Muscarinic Modulation of SK2-Type K+ Channels Promotes Intrinsic Plasticity in L2/3 Pyramidal Neurons of the Mouse Primary Somatosensory Cortex.

Muscarinic acetylcholine receptors (mAChRs) inhibit small-conductance calcium-activated K+ channels (SK channels) and enhance synaptic weight via this mechanism. SK channels are also involved in activity-dependent plasticity of membrane excitability ("intrinsic plasticity"). Here, we investigate whether mAChR activation can drive SK channel-dependent intrinsic plasticity in L2/3 cortical pyramidal neurons. Using whole-cell patch-clamp recordings from these neurons in slices prepared from mouse primary somatosensory cortex (S1), we find that brief bath application of the mAChR agonist oxotremorine-m (oxo-m) causes long-term enhancement of excitability in wild-type mice that is not observed in mice deficient of SK channels of the SK2 isoform. Similarly, repeated injection of depolarizing current pulses into the soma triggers intrinsic plasticity that is absent from SK2 null mice. Intrinsic plasticity lowers spike frequency adaptation and attenuation of spike firing upon prolonged activation, consistent with SK channel modulation. Depolarization-induced plasticity is prevented by bath application of the protein kinase A (PKA) inhibitor H89, and the casein kinase 2 (CK2) inhibitor TBB, respectively. These findings point toward a recruitment of two known signaling pathways in SK2 regulation: SK channel trafficking (PKA) and reduction of the calcium sensitivity (CK2). Using mice with an inactivation of CaMKII (T305D mice), we show that intrinsic plasticity does not require CaMKII. Finally, we demonstrate that repeated injection of depolarizing pulses in the presence of oxo-m causes intrinsic plasticity that surpasses the plasticity amplitude reached by either manipulation alone. Our findings show that muscarinic activation enhances membrane excitability in L2/3 pyramidal neurons via a downregulation of SK2 channels.

Nicotine aversion is mediated by GABAergic interpeduncular nucleus inputs to laterodorsal tegmentum.

Nicotine use can lead to dependence through complex processes that are regulated by both its rewarding and aversive effects. Recent studies show that aversive nicotine doses activate excitatory inputs to the interpeduncular nucleus (IPN) from the medial habenula (MHb), but the downstream targets of the IPN that mediate aversion are unknown. Here we show that IPN projections to the laterodorsal tegmentum (LDTg) are GABAergic using optogenetics in tissue slices from mouse brain. Selective stimulation of these IPN axon terminals in LDTg in vivo elicits avoidance behavior, suggesting that these projections contribute to aversion. Nicotine modulates these synapses in a concentration-dependent manner, with strong enhancement only seen at higher concentrations that elicit aversive responses in behavioral tests. Optogenetic inhibition of the IPN-LDTg connection blocks nicotine conditioned place aversion, suggesting that the IPN-LDTg connection is a critical part of the circuitry that mediates the aversive effects of nicotine.

Identification and validation of genetic variants that influence transcription factor and cell signaling protein levels.

Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

Protein quantitative trait loci identify novel candidates modulating cellular response to chemotherapy.

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p ≤ 0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.