPA. These data indicate that approaches to regulate GRK6 activity could be useful in modulating both therapeutic and side-effects of L-DOPA.

The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.

Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.