Leveraging Optogenetic-Based Neurovascular Circuit Characterization for Repair

Abstract

Optogenetic techniques are a powerful tool for determining the role of individual functional components within complex neural circuits. By genetically targeting specific cell types, neural mechanisms can be actively manipulated to gain a better understanding of their origin and function, both in health and disease. The potential of optogenetics is not limited to answering biological questions, as it is also a promising therapeutic approach for neurological diseases. An important prerequisite for this approach is to have an identified target with a uniquely defined role within a given neural circuit. Here, we examine the retinal neurovascular unit, a circuit that incorporates neurons and vascular cells to control blood flow in the retina. We highlight the role of a specific cell type, the cholinergic amacrine cell, in modulating vascular cells, and demonstrate how this can be targeted and controlled with optogenetics. A better understanding of these mechanisms will not only extend our understanding of neurovascular interactions in the brain, but ultimately may also provide new targets to treat vision loss in a variety of retinal diseases.

Keywords: Acetylcholine; Amacrine cell; Neurovascular unit; Optogenetics; Pericyte.

Fig. 1

Pericyte control of capillary diameter. (A) Simplified schematic of blood vessel regulation components in retina (modified from [15]). In response to light stimulation of the retina, cholinergic amacrine cells release neurotransmitter that activates acetylcholine receptors (AChRs) on pericytes and endothelial cells. Activity from an individual pericyte can spread via gap junctions across the vascular network to regulate upstream blood vessels. (B) Live fluorescent image of intact retina in NG2-DsRed mice, revealing pericyte bodies and processes. Boxed region is enlarged in (C). White arrowheads point to individual pericyte cell bodies. (D) An example shows how a single pericyte is stimulated using an electrode (dotted lines) to assess changes in diameter of blood vessel, or its vasomotor response. (E) Focal electrical stimulation of a pericyte induces a robust and reversible vasomotor response (constriction). Scale bars = 25 μm

Fig. 2

Retina as a model to study function of the central nervous system in health and disease. (A) Mouse retina wholemount placed on a Millicell Biopore membrane filter. Once immersed in physiological media, filter becomes transparent. Scale bar = 2 mm. (B) Confocal live imaging of a whole intact retina illustrating retinal vasculature stained with endothelial cell-specific marker added to perfusion media (SRH, red, modified from [39]). Scale bar = 1 mm. (C) A magnified section of the retina illustrating individual components of the hypothetical neurovascular unit. Blood vessels (isolectin, green), neurons, cholinergic amacrine cells [choline acetyltransferase (ChAT), red], and astroglia [glial fibrillary acidic protein (GFAP), blue]. Scale bar = 25 μm

Fig. 3

Cholinergic neurovascular interaction sites in the retina. (A) Schematic of the laminar neurovascular network showing the first-order arteriole (A) and venule (V) and the connectivity of the (1) superficial, (2) intersublaminar, (3) intermediate, and (4) deep vascular layers (left) and their locations within the retina (middle). Blood vessels are labeled with isolectin (right, green). In red are cell bodies and the processes of cholinergic amacrine cells, a source of neuro- and vasomodulator, acetylcholine. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer. Drawing is modified from [37]. (B) Confocal images of the (1) superficial, (2) intersublaminar, (3) intermediate, and (4) deep vascular layers, labeled with isolectin (green) and choline acetyltransferase (ChAT; red). Numbers are vascular layers as in (a). Panel (B2) illustrates potential sites of neurovascular interactions between cholinergic cells blood vessels. Scale bar = 50 μm. (c) Z-stack reconstruction from a magnified area in panel (B2). (D) Intensity profiles across an imaginary line between arrowheads in (c). Scale bar = 50 μm in (A) and (B). Scale bar = 5 μm in (C). (Modified from [46])

Fig. 4

Channelrhodopsin-2 (ChR2)-assisted probing of the cholinergic pathways in living mammalian retina. (A) Confocal images in choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-YFP mouse retina showing selective and robust expression of ChR2 (green, left) in all ChAT-positive starburst amacrine cells in ganglion cell layer (blue, middle) and their processes in the inner plexiform layer (IPL, right). As shown in Z-stack reconstructions of the IPL, both ON and OFF ChAT-positive cells contain ChR2. Arrows identify the cell targeted for recording. (B) In living retinal wholemount, the green fluorescent protein-positive ChR2-expressing cells were targeted for electrophysiological recordings and backfilled with Alexa 568 included in recording pipette (arrow, red). Note a characteristic starburst appearance of ChAT-positive amacrine cell [see also (A), right]. An asterisk indicates a Muller cell damaged during the recording procedure. (C) Spiking activity (left) and excitatory currents (right) from cell in (B) in presence of synaptic blockers (LAP4, NBQX). Flashing spots of light of varying intensity and frequency produce characteristic sustained and nondesensitizing ChR2-driven activity. Neutral density (ND) values represent the ND filter attenuation of light from the light source. In right panel, the number represents the frequency in Hz of a flashing 50-ms-long spot of light. Scale bars = 40 μm

Fig. 5

Channelrhodopsin-2 (ChR2)-assisted light activation of cholinergic amacrine cells produces dilation of capillaries and precapillary arterioles. (A) Cholinergic receptor activation leads to dilation of capillaries and arterioles. Bath application of with acetylcholine analog carbachol (100 μM) leads to dilation of both capillaries (arrowhead) and precapillary arterioles (arrow). (B) Quantification of vasomotor response across 40 samples showing consistent and significant changes in diameter following activation of acetylcholine receptors in intact wholemount retina. Error bars ± SEM; **p < 0.001, paired t test. (C) Vasomotor response was measured when photoreceptor inputs were blocked by a mix of NBQX and L-AP4. Arrowhead points to region of dilation, allowing passage of a blood cell. (D) Quantification of vasomotor response across 15 samples each. Paired t test p = 0.01 and p = 0.03 for arteriole and capillary, respectively. Error bars ± SEM. Scale bars = 30 μm