( ircuitry that underlies brain function....)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

ircuitry that underlies brain function. Retrograde transport of fluorescent molecules has become a standard method to delineate neural circuits and their component neurons, and new connections between known brain areas continue to be discovered with its use (Kobbert et al., 2000). Here we describe a method that uses fluorescent neuronal tracers as markers for microdissecting cell bodies by LCM (Lombardino et al., 2005). The RNA purified from these captured cells was amplified using RiboAmp HS and applied to a cDNA microarray made from songbird brain. Differentially expressed genes were discovered from each of two interdigitating projection neuron types, and the results were verified by two other gene expression techniques. This suite of techniques has permitted the molecular characterization of identified neurons as they exist within neural circuits, both in terms of their constitutive gene expression and the regulated transcription of genes induced by behavior.

Equipment and Reagents
I. STEREOTAXIC INJECTION OF TRACERS
◊ Stereotaxic apparatus for the animal subject (varies depending on species used; e.g. David Kopf Instruments)
◊ Anesthesia: Ketalar (Monarch Pharmaceuticals), Rompun (Bayer)
◊ Cholera toxin conjugated to Alexa-fluor dyes (Molecular Probes, Cat. #C-22841, C-22842)
◊ Glass micropipettes (Drummond Wiretrol, 1-5 uL Cat. #5-000-1001)
◊ Microinjection apparatus (Narishige PE-2)

II. FREEZING BRAIN AND CRYOSECTIONING
◊ Tissue embedding molds (ThermoShandon, Cat. #2219)
◊ Optimal cutting temperature (OCT) compound (Tissue-Tek, Cat. #4583)
◊ RNAlater (Sigma cat. #R0901)
◊ Precleaned glass slides (Becton Dickinson and Co., Cat. #3050)

III. LASER CAPTURE MICRODISSECTION
◊ Pixcell IIe with fluorescence (Arcturus, Ca
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