It matters how you slice it

Abstract

Communications from neurons to the vascular system, termed neurovascular coupling, have been of interest since the nineteenth century (Roy & Sherrington, 1890). Although important contributions to our understanding of neurovascular coupling were made during the twentieth century, development of intracellular calcium imaging techniques, patch clamp electrophysiology, confocal or two-photon microscopy, and the release of caged-compounds have enabled us to probe much deeper into the complexities of neurovascular coupling in the past decade (Zonta et al. 2003; Filosa et al. 2004, 2006; Mulligan & MacVicar, 2004). These new technologies have been combined with ex vitro slices of brain to not only demonstrate a role for calcium signalling, vasoactive substances and ion channels in neurovascular coupling but also to determine the cellular location of each step in the process. The ‘brain slice’ preparation is significant in that it allows for cells that are normally below the brain surface to be visualized in a volume of tissue (several hundred microns thick) which can be imaged using confocal or two-photon microscopy. Thus, functioning neurons, astrocytes and arterioles forming a linear circuit can be individually monitored and/or activated. Notwithstanding the importance of the slice preparation in our understanding of neurovascular coupling, the meaning of diameter changes in arterioles in the slice has been questioned given that the arterioles are neither pressurized nor perfused. In the intact system, pressure and the flow of blood create important forces that produce a physiological response in the arteriole. Pressures in the arteriole attempt to expand the vessel and increase the radius. However, vascular smooth muscle cells in the vessel wall counteract this force by contracting to physiological pressures with the net result being constriction of the arteriole (Golding et al. 1998). Additionally, the flow of blood through the arteriole creates a force, shear stress, which acts on the vessel wall in a direction paralleling that of the flowing blood. Shear stress further constricts cerebral, but not peripheral, arterioles through a different mechanism than the one involved with pressure (Bryan et al. 2001). Thus, during physiological conditions, cerebrovascular arterioles have resting ‘tone’, i.e. the state of being partially constricted, as a background upon which dilatations or further constrictions occur. Arterioles that do not have tone will not dilate since dilatations are a passive response when smooth muscle relaxes. In the slice preparation the arterioles are neither pressurized nor perfused. Unless the arterioles are contracted by a constrictor agent, they cannot dilate. The lack of tone on the arterioles in the slice preparation has undoubtedly created discrepancies in the literature. Both arteriolar constrictions and dilatations have been reported during the process of neurovascular coupling (Zonta et al. 2003; Filosa et al. 2004, 2006; Mulligan & MacVicar, 2004). In a recent issue of The Journal of Physiology, Kim and Filosa (2012) have addressed the problem by pressurizing and perfusing a single arteriole in the brain slice preparation. A glass pipette was inserted into the proximal end of an arteriole in a brain slice and buffer from the pipette was perfused into the arteriole. A second pipette was pressed against the arteriole at the distal end to partially occlude and manipulate the outflow resistance. By adjusting the flow through the pipette and the output resistance, the desired flow and pressure combination was achieved in the arteriole. When arterioles were pressurized and perfused to shear stresses similar to those expected in the intact animal, arterioles constricted by 28% from their maximum diameter. This new method undoubtedly requires great skill and patience; however, the difficulty is offset by providing a more physiological slice preparation for the study neurovascular coupling. When astrocytes, the intermediary cells between neurons and arterioles were activated, Kim and Filosa (2012) found that arterioles dilated significantly. Ideally, we would like to study neurovascular coupling in the intact animal and in this regard significant strides are being made (Takano et al. 2006). Nevertheless, we, as scientists, must often move to in vitro preparations in order to make certain measurements and/or to control experimental conditions. Thus, biological preparations will undoubtedly continue in the ‘dish’ for the foreseeable future. However, when studying biological preparations in the dish, it is important to mimic conditions in vivo as much as possible. Therefore, as preparations, such as the brain slice, evolve we need to make efforts to more closely mimic those in vivo. In this regard, Kim & Filosa are to be commended for adding a new dimension to the brain slice preparation for studies involving neurovascular coupling. This addition of pressure and shear stress to arterioles should help to resolve discrepancies in the literature and help further our understanding of mechanisms involved with neurovascular coupling.