Assaying activity-dependent arteriole and capillary responses in brain slices

Abstract

Significance: Neurovascular coupling (NVC) is the process that increases cerebral blood flow in response to neuronal activity. NVC is orchestrated by signaling between neurons, glia, and vascular cells. Elucidating the mechanisms underlying NVC at different vascular segments and in different brain regions is imperative for understanding of brain function and mechanisms of dysfunction. Aim: Our goal is to describe a protocol for concurrently monitoring stimulation-evoked neuronal activity and resultant vascular responses in acute brain slices. Approach: We describe a step-by-step protocol that allows the study of endogenous NVC mechanisms engaged by neuronal activity in a controlled, reduced preparation. Results: This ex vivo NVC assay allows researchers to disentangle the mechanisms regulating the contractile responses of different vascular segments in response to neuronal firing independent of flow and pressure mediated effects from connected vessels. It also enables easy pharmacological manipulations in a simplified, reduced system and can be combined with ${\mathrm{Ca}}^{2+}$ imaging or broader electrophysiology techniques to obtain multimodal data during NVC. Conclusions: The ex vivo NVC assay will facilitate investigations of cellular and molecular mechanisms that give rise to NVC and should serve as a valuable complement to in vivo imaging methods.

Keywords: acute brain slice; arteriole; capillary; ex vivo; neurovascular coupling.

Fig. 1

Examples of blood vessels in cortical slices. (a), (b). Examples of arterioles demonstrating the larger vessel diameter and thick abluminal walls (marked with brackets) where the VSMC layer is visible. Note the size of the vessels compared with the red blood cells visible within the lumen. (c). A transition zone between arteriole and capillary depicting the smaller vessel diameter and thinner wall compared with arterioles. Although the VSMC layer is not clearly visible, the abluminal wall is thicker than that expected of capillaries. (d), (e). Example capillaries depicting their narrower lumen and thin endothelial wall. Pericytes can often be observed as individual cell bodies attached to the outer wall (arrowheads). (f) Venules have a large diameter and thin endothelial walls with sparse VSMCs. Scale bars: $10\mu \mathrm{m}$.

Fig. 2

Construction of custom slice storage chambers. These custom handmade chambers contain a raised nylon mesh platform on which the slices sit, thus exposing the tissue to gassed solutions evenly on both sides. These can be easily prepared with a beaker or similar container, a plastic funnel, and a plastic petri dish. (a) Select a funnel that is narrower than the beaker by about 1 cm. Cut the top two-thirds of the funnel away from the stem and punch several holes into the wall of this segment. Attach it to the bottom of the beaker using hot glue. (b) Select a Petri dish that is roughly the same circumference as the funnel mouth. Cut the bottom of the Petri dish away (this can be accomplished quite smoothly using a scalpel blade heated over a Bunsen burner) and glue a nylon mesh over the opening. Carefully glue this dish (nylon mesh facing down) to the mouth of the funnel and let it dry completely. (c) Place the slices on the nylon mesh and a bubbling tube or needle on the side of the chamber. We use a short version of a similar chamber for the warm recovery step so as to use less solution.

Fig. 3

Dissection tools and steps for obtaining cortical brain slices. (a) Dissection tool setup. (1) Ice bath, (2) tissue slicer (Precisionary Compresstome VF-300-0Z shown), (3) warm water bath, (4) various scissors (large scissors, iris scissors, and fine scissors), (5) forceps (curved forceps with fine tip and blunt forceps), (6) spatulas (one flat and one angled end), (7) Rongeurs tool, (8) slice transfer pipette, (9) specimen holder, (10) Sylgard-coated Petri dish, (11) cyanoacrylate glue, (12) small beaker, (13) razor blade, (14) slice recovery (inside warm bath, not visible) and storage chambers (at room temperature), (15) warmed magnetic stir plate for keeping agar solution molten. (b), (c). After cutting the skin with a pair of sharp scissors to expose the skull, cut the skull bone with scissors or Rongeurs tool (depending on skull thickness) to expose the brain. (b) In mice and young rats, the skull bone is cut along the midline and on each side from the base of the neck, then the bones pulled back laterally to expose the brain. (c) In adult rats, it is better to cut the skull laterally and at the bottom on each side and remove it in one piece from the top to avoid damaging the cortex. (d) Using a razor blade, cut away the bottom of the brain (cerebellum plus a small segment of the occipital lobes) and separate the hemispheres along the sagittal midline. (e) Attach one hemisphere upright on the specimen block using cyanoacrylate glue. Arrows indicate slicing direction for obtaining coronal slices. (f) Cut brain slices to appropriate thickness, for example, $300\mu \mathrm{M}$. (b)–(f) Created with BioRender.com.

Fig. 4

Placement of electrodes for cortical stimulation and field potential recording. (a) Schematic illustration of the cortical slice preparation for simultaneous imaging of blood vessels and electrophysiology. The slice is depicted in the imaging chamber as an orange slab, with a red dot representing the vessel of interest focused under the objective. (b) The slice is secured with a harp. The recording electrode is placed adjacent to the vessel of interest and the stimulating electrode is placed in layer I/II close to the pial surface. (c), (d). Detailed schematic illustration (c) and low magnification image (d) of a slice demonstrating appropriate electrode placement. Inset on upper right in (d) shows the recording electrode relative to the capillary of interest. (e) An example recording demonstrating the stimulation-evoked fiber volley and fEPSPs (measured as the current needed to hold the electrode at 0 mV; labeled fEPSCs), representing pre- and postsynaptic activity of neurons, respectively. (c)–(e) Adapted from Ref. .

Fig. 5

Placement of electrodes depends on direction of dendritic projections. (a)–(c). In a coronal slice, the direction of dendrites of neurons in deeper cortical layers is always perpendicularly toward the pial surface, but the relative direction under imaging optics will change depending on the location of the ROI. Dendrites of neurons in the ROI may be (b) extending straight or (c) diagonally depending on slice orientation. (d) The dendritic processes will also not always be in the same plane as the slice (top panel) but may be angled toward the top surface (middle panel), or deeper into the tissue (toward the bottom surface; bottom panel). The direction of dendrites in all three dimensions must be considered when placing the stimulating electrode to successfully activate the ROI. Created with BioRender.com.

Fig. 6

Stimulation intensity required to induce maximal field activity in different slices. Ten different slices were stimulated with 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, $1000\mu \mathrm{A}$ stimulus pulses, until a maximum peak was reached. Maximum intensity was determined as two stimulation intensities resulting in a similar field potential peak amplitude. Stimulation protocol was a train of 5 pulses at 1 Hz at each intensity with $\sim 10\mathrm{s}$ of rest between trains. The average peak amplitude was plotted at each stimulation intensity. The scatter plot for each slice was fit with a nonparametric loess regression in R. Solid black line depicts the 50% and dashed black line depicts the 80% peak intensity. Note that the stimulation intensity required to reach 50% peak intensity (solid black line) or 80% peak intensity (dashed black line) differs in each slice. As examples, three sets of vertical lines are shown in red, orange, and teal, depicting the stimulus intensity required to reach 50% (solid lines) or 80% (dashed lines) peak for three slices.

Fig. 7

Arteriole and capillary responses to cortical stimulation. (a) An example arteriole at baseline, following U46619-induced constriction, and after stimulation (STIM). (b) The diameter of the vessel shown in (a) plotted over time, showing the change in arteriole diameter in response to U46619 and neural stimulation. A video of the same arteriole is available online (Video 1, MOV, 1.61 MB [URL: https://doi.org/10.1117/1.NPh.9.3.031913.1]). U46619 and neuronal stimulation (red circle) were applied at the frames indicated. Video is played at $80\times$ speed. (c) An example capillary at baseline, after U46619-induced constriction, and after stimulation and (d) the corresponding diameter trace demonstrating the response of the capillary to each manipulation. A video of the same capillary is available online (Video 2, MOV, 1.23 MB [URL: https://doi.org/10.1117/1.NPh.9.3.031913.2]). U46619 and neuronal stimulation (red circle) were applied at the frames indicated. Video is played at $50\times$ speed. Yellow lines flanked by red triangles in (a) and (c) indicate the diameter of the vessels in each condition. The arteriole response plotted in (b) was averaged from measurements from both marked locations. Two regions marked with white lines in (c) depict regions of the capillary that do not change in diameter. Note that this is typical of capillaries, as pericytes are spatially separated and their processes do not contiguously cover the vessel.