Two photon imaging of calcium responses in murine Purkinje neurons
Purkinje neurons (PNs) in vertebrate cerebella are required for motor learning and motor co-ordination. Dysregulation of intracellular calcium signaling in PNs can be a causative pathogenic factor for Spino-cerebellar Ataxias. Our protocol describes a method for measuring Ca2+ transients in PNs from adult mice. This protocol can measure neuronal excitability and agonist mediated Ca2+ signaling in cerebellar slices. As maintaining healthy PNs in cerebellar slices and recording their Ca2+ transients are challenging, we provide detailed descriptions and problem-solving tips.
Steps to reproduce
1) Parasagittal cerebellar slices of about 250 μm are obtained from Purkinje specific GCaMP6f expressing mice of 17 weeks old using vibratome (Leica, VT1200) following isoflurane anesthesia and decapitation. The slices are sectioned in ice-cold cutting solution containing the following (in mM): 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 75 sucrose and 10 glucose, bubbled with 95% O2 and 5% CO2. 2) The slices are immediately placed in artificial CSF (ACSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3 and 20 glucose bubbled with 95% O2 and 5% CO2. For recovery, slices are incubated at 34°C for 45mins and then kept at room temperature (approx. 22 °C) till imaging. 3) For two photon calcium imaging, acute slices are placed in a recording chamber under a custom built two-photon laser scanning microscope equipped with a laser (Coherent Chameleon Ultra II). Microscopes are equipped with 20X water-immersion objective (Olympus XLUMPLFLN20XW, 1.0 NA). 4) Purkinje neurons are stimulated with 75 mM KCl or 200 µM DHPG (Tocris Cat# 0805). An mGluR1 antagonist, CPCCOEt (Tocris Cat# 1028) of 200 μM is applied to test for the specificity of mGluR1 activation. 5) Images are recorded with frame size of 512 x 512 pixels using Scan Image 3.8 and further analysed using ImageJ. 6)For quantifying the changes in fluorescence, region of interest (ROI) are drawn around each cell and the fluorescence intensity at each time points is determined. ΔF/F [ΔF/F=(Ft-Fbasal)/Fbasal] is measured for each time point, where Ft is fluorescence a particular time point and Fbasal is the fluorescence of the cell when starting the experiment. 7)Data are plotted using the Origin 8.0 software. Peak values of ΔF/F and area under the curve are obtained for every cell and the data are plotted as histogram. 8)Rate of Ca2+ entry during KCl stimulation are calculated by computing the average rate of change in fluorescence intensity (ΔF) between the time at which Fmax (ΔF/F) occurs and 11th sec time points and expressed as ΔF/t.