An immunohistochemical analysis of fluoxetine effects in the parvalbumin interneuron network

Parvalbumin (PV) interneurons are GABAergic inhibitory neurons that shape neuronal network activity and plasticity. They are involved in both developmental and adult plasticity and have recently been divided into subpopulations that differ in birthdate, intrinsic properties and are involved in different types of learning; while late born PV neurons, expressing low levels of PV, are required for the acquisition of new information, early born PV neurons, expressing high levels of PV, are involved in the consolidation of the information. PV cells can be enwrapped with perineuronal nets (PNNs), an extracellular matrix structure that stabilizes synapses and indicates a mature state of the cell. The development of PNNs correlates with the closure of critical period of plasticity in development, and the enzymatic removal in adulthood can reopen those periods. Similarly, antidepressants like fluoxetine have been proven to reopen critical periods of plasticity in adulthood (iPlasticity) and decrease PNN structures in PV cells. However, whether the effect of fluoxetine is restricted to a subpopulation of PV interneurons is unknown. In addition, no previous studies have yet investigated the maturity state of the PV subpopulation by analyzing its PNN structures. In this thesis we aimed to elucidate differences in the maturity state of the subpopulations and the fluoxetine effect in those. To do that, we treated a cohort of adult mice with a chronic fluoxetine treatment previously reported to be capable of the reopening of critical periods. Following, we performed an immunohistochemistry analysis to detect PV and PNN levels in the CA3b hippocampal area. In addition, our mice line expressed TdTomato (TdT) in PV cells which allowed a more sensitive detection of PV neurons. After imaging the slices with a confocal microscope, we analyzed the PV and PNN intensity both by manual counting and with a semi-automatic macro script in ImageJ software that we developed and validated. The PV intensity of control mice was used to divide the cells in two groups; low PV and high PV expressing cells. PNNs in those subpopulations in both the control and fluoxetine treated group were analyzed and statistically compared. The low PV subpopulation showed a significantly low PNN intensity compared to the high PV subpopulation, indication a plastic or immature low PV subpopulation and a mature or consolidated high PV subpopulation. Interestingly, fluoxetine selectively decreased the PNN structures in the high PV subpopulation, by bringing the PNN intensity to comparable levels found in the low PV network. No effect of fluoxetine in the low PV network was detected. Fluoxetine induced a change towards a plastic state in the network believed to be involved in memory consolidation by decreasing its PNNs structures. This discovery gives new insights on the understanding of antidepressant plastic actions, suggesting that a chance for strong memories to change could be facilitated with the drug, and explain the antidepressant’s effects when combined with psychotherapy. However, supplementary experiments to compare and define PV subpopulations and a confirmation of the selective effect of fluoxetine are needed to confirm the preliminary hypothesis suggested by our data