Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

Machine learning-based automated phenotyping of inflammatory nocifensive behavior in mice.

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.

Using the Tools of Behavioral Neuroscience to Determine the Identity of Different Mouse Strains in a Laboratory Course.

Understanding the neural mechanisms underlying behavior depends on our ability to define and to measure these behaviors in the model animal. We describe an upper-level course which provides students with hands-on experience in the methods of behavioral neuroscience. There are many well-established behavioral tests which are relatively easy for students to conduct that can be used to determine the performance of animals in such tasks as anxiety, motor performance and memory. Laboratory mice bred specifically to exhibit particular behavioral characteristics are readily available from vendors along with well documented behavioral profiles for these strains. We used two albino strains CD1 and BALBc as our model animals. Students were given the task of identifying the strains based on the results of a battery of behavioral tests but were not given information about the mice. These two strains were chosen for their clear differences particularly in tests of anxiety. Students conducted elevated plus maze and zero maze tests, open field test, light-dark exploratory task, rotarod, balance beam test, spatial or novel object learning. Students were able to correctly identify the two strains by comparing their own data with the published literature in the field. The course structure encouraged students to work in teams to design protocols, and then to collect and explore data. Students were enthusiastic about the hands-on laboratory experience and were able to demonstrate an appreciation for and understanding of these methods in behavioral neuroscience.

Evaluation of tarsal injuries in C57BL/6J male mice.

Tarsal joint abnormalities have been observed in aged male mice on a C57BL background. This joint disease consists of calcaneal displacement, inflammation, and proliferation of cartilage and connective tissue, that can progress to ankylosis of the joint. While tarsal pathology has been described previously in C57BL/6N substrains, as well as in STR/ort and B10.BR strain, no current literature describes this disease occurring in C57BL/6J mice. More importantly the behavioral features that may result from such a change to the joint have yet to be evaluated. This condition was observed in older male mice of the C57BL/6J lineage, around the age of 20 weeks or older, at a frequency of 1% of the population. To assess potential phenotypic sequela, this study sought to evaluate body weight, frailty assessment, home cage wheel running, dynamic weight bearing, and mechanical allodynia with and without the presence of pain relief with morphine. Overall mice with tarsal injuries had significantly higher frailty scores (p< 0.05) and weighed less (p<0.01) compared to unaffected mice. Affected mice had greater overall touch sensitivity (p<0.05) and they placed more weight on their forelimbs (p<0.01) compared to their hind limbs. Lastly, when housed with a running wheel, affected mice ran for a shorter length of time (p<0.01) but tended to run a greater distance within the time they did run (p<0.01) compared to unaffected mice. When tested just after being given morphine, the affected mice performed more similarly to unaffected mice, suggesting there is a pain sensation to this disease process. This highlights the importance of further characterizing inbred mouse mutations, as they may impact research programs or specific study goals.

Identifying genetic determinants of inflammatory pain in mice using a large-scale gene-targeted screen.

ABSTRACT: Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

Extensive identification of genes involved in congenital and structural heart disorders and cardiomyopathy.

Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease.

Responses to sounds in the central auditory system of the frog: an advanced electrophysiology laboratory in sensory processing.

Frogs rely upon vocal communication to advertise for potential mates, to defend territory and to alarm neighbors of danger. Cells in the auditory midbrain of an awake frog display tuning to the spectral energy present in calls based upon discharge rate and encode the temporal properties of calls in the timing of their discharges. This laboratory experiment is designed to allow students to explore the relationship between stimulus amplitude or frequency and response rate, and how the timing of responses can also be used to encode behaviorally relevant features of the stimulus. Action potentials in the midbrain auditory nucleus, the torus semicularis, are evoked by delivery of free field sounds and recorded. Most cells are broadly tuned to frequency, yet some can be fairly precise in preserving periodic structure. The use of a comparative model of study should help students understand principles common among all sensory systems, and an appreciation that the architecture of each system is adaptively matched to the ethological task at hand.

Ambiguous animals: mutable mammal biases bird.

Ambiguous figures were primed with picture context, movement, and by presentation of a prior ambiguous figure. We tested two mammal/bird figures to determine if the multiple primes would add or interfere. Picture priming was effective for both figures but diminished with the presentation of a prior ambiguous figure. For the 'swan/squirrel' there was little movement priming. However, 'rabbit/duck' showed strong movement priming which cancelled the reducing effect of prior presentation and added to the picture effect.

The emergence of temporal hyperacuity from widely tuned cell populations.

Typically, individual neural cells operate on a millisecond time scale yet behaviorally animals reveal sub-microsecond acuity. Our model resolves this huge discrepancy by using populations of many widely tuned cells to attain sub-microsecond resolution in a temporal discrimination task. An echolocating bat uses its auditory system to locate objects and it demonstrates remarkable temporal precision in psychophysical tasks. Auditory cells were simulated using realistic parameters and connected in three ascending layers with descending projections from auditory cortex. Coincidence detection of firing collicular cells at thalamus and subsequent integration of multiple inputs at cortex, produce an estimate of time represented as the mean of the active cortical population. Multiple estimates allow the model bat to use memory to recognize predictable change in stimuli values. The best performance is produced using cortical feedback and a computation of target time based on combining the current and previous estimates. Temporal hyperacuity is attained through population coding of physiologically realistic cells but depends on the inherent properties of the psychophysical task.

Transformation of external-ear spectral cues into perceived delays by the big brown bat, Eptesicus fuscus.

The external-ear transfer function for big brown bats (Eptesicus fuscus) contains two prominent notches that vary from 30 to 55 kHz and from 70 to 100 kHz, respectively, as sound-source elevation moves from -40 to +10 degrees. These notches resemble a higher-frequency version of external-ear cues for vertical localization in humans and other mammals. However, they also resemble interference notches created in echoes when reflected sounds overlap at short time separations of 30-50 micros. Psychophysical experiments have shown that bats actually perceive small time separations from interference notches, and here we used the same technique to test whether external-ear notches are recognized as a corresponding time separation, too. The bats' performance reveals the elevation dependence of a time-separation estimate at 25-45 micros in perceived delay. Convergence of target-shape and external-ear cues onto echo spectra creates ambiguity about whether a particular notch relates to the object or to its location, which the bat could resolve by ignoring the presence of notches at external-ear frequencies. Instead, the bat registers the frequencies of notches caused by the external ear along with notches caused by the target's structure and employs spectrogram correlation and transformation (SCAT) to convert them all into a family of delay estimates that includes elevation.