In vivo two-photon fluorescence imaging studies of cerebellar-dependent learning and memory. Classical or Pavlovian conditioning is one of the simplest and earliest known forms of associative memory. A modern version of such conditioning that is suitable for use in mice and that depends critically on cerebellar function is classical eyeblink conditioning, in which a subject is trained to blink in response to a conditioning stimulus such as an audible tone. Many theories of how this cerebellar-dependent form of learning occurs focus on cerebellar Purkinje neurons, which exhibit highly regular anatomical patterns of neural connections. The Schnitzer lab has shown that they can image up to ~50 Purkinje cells simultaneously in live mice using in vivo two-photon fluorescence imaging. By combining in vivo imaging and electrophysiological techniques with behavioral, computational, and trans-synaptic circuit tracing approaches, the lab seeks to understand the neural circuit dynamics in the cerebellar cortex that underlie learning, memory, and forgetting.

Fiber optic fluorescence microendoscopy. The Schnitzer group has recently invented two forms of fiber optic fluorescence imaging, respectively termed one- and two-photon fluorescence microendoscopy, which enable minimally invasive in vivo imaging of cells in deep (brain) areas that have been inaccessible to conventional microscopy. Such areas that the group has studied include the hippocampus, thalamus, and inner ear. The group has developed the capability for repeated microendoscopy imaging of hippocampal neurons and dendrites over the long-term using a chronic mouse preparation. This preparation has proved highly applicable for extended imaging studies over the progression of brain disease in animal model systems. Such ability to image cells deep within the live mammalian brain also promises to permit studies of how cellular properties are impacted by environment, training, or life experience. Moreover, the Schnitzer group has created portable, miniaturized microendoscopy devices based on flexible fiber optics for use in freely moving mice. The Schnitzer group now seeks to develop and apply these microendoscopy techniques to applications in both basic neurobiology and clinical settings, and has begun to examine human nervous tissues. For example, microendoscopy has recently provided the first images of sarcomeres in live human subjects, and we are now working with collaborators to bring this imaging capability into the neurology clinic for applications regarding neuromuscular disorders.

Massively parallel brain imaging in live fruit flies. Because the study of neural circuits remains deeply limited by a paucity of data, we need massively parallel approaches to brain imaging that will raise data acquisition rates by over two orders of magnitude. High-throughput technologies have already revolutionized certain areas of biology such as genomics and proteomics, but neuroscience has yet to experience a growth spurt of comparable magnitude. We are constructing instrumentation allowing the brain volumes of ~100 alert flies to be imaged simultaneously by two-photon fluorescence microscopy. We have chosen the fruit fly, Drosophila melanogaster, because of its small brain, its sophisticated behavioral repertoire, the large number of strains with genetically targeted alterations to brain circuitry, the utility of fluorescence imaging of neural activity in this species, and the importance of the fly as a model for the study of many brain diseases. Massively parallel brain imaging will open new resesarch avenues: 1) The ability to track neural dynamics across the brains of large numbers of normal flies and those with genetically induced neural circuit perturbations will transform our understanding of how neural circuits produce animal behavior; 2) The now prominent role of the fruit fly as a model system for the study of developmental disorders, neurodegenerative diseases, and addiction implies we will gain significant medical insights into devastating conditions; 3) Our technology will have important applications to drug screening, allowing the cellular effects of new compounds to be assessed rapidly in vivo; 4) The ability to perform high-throughput time-lapse imaging of cellular events during the maturation of fly embryos will greatly benefit developmental neurobiology. Applications of our technology will also be plentiful in other model organisms such as nematodes and zebrafish, impacting multiple areas of biomedicine.