A vibrating microtome attachment for cutting brain slice preparations at reproducible compound angles relative to the midline

Abstract

Slice preparations isolate functional networks, permitting single unit recording under visual control, and the use of fluorescent indicators. Circuits of interest often lie at a tilt in both the rostrocaudal and ventrodorsal axis, thus exposing circuits of interest at the cut surface of a slice would require a device for tilting a preparation along two orthogonal axes relative to the blade. Such a device, designed to be used in conjunction with a vibrating microtome, permitting the isolation of slice preparations at reproducible angles, is described here. Because the two orthogonal axes of tilt can be independently and continuously adjusted, it is possible to use this device to successively refine tilt parameters from preparation to preparation for optimal exposure of circuits of interest, facilitating the development of new slice preparations. Its use in cutting a thick medullary slab preparation, isolated from the neonate rat, which exposes respiratory networks at the cut surface is described.

Functional Anatomical Evidence for Respiratory Rhythmogenic Function of Endogenous Bursters in Rat Medulla

“…A tilted sagittal slab preparation was cut at a compound angle of 2.2° rostrocaudal, and 12° ventrodorsal tilt, using a device developed for this purpose (Mellen, 2008). Level of section was determined as a ratio of brainstem width: respiratory networks were exposed by cutting at 0.34 of the distance from the midline to the lateral edge of the preparation; the blind side was cut at 0.7-0.8 of the full width of the brainstem.…”

Synchronization of inspiratory burst onset along the ventral respiratory column in the neonate mouse is mediated by electrotonic coupling

“…The hindbrain was transected at the level of the sensory portion of the trigeminal nerve, just rostral to the facial nucleus (VIIn). The preparation was then mounted with the dorsal side down on an attachment permitting sectioning at compound angles [ 57 ], using the basilar artery to reproducibly align the preparation along the midline of the chuck. A sagittal section was cut at the lateral edge of the VIIn, visible at the surface of the preparation, at 17.7° ventrodorsal tilt and 3.7° rostrocaudal tilt relative to the midline, to expose the VRC along its major axis.…”

Semi-automated region of interest generation for the analysis of optically recorded neuronal activity

“…In accordance with methods approved by the Institutional Animal Care and Use Committee, neonate Sprague-Dawley rat pups (P0–P4) were anesthetized with isoflurane, and, according to methods described elsewhere (Barnes et al, 2007; Mellen, 2008), the neuraxis was isolated, in chilled aCSF made up of (in mM) 128.0 NaCl, 3.0 KCl, 1.5 CaCl 2 , 1.0 MgSO 4 , 21.0 NaHCO 3 , 0.5 NaH 2 PO 4 , and 30.0 glucose, equilibrated with 95% O 2 -5% CO 2 , and a thick sagittal slab was cut (Mellen, 2008), exposing respiratory networks at the surface, with the highest concentration of respiratory neurons ventral, dorsal, and caudal to the facial nucleus (VIIn), and approximately 500 µm caudally in the pre-Bötzinger Complex (preBötC). The preparation was then incubated for 2 hours in an aerated solution containing the high affinity cell-permeant Ca 2+ indicator fluo-4 AM (50 µg, K d = 350 nM; Invitrogen), or the lower affinity fluo-8L (50 µg, K d = 1.86 µM, ABD Bioquest), solubilized in 25 µL of the surfactant pluronic F-127 (2g/10 ml DMSO; Invitrogen), and diluted in 750 µL aCSF for a final concentration of 60 µM.…”

“…The specific problem of fitting ROIs to somatic Ca 2+ signals is a special case of the more general problem of image change detection (Radke et al, 2005), in which changes in fluorescence as a function of time in an otherwise static image series are targeted. We apply our methods to the activity of medullary networks responsible for respiratory rhythm generation, recorded in vitro from a neonate rat tilted slab preparation (Barnes et al, 2007; Mellen, 2008). This preparation provides access to medullary networks involved in respiratory rhythm generation, as well as phrenic motor output, recorded from ventral roots C1–C4, which serves as the criterion signal for the identification of optically recorded respiratory neurons.…”