Rats have the ability to learn and perform sophisticated behavioral tasks, making them very useful for investigating neural circuit functions. In contrast to the extensive mouse genetic toolkit, the paucity of recombinase-expressing rat models has limited the ability to monitor and manipulate molecularly-defined neural populations in this species. Here we report the generation and validation of two knock-in rat strains expressing either Cre or Flp recombinase under the control of Parvalbumin (Pvalb), a gene expressed in the critical "fast-spiking" subset of inhibitory interneurons (FSIs). These strains were generated with CRISPR-Cas9 gene editing and show highly specific and penetrant labeling of Pvalb-expressing neurons, as demonstrated by in situ hybridization and immunohistochemistry. We validated these models in both prefrontal cortex and striatum using both ex vivo and in vivo approaches, including whole-cell recording, optogenetics, extracellular physiology and photometry. Our results demonstrate the utility of these new transgenic models for a wide range of neuroscience experiments. IntroductionThe extensive library of recombinase driver mouse models [1,2] has enabled precise functional and structural dissection of neural circuits by providing specific and reliable genetic access to molecularly defined neural populations. When used in combination with recombinase-dependent reporter models or viruses, reporter genes can be expressed in specific neural populations [3]. For complex behavioral and large-scale in vivo electrophysiological experiments, rats are the model of choice. Yet their usefulness is restricted by the limited number of transgenic recombinase driver models to label molecularly defined neural populations. Thus, reliable rat recombinase driver models have great potential for accelerating progress in neuroscience.One important application of recombinase driver models is to suppress local brain activity in real time. This can be effectively achieved by optogenetic stimulation of local inhibitory interneurons, as demonstrated in mice using inhibitory neuron-specific Cre models [2,4]. In both cortical [5] and subcortical [6] regions, Parvalbumin (Pvalb) is expressed by GABAergic interneurons that provide perisomatic inhibition to projection neurons [7]. A rat recombinase driver model that directs transgene expression to Pvalb+ interneurons will therefore be a powerful tool for temporally-precise control of local circuit function.Until recently, it has been difficult to insert exogenous genes of interest into specific genomic locations in rats, primarily due to the lack of pluripotent stem cells for in vitro genomic modification [8]. To overcome this limitation, genetically modified rats have been predominantly generated using Bacterial Artificial Chromosome (BAC) transgenic technology [9][10][11]. This involves inserting a large chunk of chromosomal DNA (200-300kb) into the genome that includes the promoter of interest, coupled with an additional coding sequence for the desired recombinase. Expres...

Danforth's short tail (Sd) mice provide an excellent model for investigating the underlying etiology of human caudal birth defects, which affect 1 in 10 000 live births. Sd animals exhibit aberrant axial skeleton, urogenital and gastrointestinal development similar to human caudal malformation syndromes including urorectal septum malformation, caudal regression, vertebral-anal-cardiac-tracheo-esophageal fistula-renal-limb (VACTERL) association and persistent cloaca. Previous studies have shown that the Sd mutation results from an endogenous retroviral (ERV) insertion upstream of the Ptf1a gene resulting in its ectopic expression at E9.5. Though the genetic lesion has been determined, the resulting epigenomic and transcriptomic changes driving the phenotype have not been investigated. Here, we performed ATAC-seq experiments on isolated E9.5 tailbud tissue, which revealed minimal changes in chromatin accessibility in Sd/Sd mutant embryos. Interestingly, chromatin changes were localized to a small interval adjacent to the Sd ERV insertion overlapping a known Ptf1a enhancer region, which is conserved in mice and humans. Furthermore, mRNA-seq experiments revealed increased transcription of Ptf1a target genes and, importantly, downregulation of hedgehog pathway genes. Reduced sonic hedgehog (SHH) signaling was confirmed by in situ hybridization and immunofluorescence suggesting that the Sd phenotype results, in part, from downregulated SHH signaling. Taken together, these data demonstrate substantial transcriptome changes in the Sd mouse, and indicate that the effect of the ERV insertion on Ptf1a expression may be mediated by increased chromatin accessibility at a conserved Ptf1a enhancer. We propose that human caudal dysgenesis disorders may result from dysregulation of hedgehog signaling pathways.

The COPII component SEC24 mediates the recruitment of transmembrane cargoes or cargo adaptors into newly forming COPII vesicles on the ER membrane. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/dgenes rather than differences in cargo function explain the early embryonic requirements for SEC24C and SEC24D.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Danforth's short tail (Sd) mice provide an excellent model for investigating the underlying etiology of human caudal birth defects, which affect 1 in 10,000 live births. Sd animals exhibit aberrant axial skeleton, urogenital, and gastrointestinal development similar to human caudal malformation syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca. Previous studies have shown that the Sd mutation results from an endogenous retroviral (ERV) insertion upstream of the Ptf1a gene resulting in its ectopic expression at E9.5. Though the genetic lesion has been determined, the resulting epigenomic and transcriptomic changes driving the phenotype have not been investigated. Here, we performed ATAC-seq experiments on isolated E9.5 tailbud tissue, which revealed minimal changes in chromatin accessibility in Sd/Sd mutant embryos. Interestingly, chromatin changes were localized to a small interval adjacent to the Sd ERV insertion overlapping a known Ptf1a enhancer region, which is conserved in mice and humans.Furthermore, mRNA-seq experiments revealed increased transcription of PTF1A target genes and, importantly, downregulation of hedgehog pathway genes. Reduced sonic hedgehog (SHH) signaling was confirmed by in situ hybridization and immunofluorescence suggesting that the Sd phenotype results, in part, from downregulated SHH signaling. Taken together, these data demonstrate substantial transcriptome changes in the Sd mouse, and indicate that the effect of the ERV insertion on Ptf1a expression may be mediated by increased chromatin accessibility at a conserved Ptf1a enhancer. We propose that human caudal dysgenesis disorders may result from dysregulation of hedgehog signaling pathways.

Background: The relevance of the work lies in the search for new hemocontact drugs with hemocompatibility and a pronounced activation effect on the cellular and humoral blood systems for their possible use in clinical practice during low-volume hemoperfusion.The aim of this work was to assess the activation capabilities of three granular hemosorbents by the rate of adhesion of blood cellular elements to the surface of granules in vitro.Materials and methods. When using the method of low-volume hemoperfusion (LVH) in the clinic it is important to take into account the activation properties of solid-phase granular drugs. Blood-contact interaction was carried out in bench conditions with the use of donated blood in rotary mode. Blood samples were taken before the experiment and after 5, 20, 40 and 60 minutes. Changes in blood cell and subcellular populations were evaluated using the Sysmex XT 1800i hematological analyzer (26 parameters), which made it possible to indirectly judge the activation of blood cells. 30 experiments were conducted. To analyze the activation functions of the hemocontact preparations the speed-time adhesive profile of blood cells on the sorbent was used.Results. The effect of using the preparations Silochrome S-120 and SPS in comparison with SСT-6A HP as contact hemoactivators can be more pronounced, since the activation potential of these sorbents for blood cells is much higher. Silochrome S-120 has the highest activation capabilities compared to SPS and SKT-6A HP.Conclusion. Adhesion rate indicators can be indicators of the activation of blood cells upon contact with foreign surfaces and serve as a criterion for assessing the activation capabilities of these surfaces when using the LVH method in the clinic.