Type 7 Adenylyl Cyclase is Involved in the Ethanol and CRF Sensitivity of GABAergic Synapses in Mouse Central Amygdala

The GABAergic system in the central amygdala (CeA) plays a major role in ethanol dependence and in the anxiogenic response to ethanol withdrawal. Previously, we found that both ethanol and corticotropin releasing factor (CRF) increase GABAergic transmission in mouse and rat CeA neurons, in part by enhancing the release of GABA via activation of presynaptic CRF1 receptors. CRF1 receptors are coupled to the enzyme adenylyl cyclase (AC), which produces the second messenger cyclic AMP. There are nine isoforms of AC, but we recently found that CRF1 receptors in the pituitary were coupled to the Type 7 AC (AC7). Therefore, using an in vitro electrophysiological approach in brain slices, here we have investigated a possible role of the AC7 signaling pathway in ethanol and CRF effects on CeA GABAergic synapses of genetically modified mice with diminished brain Adcy7 activity (HET) compared to their littermate male wild-type (WT) mice. We found no significant differences in basal membrane properties, mean baseline amplitude of evoked GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs), or paired-pulse facilitation (PPF) of GABAA-IPSPs between HET and WT mice. In CeA neurons of WT mice, ethanol superfusion significantly augmented (by 39%) GABAA-IPSPs and decreased PPF (by 25%), suggesting increased presynaptic GABA release. However, these effects were absent in HET mice. CRF superfusion also significantly augmented IPSPs (by 38%) and decreased PPF (by 23%) in WT CeA neurons, and still elicited a significant but smaller (by 13%) increase of IPSP amplitude, but no effect on PPF, in HET mice. These electrophysiological data suggest that AC7 plays an important role in ethanol and CRF modulation of presynaptic GABA release in CeA and thus may underlie ethanol-related behaviors such as anxiety and dependence

A long non‐coding RNA (Lrap) modulates brain gene expression and levels of alcohol consumption in rats

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype

Genomic landscape of rat strain and substrain variation

Background: Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004). Results: Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs. Conclusions: In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs