Neuronal circuits consist of diverse cell types, and there is increasing evidence that each cell type often displays stereotyped connectivity and carries out specialized functions. To understand the organization and operation of neuronal circuits, it is therefore necessary to be able to visualize the structure and connectivity of different cell types at high resolution and to manipulate the function of specific cell types with precision. Of particular relevance are the GABAergic inhibitory circuits in the neocortex. GABAergic inhibition is crucial in all aspects of neural circuit operation in the cortex and is mediated by diverse interneuron cell types. Because different cell types are highly intermingled and even neighboring neurons differ in their connectivity and function 
, such heterogeneity and complexity has been difficult to penetrate by conventional anatomical and physiological techniques. For example, there is increasing evidence that GABAergic synapses are structurally modified by sensory experience and neural activity 
, potentially leading to significant reconfiguration of neural circuits. However, there has been no study that examines the structural dynamics of defined classes of cortical inhibitory neurons and synapses in the intact brain. This gap in knowledge is largely due to the heterogeneity of cortical GABAergic cell types and the lack of a high resolution labeling method.
Genetic strategies can significantly contribute to studying GABAergic circuits and neural circuits in general because they tap into the intrinsic gene regulatory mechanisms that generate and maintain the cellular diversity of the nervous system 
. Because different cell types often display distinct gene expression profiles 
, transcriptional promoters provide genetic access to visualize and manipulate different cell types. Gene knockin and transgenesis using bacterial artificial chromosomes (BAC; 
) are two useful techniques to introduce exogenous genes into a cell type of interest defined by the expression of an endogenous gene. In particular, Cre/loxP recombination-regulated gene expression is an efficient and powerful approach to systematically label and manipulate defined cell types 
. This binary gene expression strategy involves the combination of two mouse strains: a “driver” strain expressing Cre-recombinase in specific cell types and/or brain regions, and an “indicator” strain capable of expressing a gene(s) of interest upon Cre/loxP recombination. To date, an increasing number of cell-type restricted driver lines have been generated 
. However, the current implementation of this strategy suffers three major shortcomings. First, the spatial and temporal expression pattern of any one single gene may not be ideal to manipulate a cell type at a restricted developmental stage and brain region. Second, with only a few exceptions, the expression level of fluorescent markers introduced by either knockin or BAC trangenics are often insufficient to label fine neuronal structures such as neuronal axons and synapses; expression levels from available indicator lines are orders of magnitude lower than what is necessary for high resolution imaging in vivo. Third, mouse genetic engineering, especially when involving multiple strains, is time consuming and costly.
Viral-mediated gene delivery represents an alternative and powerful strategy to label and manipulate neurons in the mammalian brain. Because of their multi-copy transfection of a single neuron and the use of strong and ubiquitous transcription promoters, viral-mediated delivery can often achieve high-level gene expression and thus bright labeling of fine structures such as neuronal synapses 
. In addition, viral transfection can be targeted to specific brain regions and developmental stages by stereotactic injection 
. Furthermore, neurotrophic viruses suitable for longitudinal studies have been well characterized and can now be efficiently engineered at low cost 
. However, a major drawback of viral-mediated gene delivery is the lack of cell-type specificity - currently there is no general strategy to restrict viral-mediated gene expression to defined cell types for a prolonged time period. Here we describe a method that combines Cre-recombinase knockin mice and Cre-activated adeno-associated viral vectors to achieve high-level, stable, and cell-type specific gene expression. This method is simple, highly efficient, and allows chronic live imaging of defined classes of synapses in vivo and light activation of neuronal spiking. With the establishment of increasing number of Cre knockin and transgenic lines (Cre drivers), this method represents a general strategy to systematically visualize and manipulate specific cell types in-vivo.
Using this method to label a specific class of inhibitory cell, the neocortical parvalbumin (Pv) inhibitory interneuron, we were able to chronically image, for the first time, the fine axonal structures and dynamics of Pv inhibitory axon boutons in vivo. We found that although the majority of putative Pv presynaptic boutons were stable in young adult mice, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex.