Optogenetics as a means of cell-type specific neuromodulation in brain plasticity.

Neuroplasticity allows the brain to organize and reorganize itself structurally and functionally based on sensory information, a well-studied phenomenon in the mammalian visual cortex. Previously, our research lab observed after a period of seven weeks of unilateral vision loss through monocular enucleation (7wME) in adult mice, that the contralateral visual cortex almost completely reactivated, by measuring the expression of the immediate early gene zif268. In the binocular region, open-eye potentiation serves this reactivation, whereas the monocular zone reactivates partially due to cross-modal recruitment by other intact sensory modalities. A key regulator of these experience-dependent modifications is the balance between excitation and inhibition, yet many distinct inhibitory subtypes populate the cortex, which challenges our understanding of the cellular and molecular basics of cortical plasticity. In this dissertation we aimed to investigate by means of cell type-specific neuromodulation strategies, how a particular inhibitory cell type regulates adult, ME-induced, cortical plasticity. We targeted somatostatin (SST)-interneurons due to their central position within the cortical circuit where they can precisely regulate the integration of incoming signals onto dendritic trees of pyramidal, excitatory neurons.

In order to achieve cell type-specific expression of neuromodulatory transgenes in the cell population and cortical area of interest, we first validated a recombinant adeno-associated viral (rAAV) vector approach to transduce neurons within the visual cortex of C57BL/6J mice. Several rAAV serotypes, each resulting in different transduction efficiencies and  tropisms, were tested for expression confined to the primary visual cortex, V1. Three promoter-sequences were tested for cell type-specific transgene expression: the cytomegalovirus (CMV) promoter, and two versions (0.4 kb and 1.3 kb) of Ca2+/Calmodulin dependent kinase a (CaMKIIa) promoter. The results indicated that rAAV2/7 resulted in a reproducible, wide expression pattern, confined within V1. CMV resulted in widespread expression in both excitatory and inhibitory cell types, whereas CaMKIIa resulted in expression predominantly in excitatory neurons with the highest excitatory specificity for CaMKIIa 0.4.

Next we investigated the contribution of SST-interneuron activity to cortical plasticity, by using the rAAV2/7 construct with a CMV promoter carrying a floxed transgene to achieve cell type-specific neuromodulation in SST-Cre transgenic mice. A stable-step function opsin (SSFO) was used to achieve optogenetic activation upon intracranial light delivery. The mutant G-protein coupled receptor hM4Di, a designer receptor exclusively activated by designer drugs (DREADD), was used to achieve chemogenetic silencing of SST-interneurons upon intraperitoneal injection of the designer drug clozapine N-oxide (CNO). Short-term activation or silencing of SST-interneurons prior to ME, resulted either in a strongly reduced, versus an increased reactivation of visual cortex after 7wME, respectively, as measured by zif268-mRNA expression. Furthermore, we combined these experiments with dark exposure (DE) pretreatment, a strategy known to affect the potential for cross-modal plasticity by altering the excitation/inhibition balance. DE combined with activating SST-interneurons resulted in the most severe lack of reactivation, whereas DE combined with silencing SST-interneurons resulted in a recovery profile comparable to non-perturbed 7wME mice. These results suggest that SST-interneuron activity and DE each address distinct mechanisms both working in the same direction to block reactivation in the ME-affected visual cortex. On the other hand, SST-interneuron silencing overcame the effects of DE, suggesting that even though DE is a non-invasive strategy to modulate the cortical response to sensory loss, directly manipulating the activity of specific neuronal subsets modulates the brain's ability to tap into its plasticity-potential in a more powerful way.

These results are a first indication that SST-interneurons are pivotal players in regulating adult cortical plasticity upon a complete, unilateral loss of a sensory modality, which is characterized by cross-modal recruitment via intact sensory modalities. Functional assessment of cross-modal processing in these recovered areas, and neuromodulation of upstream input sources of SST-interneurons, will be required to further elucidate how manipulating the neuronal cortical circuit can switch cross-modal plasticity on or off. In all, the advances in cell type-specific neuromodulation strategies offer new opportunities both for fundamental research as well as clinical therapies to help a damaged or sensory deprived brain to restore its functionality throughout life. This holds an invaluable potential to understand, and treat, a wide range of plasticity-related neurological disorders.