Biology subjects

Lusk, S.

Publications and source records attributed to Lusk, S..

3 recordsLinked to original sources

bioRxiv2023

Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for normal breathing and chemosensory reflexes

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders including Congenital Central Hyperventilation Syndrome (CCHS), Sudden Infant Death Syndrome (SIDS) and Rett Syndrome. The current unchallenged paradigm in the field, supported by multiple studies, is that glutamate co-transmission in subsets of central NA neurons plays a role in breathing control. If true, NA-glutamate co-transmission may also be mechanistically important in respiratory disorders. However, the requirement of NA-derived glutamate in breathing has not been directly tested and the extent of glutamate co-transmission in the central NA system remains uncharacterized. Therefore, we fully characterized the cumulative fate maps and acute adult expression patterns of all three Vesicular Glutamate Transporters (Slc17a7 (Vglut1), Slc17a6 (Vglut2), and Slc17a8 (Vglut3)) in NA neurons, identifying a novel, dynamic expression pattern for Vglut2 and an undescribed co-expression domain for Vglut3 in the NA system. In contrast to our initial hypothesis that NA derived glutamate is required to breathing, our functional studies showed that loss of Vglut2 throughout the NA system failed to alter breathing or metabolism under room air, hypercapnia, or hypoxia in unrestrained and unanesthetized mice. These data demonstrate that Vglut2-based glutamatergic signaling within the central NA system is not required for normal baseline breathing and hypercapnic, hypoxic chemosensory reflexes. These outcomes challenge the current understanding of central NA neurons in the control of breathing and suggests that glutamate may not be a critical target to understand NA neuron dysfunction in respiratory diseases.

neuroscience↗

bioRxiv2022

Breathe Easy, an automated respiratory data pipeline for waveform characteristic analysis

Comprehensive and accurate analysis of respiratory and metabolic data is crucial to modelling congenital, pathogenic, and degenerative diseases converging on autonomic control failure. A lack of tools for high-throughput analysis of respiratory datasets remains a major challenge. We present Breathe Easy, a novel open-source pipeline for processing raw recordings and associated metadata into operative outcomes, publication-worthy graphs, and robust statistical analyses including QQ and residual plots for assumption queries and data transformations. This pipeline uses a facile graphical user interface for uploading data files, setting waveform feature thresholds, and defining experimental variables. Breathe Easy was validated against manual selection by experts, which represents the current standard in the field. We demonstrate Breathe Easys utility by examining a 2-year longitudinal study of an Alzheimers Disease mouse model to assess contributions of forebrain pathology in disordered breathing. Whole body plethysmography has become an important experimental outcome measure for a variety of diseases with primary and secondary respiratory indications. Respiratory dysfunction, while not an initial symptom in many of these disorders, often drives disability or death in patient outcomes. Breathe Easy provides an open-source respiratory analysis tool for all respiratory datasets and represents a necessary improvement upon current analytical methods in the field.

physiology↗

bioRxiv2021

A CRISPR toolbox for generating intersectional genetic mice for functional, molecular, and anatomical circuit mapping

BackgroundA full understanding of circuits and cellular mechanisms governing health and disease requires the dissection and multi-faceted study of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic response alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical study. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allow for more defined target expression. Although the application of this method is becoming increasingly popular, the expense and difficulty associated with production of customized intersectional mouse lines have limited widespread application to more common allele manipulations that are often commercially produced at great expense. ResultsWe present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study how functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. ConclusionsLines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

genetics↗