Junctional membrane complexes facilitate excitation-contraction coupling in skeletal and cardiac muscle cells by forming subcellular invaginations that maintain close (≤20 nm) proximity of ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) with voltage-dependent Ca²⁺ channels in the plasma membrane. In fully differentiated smooth muscle cells, junctional membrane complexes occur as distributed sites of peripheral coupling. We investigated the role of the cytoskeleton in maintaining peripheral coupling and associated Ca²⁺ signaling networks within native smooth muscle cells of mouse and rat cerebral arteries. Using live-cell confocal and superresolution microscopy, we found that the tight interactions between the SR and the plasma membrane in these cells relied on arching microtubule structures present at the periphery of smooth muscle cells and were independent of the actin cytoskeleton. Loss of peripheral coupling associated with microtubule depolymerization altered the spatiotemporal properties of localized Ca²⁺ sparks generated by the release of Ca²⁺ through type 2 RyRs (RyR2s) on the SR and decreased the number of sites of colocalization between RyR2s and large-conductance Ca²⁺-activated K⁺ (BK) channels. The reduced BK channel activity associated with the loss of SR–plasma membrane interactions was accompanied by increased pressure–induced constriction of cerebral resistance arteries. We conclude that microtubule structures maintain peripheral coupling in contractile smooth muscle cells, which is crucial for the regulation of contractility and cerebral vascular tone.

Pericytes in the brain are candidate regulators of microcirculatory blood flow because they are strategically positioned along the microvasculature, contain contractile proteins, respond rapidly to neuronal activation, and synchronize microvascular dynamics and neurovascular coupling within the capillary network. Analyses of mice with defects in pericyte generation demonstrate that pericytes are necessary for the formation of the blood-brain barrier, development of the glymphatic system, immune homeostasis, and white matter function. The development, identity, specialization, and progeny of different subtypes of pericytes, however, remain unclear. Pericytes perform brain-wide ‘transportation engineering’ functions in the capillary network, instructing, integrating, and coordinating signals within the cellular communicome in the neurovascular unit to efficiently distribute oxygen and nutrients (‘goods and services’) throughout the microvasculature (‘transportation grid’). In this review, we identify emerging challenges in pericyte biology and shed light on potential pericyte-targeted therapeutic strategies.

Several hypotheses have been tested to understand whole organ regulation in other organs such as the brain and kidney, but no such hypothesis has yet been proposed for ocular circulations. To some extent resolve this deficit our ex vivo mouse eye perfusion model takes the first step in elucidating the mechanisms controlling the individual components of the ocular circulation. Various isolated ocular vascular preparations have been utilized in studies of ocular vascular biology, physiology, and pharmacology, including studies on both normal and pathological conditions. However, there is still significant potential for further studies to improve our understanding of ocular circulation and its regulation. The choroid specifically is inaccessible to direct visualization due to the retina's high metabolic requirement with a transparency that cannot be compromised by an overly rich vascular network on the inner retinal side hindering the visualization of the choroid. In this technical paper, we provide a detailed description of all the steps to be followed from the enucleation of mouse eyes to cannulation of the ophthalmic artery and perfusion and ex vivo confocal microscopy imaging of the dynamic nature of the choroid circulation.

The choroid is the thin, vasculature-filled layer of the eye situated between the sclera and the retina, where it serves the metabolic needs of the light-sensing photoreceptors in the retina. Illumination of the interior surface of the back of the eye (fundus) is a critical regulator of subretinal fluid homeostasis, which determines the overall shape of the eye, but it is also important for choroidal perfusion. Noted for having some of the highest blood flow rates in the body, the choroidal vasculature has been reported to lack intrinsic, intravascular pressure-induced (myogenic) autoregulatory mechanisms. Here, we ask how light directly regulates choroid perfusion and ocular fluid homeostasis, testing the hypothesis that light facilitates ocular fluid absorption by directly increasing choroid endothelial permeability and decreasing choroid perfusion. Utilizing ex vivo pressurized whole-choroid and whole-eye preparations from mice expressing cell-specific Ca ²⁺ indicators, we found that the choroidal vasculature has two intrinsically light-sensitive Ca ²⁺ -signaling mechanisms: One increases Ca ²⁺ -dependent production of nitric oxide in choroidal endothelial cells; the other promotes vasoconstriction through Ca ²⁺ elevation in vascular smooth muscle cells. In addition, we found that choroidal flow, or pressure, modulates endothelial and smooth muscle photosensitivity and trans-retinal absorption of fluid into the choroid. These results collectively suggest that the choroid vasculature exhibits an inverted form of autoregulatory control, where pressure- and light-induced mechanisms work in opposition to regulate blood flow and maintain fluid balance in response to changes in light and dark, aligning with the metabolic needs of photoreceptors.

Localized control of blood flow and arterial constriction in response to changes in intraluminal pressure are dependent on the intrinsic regulation of smooth muscle cell membrane potential. The melastatin transient receptor potential (TRP) channel TRPM4 is highly selective for monovalent cations, is activated by elevated levels of intracellular Ca ²⁺ , and is a critical regulator of smooth muscle membrane depolarization, which serves to open voltage dependent Ca ²⁺ channels (VDCC), and resulting in myocyte contraction. Interestingly, Ca ²⁺ activates and inactivates TRPM4, causing the channel to rapidly inactivate under conventional whole‐cell patch clamp conditions. When the slow Ca ²⁺ buffer, ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA), was included in the pipette solution, we identified a transient tetraethylammonium (TEA)‐insensitive inward Na ⁺ current in freshly isolated cerebral arterial myocytes. siRNA‐mediated silencing of TRPM4 and the recently described TRPM4 inhibitor, 9‐phenanthrol, both diminish the open probability (NP _o ) compared to controls, strongly suggesting the molecular identity of the channel to be TRPM4. This study demonstrates a practical method to record sustained TRPM4 activity and allows for further investigation into mechanisms of channel regulation. AHA0535226N; F31HL094145‐01.