Techniques
Cellular Neurophysiology
We use whole-cell patch-clamp recording, and other core techniques of in vitro electrophysiology, to study the cellular and synaptic physiology of neurons in acute brain slices. We frequently combine electrophysiological approaches with quantitative optical imaging, optogenetics, viral transformation, and immunohistochemistry as further described below.
Multiphoton Microscopy
Using a combination of multi-photon-excitable calcium sensitive and calcium-insensitive dyes loaded into a neuron through the patch pipette, we can simultaneously record electrophysiological properties of neurons while measuring changes in sub-cellular calcium concentration with high spatial and temporal resolution.
The sensitivity of this method is great enough to facilitate measurement of calcium transients produced in individual dendritic spines in response to synaptically released transmitter (below). We also use a similar approach to measure activity induced calcium influx in distal dendrites as produced by back propagating action potentials.
Optogenetics
Channelrhodopsin-2 (ChR2) is a light-activated ion channel. Selective expression of ChR2 (see section on viral transformation below) can facilitate very selective stimulation of specific cell types (or their axons). Conversely, we can drive cell type specific expression of inhibitory opsins, such as halorhodopsin, in order to facilitate experiments the require selective silencing of specific types of neurons or of afferent inputs from specific locations.
High throughput calcium imaging
GCaMP is a calcium indicator which can also be selectively delivered to specific cell types using viral transformation. We often use cell type specific expression of GCaMP to screen specific types of cells for responsiveness to bath applied agents. This approach is higher throughput that single cell recording because multiple GCaMP positive neurons can be imaged simultaneously while bath conditions are changed. In some cases, responsive neurons are identified using GCaMP and then further analyzed using techniques for whole-cell recording.
Viral transformation
Our laboratory frequently performs experiments in tissue from transgenic animals. In some cases animals with selective expression of Cre recombinase are crossed with floxed reporter animals (animals a loxP -flanked STOP cassette preventing transcription of a CAG promoter driven flurophore) to create an animal model with cell type specific expression of a specific fluorophore. In other cases (some of which are noted above) AAV is used to deliver fluorophores or other functional proteins (e.g. opsins, or calcium indicators) in a brain region and/or cell type specific manner. We also sometimes use similar approaches to facilitate cell-type specific knockout of specific endogenous proteins.
Immunohistochemistry and epifluoresence microscopy
Our laboratory frequently uses immunohistochemical and other histological techniques to anatomically and chemically characterize neurons. Individual neurons can be filled with fluorophores or other makers (such as biocytin) delivered through the patch pipette during electrophysiological recording. Post-hoc anatomical and immunohistochemical characterization of neurons produces data that can be combined with core intrinsic physiological properties (obtained during electrophysiological experiments) to create a robust phenotype.
