RESUMO
The cerebellum stores associative motor memories essential for properly timed movement; however, the mechanisms by which these memories form and are acted upon remain unclear. To determine how cerebellar activity relates to movement and motor learning, we used optogenetics to manipulate spontaneously firing Purkinje neurons (PNs) in mouse simplex lobe. Using high-speed videography and motion tracking, we found that altering PN activity produced rapid forelimb movement. PN inhibition drove movements time-locked to stimulus onset, whereas PN excitation drove delayed movements time-locked to stimulus offset. Pairing either PN inhibition or excitation with sensory stimuli triggered the formation of robust, associative motor memories; however, PN excitation led to learned movements whose timing more closely matched training intervals. These findings implicate inhibition of PNs as a teaching signal, consistent with a model whereby learning leads first to reductions in PN firing that subsequently instruct circuit changes in the cerebellar nucleus.
Assuntos
Aprendizagem por Associação/fisiologia , Membro Anterior/fisiologia , Movimento/fisiologia , Plasticidade Neuronal/fisiologia , Células de Purkinje/fisiologia , Memória Espacial/fisiologia , Animais , Channelrhodopsins , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fibras Nervosas/fisiologiaRESUMO
BACKGROUND: One of the limitations when establishing an electrophysiology setup, particularly in low resource settings, is the high cost of microscopes. The average cost for a microscope equipped with the optics for infrared (IR) contrast or microfluorometry is $40,000. We hypothesized that optical elements and features included in commercial microscopes are not necessary to IR video-visualize neurons or for microfluorometry. NEW METHOD: We present instructions for building a low-cost epifluorescence upright microscope suitable for visualized patch-clamp recording and fluorescence detection using mostly catalog-available parts. RESULTS: This microscope supports applications such as visualized whole-cell recording using IR oblique illumination (IR-OI), or more complex applications such as microfluorometry using a photodiode. In both IR-OI and fluorescence, actual resolution measured with 2-µm latex beads is close to theoretical resolution. The lack of movable parts to switch configurations ensures stability when doing intracellular recording. COMPARISON WITH EXISTING METHODS: The low cost is a significant advantage of this microscope compared to existent custom-built microscopes. The cost of the simplest configuration with IR-OI is â¼$2000, whereas the cost of the configuration with epifluorescence is â¼$5000. Since this design does not use pieces discarded from commercial microscopes, it is completely reproducible. CONCLUSIONS: We suggest that this microscope is a viable alternative for doing in vitro electrophysiology and microfluorometry in low-resource settings. Characteristics such as an open box design, easy assembly, and low-cost make this microscope a useful instrument for science education and teaching for topics such as optics, biology, neuroscience, and for scientific "hands-on" workshops.