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The Morris Water Maze (MWM) is the most commonly used assay for evaluating learning and memory in laboratory mice. Despite its widespread use, contemporary reviews have highlighted substantial methodological variation in experimental protocols and that the associated testing procedures are acutely (each trial) and chronically (testing across days) stressful; stress impairs attention, memory consolidation and the retrieval of learned information. Moreover, the interpretation of behavior within the MWM is often difficult because of wall hugging, non-spatial swim strategies, floating, and jumping off the escape platform. Together, these issues may compromise the reproducibility, generalizability, and predictability of experimental results, as well as animal welfare. To address these issues, and as an initial proof-of-principle, we first narrowed the spatial dimensions of the MWM by using a T-insert, which constrained and reduced the overall length of time/distance that the animal must swim in order to navigate to the escape platform, thus reducing stress and off-task behavior. Given the robust performance observed across spatial acquisition (learning and memory) as well as during reversal learning (executive function), we further reduced (by 43%) the overall distance and time that the animal must swim in order to find the escape platform in a bespoke standalone Water T-Maze (WTM). We show, across five experiments, procedural refinements to our protocol and demonstrate robust, reliable and reproducible indicators of learning, memory and executive functioning in a task that is also significantly more efficient (3 days of testing within the WTM vs. 11 days of testing within the MWM). Taken together, our WTM apparatus and protocol are a significant improvement over other water-based apparatuses and protocols for evaluating learning, memory, and executive functioning in laboratory mice.

Adipose tissue (AT) is an endocrine organ with a central role on whole-body energy metabolism and development of metabolic diseases. Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, respectively) analyses in mice and human AT have revealed vast cell heterogeneity and functionally distinct subtypes that are potential therapeutic targets to metabolic disease. In periparturient dairy cows, AT goes through intensive remodeling and its dysfunction is associated with metabolic disease pathogenesis and decreased productive performance. The contributions of depot-specific cells and subtypes to the development of diseases in dairy cows remain to be studied. Our objective was to elucidate differences in cellular diversity of visceral (VAT) and subcutaneous (SAT) AT in dairy cows at the single-nuclei level. We collected matched SAT and VAT samples from three dairy cows and performed snRNA-seq analysis. We identified distinct cell types including four major mature adipocytes (AD) and three stem and progenitor cells (ASPC) subtypes, along with endothelial cells (EC), mesothelial cells (ME), immune cells, and pericytes and smooth muscle cells. All major cell types were present in both SAT and VAT, although a strong VAT-specificity was observed for ME, which were basically absent in SAT. One ASPC subtype was defined as adipogenic (PPARG+) while the other two had a fibro-adipogenic profile (PDGFRA+). We identified vascular and lymphatic EC subtypes, and different immune cell types and subtypes in both SAT and VAT, i.e., macrophages, monocytes, T cells, and natural killer cells. Not only did VAT show a greater proportion of immune cells, but these visceral immune cells had greater activation of pathways related to immune and inflammatory response, and complement cascade in comparison with SAT. There was a substantial contrast between depots for gene expression of complement cascade, which were greatly expressed by VAT cell subtypes compared to SAT, indicating a pro-inflammatory profile in VAT. Unprecedently, our study demonstrated cell-type and depot-specific heterogeneity in VAT and SAT of dairy cows. A better understanding of depot-specific molecular and cellular features of SAT and VAT will aid in the development of AT-targeted strategies to prevent and treat metabolic disease in dairy cows, especially during the periparturient period.