4.9 Genetic Approaches to Examine the Intact and Living Brain
4.9 Genetic Approaches to Examine the Intact and Living Brain
Labelling the Living Brain
Owing to the complexity of the mammalian brain, it has remained a major challenge to decipher the patterns of connectivity made onto and by newborn neurons as they integrate into circuits of the adult brain. With major advances in both molecular genetics and light microscopy, our ability to query not only neuronal morphologies but also the molecular and cellular composition of individual neurons and their associated synaptic networks has become possible.
Arguably, one of the most influential contributions to contemporary neuroscience has been the use of fluorescent proteins (FPs) and their targeted expression in living neurons of the mammalian brain tissue. The wide array of FPs available provides an ever-expanding toolbox of vital reporters and gene expression tags. Applications for these proteins range from vital reporters expressed throughout the cytoplasm to subcellular protein fusion tags, which together can be used to monitor the process of circuit integration in vivo using both electrophysiological methods and fluorescent imaging.
Beyond merely marking cells for identification, a number of other methods have been developed to exploit the vital properties of FPs to investigate neuronal properties. For example, super-ecliptic pHluorin fluoresces at neutral pH but is quenched at acidic pH, and thus can be used to monitor the trafficking and exchange of intracellular compartments within neurons. This variant allows direct imaging of membrane dynamics, exocytosis and endocytosis of synaptic receptors, and neurotransmitter release in vitro and in vivo. More recently, a new method termed GFP reconstitution across synaptic partners (GRASP) shows promise for revealing synaptic interactions between contacting neurons. By tethering split GFP fragments to separate pre- and post-synaptic proteins, the reconstitution of GFP fluorescence can be observed when genetically targeted cells form synaptic pairs. Although this technology has been successfully applied to reveal invertebrate synapses, it has yet to be demonstrated in rodents (Gordon and Scott, 2009).
The range of FP reporters for visualizing neuronal morphologies, cellular dynamics, and synapse function continue to expand. However, perhaps the single most limiting factor for using FPs in neuroscience is our incomplete knowledge of neuronal gene regulation. Often transgenic reporters fail to recapitulate endogenous patterns of gene expression or such patterns are too broad to identify neuronal subtypes with cellular precision.
Trans-Synaptic Circuit Tracing
A major goal toward understanding mechanisms of neuronal development, synapse formation, and circuit wiring has been to elucidate nodes and patterns of synaptic connectivity. A creative angle to address this challenge has been the incorporation of genes encoding FPs and FP-fusion proteins into neurotropic viral vectors, which show the innate ability to infec