Glutamate Transporters in Caenorhabditis Elegans: the Implications of Transporter Deletions on Behavior, Learning, Memory, and Addiction

Glutamate is a critical neurotransmitter involved in excitatory synaptic transmission, cognition, memory, and learning. Although much research has been conducted to examine glutamatergic signaling pathways, the functional role of glutamate transporters (GLTs) in behavior, learning, and memory remains largely unexplored in Caenorhabditis elegans. We tested wildtype C. elegans and C. elegans with GLT deletions on a battery of behavioral tests including analysis of spontaneous locomotion, response to a mechanosensory stimulus, response to an aversive chemical, and chemotaxis in response to a chemoattractant and a chemorepellent to examine how different transporters affect basic behaviors. Thus far, we have examined C. elegans with deletions of GLT-3 located in canal cells, GLT-1 located in muscle cells, GLT-4 located on presynaptic neurons, and GLT-5 located within the pharyngeal region. We then conducted associative and non-associative learning paradigms. Compared to wildtypes, GLT-3; GLT-1 knockouts were deficient in all basic behavioral tasks besides chemotaxis in response to a chemorepellent, GLT-4 knockouts were deficient in all basic behavioral tasks besides response to the smell of an aversive chemical, and GLT-5 knockouts were deficient in response to the smell of an aversive chemical and chemotaxis in response to a chemoattractant, however they exhibited a hyperactive chemotactic response to a chemorepellent. All mutants displayed associative learning, however GLT-4 and GLT-5 mutants had less of an extreme response to the aversive chemical prior to conditioning and expressed less association to that same chemical in the post-learning period. Mutants with GLT-3; GLT-1 or GLT-5 deletions do not display normal habituation, a type of non-associative learning, while GLT-4 mutants were not analyzed in this paradigm due to an abnormal initial response. We next examined exposure to the addictive substances nicotine and ethanol as a type of memory. We evaluated the ability for different mutant strains to express drug-seeking behavior and initial preference for these drugs. We then tested whether the mutants would associate the drugs with an aversive chemical as we demonstrated in wildtypes. Compared to wildtypes, all mutant strains expressed decreased chemoattraction toward ethanol but unaltered chemoattraction toward nicotine. Furthermore, all mutants exhibited associative learning after ethanol or nicotine conditioning but expressed less association in the post-learning period than the wildtype strain. Our data suggest that basic behaviors may rely on different glutamate transporters than learning and memory do. These differences may be attributable to differences in transporter localization

The progestin receptor interactome in the female mouse hypothalamus: Interactions with synaptic proteins are isoform specific and ligand dependent

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease