Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laserscanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions. Adequate release of insulin by pancreatic beta cells in response to changing blood glucose levels is a vital requirement for maintaining glucose homeostasis. Failure to do so is one of the major causes of type 2 diabetes mellitus, the most common metabolic disorder in humans 1 . Under physiological conditions, insulin release is regulated by the complex interplay between glucose and a plethora of additional factors-for example, nutrients, autocrine-paracrine signaling and the continuous input from hormones and neurotransmitters 2 . Beta cells, together with other pancreatic endocrine cell types, are situated within the endocrine pancreas, that is, the islets of Langerhans, which are densely vascularized 3 and abundantly innervated 4 . Pancreatic islets, constituting 1%-2% of the pancreatic volume, are difficult to access for in vivo monitoring because they are deeply embedded and scattered in the exocrine tissue of the pancreas 5 . As a consequence, the majority of functional beta cell studies have so far been conducted in vitro on isolated islets or beta cells. Isolated islets 6 , and especially pancreatic slices 7 , allow functional studies of Author Contributions: S.S., D.N. and A.C. developed the experimental transplantation platform. S.S., D.N., O.C., J.Y., R.D.M., A.P., T.M., M.K., B.L. and A.C. did the experiments. J.W. was responsible for generating the transgenic mice. C.R. was involved in designing the transplantation protocols and writing the manuscript. S.S., D.N., I.B.L. and P.-O.B. were responsible for designing the overall experimental plan and writing the manuscript. P.-O.B. was the originator of the idea of using the anterior chamber of the eye for noninvasive in vivo imaging of pancreatic islet cell biology Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions Note: Supplementary information is available on the Nature Medicine website. Laser-scanning microscopy (LSM) of isolated islets and cell preparations has been successfully applied for imaging multiple signaling pathways in the beta cell 6 . However, intravital applications of LSM for studies of beta cell physiology have not been repo...

SUMMARY The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, and thus impacts glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are not known, particularly in human islets. Here we demonstrate that the innervation of human islets is different from that of mouse islets and that it does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, in contrast to mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.

Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development.

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Efficient insulin secretion requires a well-functioning pancreatic islet microvasculature. The dense network of islet capillaries includes the islet pericyte, a cell that has barely been studied. Here we show that islet pericytes help control local blood flow by adjusting islet capillary diameter. Islet pericytes cover 40% of the microvasculature, are contractile, and are innervated by sympathetic axons. Sympathetic adrenergic input increases pericyte activity and reduces capillary diameter and local blood flow. By contrast, activating beta cells by increasing glucose concentration inhibits pericytes, dilates islet capillaries, and increases local blood flow. These effects on pericytes are mediated by endogenous adenosine, which is likely derived from ATP co-released with insulin. Pericyte coverage of islet capillaries drops drastically in type 2 diabetes, suggesting that, under diabetic conditions, islets lose this mechanism to control their own blood supply. This may lead to inadequate insulin release into the circulation, further deteriorating glycemic control.