Date of Award
Doctor of Philosophy (PhD)
Cornichon homolog-3 (CNIH3) is an AMPA receptor (AMPAR) auxiliary protein highly expressed in the dorsal hippocampus (dHPC), a region where AMPARs are critical for spatial memory and synaptic plasticity. A 2016 genome-wide association study (GWAS) by Nelson et al. identified single nucleotide polymorphisms (SNPs) in the gene CNIH3 to be associated with reduced individual risk for the development of opioid use disorder (OUD) in individuals with prior opioid exposure. We previously demonstrated a key role for AMPARs in the dHPC in opioid-associated learning and memory, therefore we hypothesized that CNIH3 in the dHPC may mediate learning and memory processes through AMPAR-mediated mechanisms. To study the role of CNIH3 on memory and related mechanisms, we bred and characterized three new C57BL/6 mouse lines: a Cnih3 knockdown (KD), a functional Cnih3 global knockout (KO, Cnih3-/-), and a humanized transgenic mouse line overexpressing human Cnih3 bearing the SNPs identified in the Nelson et al., 2016 GWAS. In addition, we also created a viral vector construct to induce localized overexpression of Cnih3.
We utilized these newly generated mouse lines to study the role of Cnih3 in spatial memory, synaptic plasticity, and AMPAR subunit configuration. In our Cnih3 KD mice, we did not observe changes due to genotype in spatial memory nor in synaptic protein expression. In mice overexpressing Cnih3 in the dHPC, we observed significantly improved short-term spatial memory in a Barnes maze spatial memory task exclusively in female animals, with no observable effect of dHPC Cnih3 overexpression in males. In Cnih3-/- mice, we observed significantly attenuated short-term spatial memory only in Cnih3-/- female mice. To investigate how CNIH3 may be affecting hippocampal function in female animals, we utilized super-resolution SEQUIN imaging to quantify synaptic loci in the dHPC, biochemical analysis to assess changes in synaptic proteins, and electrophysiological recordings in slice to measure alterations in synaptic plasticity. In SEQUIN studies, we observed decreased levels of synaptic structural proteins and reduced synaptic density in dHPC samples obtained from Cnih3-/- female mice. In biochemical studies, we detected altered AMPAR subunit composition in synaptosomal samples obtained from the dHPC of Cnih3-/- female mice. In field recordings of the Schaffer Collateral (SC) circuit in the dHPC, we observed attenuated maintenance of long-term potentiation in Cnih3-/- female mice. In behavioral and in biochemical studies of Cnih3-/- mice, we observed the most robust effects of CNIH3 during the metestrus phase of the female estrous cycle. These studies identify a previously unknown role for CNIH3, as manipulation of this gene unmasks sexually dimorphic effects in spatial memory and hippocampal synaptic plasticity.
Due to the role of AMPAR-mediated mechanisms underlying drug-associated learning and memory, we next investigated how CNIH3 may interact with sex to mediate opioid-associated behaviors. We revisited the original human genomic data to probe for correlations between CNIH3 SNPs and sex on opioid dependence risk. Separating human participants by sex, we observed that women with the protective CNIH3 SNPs were less likely than men to develop opioid use disorder after prior opioid exposure. To assess potential changes in opioid-associated learning and opioid seeking behavior due to CNIH3, we first tested several paradigms of cue-associated operant opioid self-administration in mice. Oral fentanyl self-administration resulted in inconsistent and limited drug consumption during both the light and dark cycles of wild-type mice. In pilot studies testing intravenous self-administration (IVSA) of fentanyl in male and female wild-type mice, we successfully trained mice to self-administer fentanyl and induced cue-associated reinstatement following a period of extinction. Interestingly, female mice took slightly less time to acquire the IVSA task compared male animals, but additional studies are required to confirm these results. The next step of these studies will be to test the role of CNIH3 on cue-associated IVSA in male and female Cnih3-/- mice. In addition, we will also investigate whether humanized transgenic mice overexpressing the protective human CNIH3 SNPs display attenuation of cue-associated fentanyl IVSA acquisition and reinstatement.
Taken together, these studies demonstrate a role for CNIH3 in mediating sexually dimorphic hippocampal mechanisms underlying spatial memory. In addition, preliminary studies also support an interaction between CNIH3 and sex on opioid use disorder risk. We aim to continue these studies to further dissect how CNIH3 mediates opioid-associated learning and memory. The findings presented in this thesis support a role of hippocampal CNIH3 in mediating AMPAR-dependent memory processes and illustrate the importance of considering sex as a biological factor in biomedical research.
Chair and Committee
Jose A. Moron-Concepcion
Michael R. Bruchas, Joseph D. Dougherty, Steven J. Mennerick, Elliot C. Nelson,
Frye, Hannah Elizabeth, "Sex Differences in the Role of Cornichon Homolog-3 on Spatial Memory and Synaptic Plasticity" (2021). Arts & Sciences Electronic Theses and Dissertations. 2414.