The cerebral cortex is structured into functional areas and six horizontal layers. Within the cortex, different subtypes of neurons have distinct projections patterns and functions. To better understand how information is processed, and how the pyramidal cell output signal is formed, we need specific markers to investigate the function of more homogenous subpopulations of interneurons. In this study we used Chrna2 to specifically label a subset of Martinotti cells. With rabies viral tracers, combined with immunohistochemistry, we investigated the presynaptic connectivity of these cells, in different cortical areas. Further, we investigated their functional role in the primary motor cortex using chemogenetic neuronal modulation during motor learning and execution. With recordings of calcium signals, we investigated the role of increased Chrna2 Martinotti cell activity on pyramidal cell assemblies in cortical layer 5 during execution of a single pellet prehension task in freely moving animals using small portable widefield microscopes, so called miniscopes. We found that layer 5 Martinotti cells, in different cortical areas, have distinct cortical input patterns and receive direct input from different thalamic and cholinergic nuclei. Increased activity of Chrna2 Martinotti cells improved motor performance in the single pellet prehension task, and decreased plasticity of pyramidal cell assemblies. We will discuss these findings and the role of Chrna2 Martinotti cells in preserving and refining acquired skills. Further studies are needed to investigate if this function also applies to other cortical areas and other forms of learning.