Ultrathin CNS slices: Enhanced visualization of neuron structure for electrophysiology and imaging
L. Song, M. Sawchuk, & S. Hochman. Dept. Physiology, Univ. of Manitoba. Winnipeg, MB, Canada R3E 0W3. E-mail: firstname.lastname@example.org) Electrophysiological studies using patch clamp recordings in CNS slice preparations involve either ‘blind’ or visual targeting strategies. Recordings from visually-identified neurons generally require specialized upright microscopes equipped with Nomarski optics (DIC) (e.g. Konnerth et al Pflugers Arch.
414:600, 1989). Further image optimization to observe neuronal processes employ video-enhanced infrared wavelength illumination (e.g. Stuart et al Pflugers Arch. 423:511, 1993). Though powerful, these approaches require specialized, expensive equipment. Image: Live (green) and dead (red) cell assay showing abundance of living cells throughout slice in a P8 rat (scale bar = 100µm)
Since the ability to resolve detailed cellular features is generally limited to 40-50 mm from the slice surface, we have developed an alternate strategy to visualize neurons using a semi-transparent ‘ultrathin’ slice preparation (20-50 mm) visualized on an inverted microscope equipped with Hoffman modulation optics.
Isolated cerebellum, hippocampus or spinal cord from neonatal rats (P1- P14) were embedded in AGAR (2.5% w/v) then sectioned with a Leica VT1000E vibrating blade microtome. After incubation at 32°C, slices were fixed to the bottom of the recording chamber and maintained at room temperature.
The enhanced transparency due to reduced slice thickness permitted superior optical resolution of dendritic and axonal processes and cells were visible throughout the slice thickness. Image: Live cell indicator reveals abundance of living cells throughout slice (P1 rat) (scale bar = 50 µm)
ve/dead cell staining revealed that many cells remained viable for imaging and electrophysiological experimentation. In all CNS regions examined, neurons were easily identified for successful patch recordings.
While not yet tested, use of an inverted microscope should permit high numerical aperture oil-immersion objectives to be used for simultaneous imaging experiments and the reduced slice thickness would hasten drug equilibration and washout times. Supported by the Canadian Neuroscience Network.
Image: Immunostaining of CAA1 glial cells with GFAP (scale bar = 50 µm) (for slice culture procedures, see Parsley, C.P. et al. Society for Neurosci. Abstr. 230.5, 1996)