Presenting Author:

Can Peng, Ph.D.

Principal Investigator:

Ryan Drenan, Ph.D.

Department:

Pharmacology

Keywords:

nicotine addiction, nicotinic acetylcholine receptors, ventral tegmental area, CRISPR/Cas9 technology

Location:

Third Floor, Feinberg Pavilion, Northwestern Memorial Hospital

B149 - Basic Science

Genome editing of nAChR subunits in VTA using CRISPR-Cas9

Nicotine addiction is a serious threat to public health worldwide. Identification of promising molecular targets to develop new smoking cessation therapeutics is highly desired. There are a variety of nicotinic acetylcholine receptor (nAChR) subtypes in the brain. Each receptor is a pentameric assembly of one or more types of subunits. Nicotine activation of nAChRs in the ventral tegmental area (VTA) is crucial for nicotine rewarding and reinforcement in rodents. Our previous findings showed that activation of alpha4alpha6beta2* nAChRs in VTA dopaminergic (DA) neurons is sufficient to initiate cellular changes involved in nicotine addiction. However, the specific role for each of these nAChR subunits in altering neuronal activity is unclear. In this study, to isolate the alpha4, alpha6, and beta2 nAChR function in the dopamine pathway, we selectively eliminated specific nAChR subunit in the VTA of adult C57BL/6 wild type mice by using the CRISPR-Cas9 technology. To perform the in vivo genome editing of these three nAChR subunits, we developed herpes simplex virus (HSV)-based viral vectors that express Streptococcus pyogenes Cas9 nuclease and a small guide RNA (sgRNA) specifically targeting the nAChR gene of interest in the first or second exon. We also produced a control HSV expressing a scrambled (scr) sgRNA sequence. Stereotaxic surgery was conducted to infuse individual virus into the VTA. Whole-cell recordings were carried out 2-3 days after the surgery to analyze the function of these nAChRs. VTA neurons infected with scr CRISPR HSV and those not infected had comparable ACh-evoked currents. Compared to VTA neurons not infected with alpha4 CRISPR HSV, the ACh-evoked currents in the virus infected neurons were significantly reduced. Similarly, alpha6 and beta2 gene editing demonstrated profound inhibitory effects on the amplitude of ACh-evoked currents. Pharmacological properties of the mutant receptors were also evaluated. Residual ACh-evoked currents in alpha4 CRISPR HSV-infected neurons are sensitive to alpha-conotoxin MII analog (alphaCtxMII[H9A;L15A], a selective alpha6 nAChR antagonist). Co-application of alphaCtxMII[H9A;L15A] and DHbetaE (a beta2 nAChR antagonist) did not further block the residual currents in alpha4 CRISPR HSV infected neurons pretreated with alphaCtxMII[H9A;L15A]. No additional currents was blocked by alphaCtxMII[H9A;L15A] in alpha6 CRISPR HSV infected neurons. For each of the Chrna4, Chrna6, and Chrnb2 gene, two independent sgRNAs were tested and both were equally effective in eliminating the alpha4, alpha6, and beta2 subunit expression. Electrophysiological characterization of virus infected and non-infected VTA neurons showed no significant difference in resting membrane potential and input resistance. Together, our data suggest that CRISPR HSV is an efficient tool for genetic manipulation of specific nAChR gene in VTA neurons to examine their function in nicotine reward-related behavior.