There are three neuron types—sympathetic, parasympathetic, and sensory— that innervate the pancreas, in addition to an astroglial population. The sympathetic and parasympathetic branches of the autonomic nervous system are involved in maintenance of blood glucose homeostasis in response to changing energy demands. Sympathetic neurons mediate the so-called “fight or flight” response through stress-induced neural activity. They inhibit insulin secretion and up-regulate glucagon release by respective β- and α-cell populations in the pancreatic islets of Langerhans, the net physiological result of which is to convert glycogen stores to blood glucose to meet immediate energy demands (Mundinger et al.2003
). Through feeding-induced neural activity, parasympathetic neurons stimulate insulin secretion from insulin-producing β-cells to promote the removal of glucose from the blood into the liver for storage as glycogen, while repressing glucagon release (Benthem et al. 2001
; Adeghate et al.2000
; Ahren 2000
). Sensory neurons are involved in pain sensation; indeed, extreme pain is a well-documented concern in pancreatitis and pancreatic cancer patients (Wick et al. 2006a
). The function of pancreatic astroglia, which encapsulate the islets of Langerhans, is not definitively known, although there is increasing evidence that astroglia are involved in synaptic transmission in the brain, and thus may be more involved in neuronal signaling than previously speculated (Halassa et al. 2007
Anatomical and physiological characteristics of the pancreas pose technical challenges to the study of innervation. Many antigens that are considered dependable neural markers within the CNS are unsuitable for use in the pancreas because various pancreatic endocrine cells also display them. Furthermore, due to the irregular morphology of islets and the network of neurons that innervate them, thin-section immunofluorescence techniques miss important 3-dimensional information. Thus, previous developmental studies have been limited in their ability to distinguish between specific pancreatic nerve populations, and to obtain high-resolution images. In this study we employ confocal fluorescence microscopy, using neuronal subtype-specific antibodies on thick sections at particular stages in embryonic development, postnatal maturation, and synthetic pancreatic disease, to gain a greater understanding of the neuronal and glial populations associated with the pancreatic islets.
One goal of the present study was to perform a descriptive analysis of the growth and development of sympathetic and sensory neurons and astroglia during pancreatic organogenesis and maturation. In addition, we aimed to assay a synthetic pancreatic disease model for subtype-specific effects of large-scale β-cell loss and repopulation on the maintenance of islet innervation. To accomplish these goals we performed confocal analysis using single, double, and triple labeling immunohistochemistry. We included embryonic, neonatal, adolescent and adult wild-type mice in our developmental study. We also took advantage of the recent development of the RIP-cmycER
line, a transgenic mouse line that conditionally expresses cmyc specifically in β-cells. This enabled us to orchestrate the death and subsequent resurgence of this subpopulation of endocrine cells (Pelengaris et al. 2002
). We used the RIP-cmycER
regeneration model to determine the effect on existing pancreatic neurons when the bulk of β-cells are ablated and replenished.
In contrast to previous research, this analysis concerns itself with the relationship of specific neural and glial pancreatic subpopulations to both the developing and mature endocrine pancreas. It explores these populations, at the level attainable by confocal fluorescent microscopy, in order to address both developmental and maturational aspects of islet innervation in the wild type mouse, as well as disease-related aspects of innervation maintenance in a synthetic adult disease model. We report that the sympathetic and sensory populations appear in the developing embryonic pancreas in a temporally discrete fashion. We show that islet innervation by distinct cell types is temporally integrated with the intrapancreatic endocrine cell migration and organization that characterize pancreatic embryonic development and postnatal maturation, respectively. Finally, we observe that the sympathetic, sensory and astroglial populations in the pancreas are affected differently by depletion and restoration of the β-cell population in RIP-cmycER mice.