The second line carries a knock-in floxed Vgat allele Vgat fx Our genotyping procedures for all of these alleles have been described before 32 , 38 , We bred the mice using standard timed pregnancies, and we designated noon on the day a vaginal plug was detected as embryonic day E 0. Mice of both sexes were studied. An gauge syringe was used to pipette the solution up and down and dissolve any remaining tamoxifen particles.
The pups were allowed to rest in a separate cage to prevent the mother from licking out the tamoxifen. Perfusion and tissue fixation were performed as previously described Briefly, mice were anesthetized by intraperitoneal injection with Avertin 2,2,2-Tribromoethanol, Sigma-Aldrich Cat T4.
Cardiac perfusion was performed with 0. Immunohistochemistry procedures on free-floating frozen tissue sections were described previously 32 , , , After staining, the tissue sections were placed on electrostatically coated slides and allowed to dry.
The integrity of the cerebellar circuitry was checked by determining the expression patterns of several synaptic and cell type-specific markers. Purkinje cells were marked with anti-calbindin ,; Cat. Purkinje cells and molecular layer interneurons were marked with rabbit polyclonal anti-parvalbumin ; Swant, Marly, Switzerland; Cat. Excitatory interneurons were marked by rabbit polyclonal anti-calretinin ; Swant, Marly, Switzerland; Cat. AB Neuronal processes were also labeled with various markers.
Mouse monoclonal anti-neurofilament heavy NFH; also called anti-SMI; ; Covance, Princeton, NJ immunolabeled the soma, dendrites, and axons of adult Purkinje cells, and the axons and terminals of basket cells. APC was also used to label basket cell axons and pinceaux terminals. However, the overall analysis of lineage marked basket cells and stellate cells, which included a quantitative assessment of marking reliability and efficiency, structure of the marked cells, cell location, gross cerebellar anatomy, and co-expression with cell type specific markers, was conducted using 9 basket cell control mice, 6 basket cell mutant mice, 22 stellate cell control mice, and 25 stellate cell mutant mice.
For fluorescence immunostaining, we used Alexa, , and secondary goat anti-mouse and anti-rabbit antibodies , , and , respectively; Molecular Probes Inc. M2 microscope or on a Zeiss Axio Zoom. Images of tissue sections were acquired and analyzed using either Zeiss AxioVision software release 4. After imaging the tissue, the raw data were imported into Adobe Photoshop CS6 and corrected for brightness and contrast levels.
The schematics were drawn in Adobe Illustrator CS6. Images of the molecular layer were acquired with 20x magnification using Zeiss Axioimager microscope, in z-stack and ApoTome mode. Using the Fiji software for analysis, the background was subtracted using the built-in rolling ball method. The same settings were used for control and mutant tissue. The molecular layer was divided dorso-ventrally into three levels, and the levels were saved as regions of interest ROI.
The area and number of puncta in each level was measured using the built-in Analyze Particles function in Fiji and the density of VGAT-positive puncta for each level was calculated. Multiple lobules were analyzed from each image. After staining, the tissue sections were dehydrated in a series of ethanol, cleared with xylene, and then mounted onto electrostatically coated glass slides with cytoseal.
The tissue sections were allowed to dry before imaging. The tissues were immunostained with mouse monoclonal or rabbit polyclonal anti-calbindin ,; Cat. Measurements for each mouse were averaged and the numbers computed from each genotype were pooled and averaged again to obtain the mean molecular layer thickness.
Mice were anesthetized with Avertin and perfused with 0. Brains were harvested and cerebella were sagittally sectioned using a rodent brain matrix while immersed in fixative. The sections were transferred with fixative to a dish and the position of the molecular layer was noted.
Procedures were performed in a Ted Pella Bio Wave microwave oven with vacuum attachment. The images were imported into ImageJ where a smoothing function was applied and then the data were assembled in Adobe Photoshop CS6. Surgery for awake recordings was performed as detailed in White et al.
Sterile surgery techniques were followed. A custom-made headplate was first attached to the Bregma region using Metabond. Drilling was stopped before the skull was completely penetrated. Another craniotomy was performed on the right side of the midline. Once the craniotomy was complete, the hole was covered in triple antibiotic ointment to prepare for the installation of the recording chamber. One end of the straw was dipped in Metabond and carefully placed on top of the craniotomy.
Once the adhesive was dry, dental cement A-M Systems dental cement powder and solvent was applied on the outer edge of the straw to fill in holes and to further secure the chamber. After the dental cement had dried, a fresh layer was applied around the straw and the Bregma region where the headplate was attached.
After the layer dried, a final layer was applied throughout the site of surgery, including the screw, the top and underside of the headplate, and along the edges of the straw. The skin surrounding the site of the surgery was fixed to the dental cement using 3M Vetbond NC after the cement has completely dried.
While the last layer of dental cement was drying, 0.
After the surgery, the mouse was placed in a warming box V, Peco Services Ltd. Once the mouse was awake and mobile, it was returned to the home cage. On the third day, training on the running wheel was started. After each recording session, the solution was removed with a cotton tip or by aspiration with a micropipette and fresh antibiotic ointment applied.
Single-unit extracellular recordings were performed as described previously 46 , 47 , During the recordings, the reference electrode tip was immersed in the saline chamber. Purkinje cells were identified by the unique presence of two types of action potentials: simple spikes, which are intrinsically generated, and complex spikes, which are generated by climbing fiber input.
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Cerebellum Conference GRC
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Cerebellar Mechanisms for Movement, Learning and Cognition in Health and Disease
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