Innervation modulates the functional connectivity between pancreatic endocrine cells

The importance of pancreatic endocrine cell activity modulation by autonomic innervation has been debated. To investigate this question, we established an in vivo imaging model that also allows chronic and acute neuromodulation with genetic and optogenetic tools. Using the GCaMP6s biosensor together with endocrine cell fluorescent reporters, we imaged calcium dynamics simultaneously in multiple pancreatic islet cell types in live animals in control states and upon changes in innervation. We find that by 4 days post fertilization in zebrafish, a stage when islet architecture is reminiscent of that in adult rodents, prominent activity coupling between beta cells is present in basal glucose conditions. Furthermore, we show that both chronic and acute loss of nerve activity result in diminished beta–beta and alpha–beta activity coupling. Pancreatic nerves are in contact with all islet cell types, but predominantly with beta and delta cells. Surprisingly, a subset of delta cells with detectable peri-islet neural activity coupling had significantly higher homotypic coupling with other delta cells suggesting that some delta cells receive innervation that coordinates their output. Overall, these data show that innervation plays a vital role in the maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.


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Tight regulation of hormone release from pancreatic islets is critical for glucose homeostasis and 56 its disruption can lead to diabetes mellitus 1 . Pancreatic islets are composed of different cell 57 types, including the hormone producing alpha, beta and delta cells, peripheral nerves, and 58 vascular endothelial and smooth muscle cells. Studies have implicated signaling from the 59 vascular scaffold 2, 3 and nerve networks 4-9 during the development and function of pancreatic 60 islet cells. However, it remains difficult to investigate the immediate effects of acute nerve 61 modulation on islet cell function. Given the alterations in islet innervation architecture in some 62 models of diabetes 10-12 , it is imperative to understand whether disruption of nervous control can 63 contribute to diabetes etiology. normalized single cell calcium traces, correlation matrices, and average correlation coefficients 109 (R avg ), we observed that homotypic coupling between beta cells is more prominent than those 110 between delta or alpha cells ( Figure 2F-H). Notably, we found a significant increase in 111 homotypic coupling for the delta cells that display neural activity connection ( Figure 2H). 112 Future studies will determine whether direct neural activity connection is critical for the 113 regulation of this delta cell subset. 116 Activity coupling between pancreatic endocrine cells can be mediated by autocrine and paracrine 117 signaling, gap junctions, and other means. To investigate whether pancreatic innervation is 118 critical for intra-islet coordination of activity, we used different approaches to chronically or 119 acutely inhibit neural signaling. We used endoderm transplantation to generate chimeric 120 zebrafish that express two GAL4/UAS systems in different germ layer-derived tissues and 121 investigated the role of chronic neural inhibition on islet function ( Figure 3A). Pan-neural 122 expression of botulinum toxin (BoTx) chronically inhibits neurotransmitter release 7, 23 and leads 123 to elevated glucose levels at 100 hpf 7 ( Figure 3B). While primary islet volume was consistently 124 greater in BoTx + larvae at 100 hpf ( Figure 3C; as we reported for earlier stages 7 ), we did not 125 observe changes in the architectural arrangement of the different islet cell types ( Figure 3D). 126 From the normalized single cell calcium traces, correlation matrices, and average correlation 127 coefficients (R avg ), we observed that the calcium dynamics in BoTx + larvae were significantly chronic neural inhibition, the delta-silent/beta-active state was decreased while the delta-146 active/beta-silent state and the delta-active/alpha-silent state were both increased ( Figure 3K). 147 Although we cannot exclude potential defects in endocrine cell development, as reported in our 148 previous study 7 , these changes were not accompanied by alterations in delta cell calcium 149 oscillation frequency nor peak height or duration (Figure 3 -Figure Suppl. 3). We also found a 150 significant decrease in the alpha-active/beta-active state upon chronic neural inhibition ( Figure   151 3I), suggesting that neural regulation is an important regulator of alpha-beta connectivity.

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Given the potential role of pancreatic innervation on islet cell maturation, we next 153 investigated the effects of acutely blocking neural activity using two different approaches. By 154 lineage tracing, we found that the neural crest derived peri-islet neurons were also labelled by the  Notably, we did not observe further impairment in beta cell coupling over increasing distance 161 ( Figure 4G), suggesting that upon ablation of peri-islet neurons, the signal that initiates beta cell 162 coupling was blunted while beta cells maintained their propensity for coupling across the islet. neurons significantly disrupted beta-beta and alpha-beta connectivity, while conclusions 172 regarding other heterotypic interactions will require further investigation into the various neural 173 subsets that were targeted. It is likely that our targeting of peri-islet neurons affected at least 174 those that guide the activity coupling between alpha and beta cells.

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Next, we took an optogenetic approach by generating a transgenic line that allows one to 176 acutely photo-inhibit the release of neurotransmitters with a single pulse of blue light. This . Surprisingly, photo-inhibition decreased glucose levels compared with transgene-negative 183 zebrafish exposed to the same light condition ( Figure 5A). Similar to peri-islet neural ablation, 184 pan-neural photo-inhibition decreased beta cell connectivity ( Figure 5B-E). Changes in delta-185 beta and alpha-beta heterotypic interactions were also observed upon acute neural inhibition 186 ( Figure 5E-H). A significant decrease in delta-silent/beta-active state reflects what we observed 187 upon chronic neural inhibition ( Figure 5H). Like with the photo-ablation of peri-islet neurons, 188 no changes in nearest alpha-delta interactions were observed ( Figure 5F-H). Notably, following 189 acute neural inhibition, alpha-beta coupling ( Figure 5E) and alpha-active/beta-active states 190 ( Figure 5F) were significantly decreased. While we cannot exclude a role for soluble factors 191 from other peripheral organs, these changes in alpha-beta interactions were consistently observed 192 upon both acute pan-neural and peri-islet inhibition, possibly reflecting a role for neurons in the 193 maintenance of alpha-beta coupling.  is often hindered by our inability to simultaneously study them in an intact organ within its 198 innate environment. Imaging calcium dynamics with genetically encoded biosensors or calcium 199 sensitive fluorescent indicators in individual islet cell types has been conducted in vitro with 200 dispersed cells 28, 29 , whole islets 24, 30 , and perfused pancreas slices 31, 32 , and in vivo with islets 201 transplanted into the anterior chamber of the eye 4, 33 , as well as intravital imaging of the mouse 202 pancreas 34 . We report a non-invasive in vivo imaging strategy to study all the different 203 pancreatic endocrine cell types within the same animal. Our three approaches to inhibit neural 204 control, ranging in temporal and spatial specificity, provided useful insights into the role of 205 neurons in regulating pancreatic islet function ( Figure 6). Whether activity coupling between 206 beta cells is in part mediated by gap junctions warrants further studies, but our data suggest that 207 neural regulation is critical for the establishment and maintenance of beta cell connectivity as we 208 consistently found decreased beta cell coupling upon chronic and acute neural inhibition. Given 209 that our targeted neural ablation approach also led to this decline in beta cell coupling, it is likely We have focused our studies on the pancreatic beta, alpha, and delta cells; however, it is 217 important to note that there are other endocrine cell types (gamma and epsilon cells) that remain 218 undefined, but do display glucose induced activity coupling with beta cells ( Figure 3F).

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Correlation analysis allowed us to study heterotypic coupling and fraction time analysis further 220 allowed us to study activity patterns of heterotypic cell pairs that are near each other, upon loss 221 of neural signaling. We found changes in delta-beta activity coupling, supporting a role for gap 222 junctions in mediating electrical coupling between delta and beta cells 17 . Both chronic and acute 223 neural inhibition also blunted the functional connectivity between alpha and beta cells suggesting 224 that neurons may have an important role in the paracrine potentiation of beta cell activity by 225 glucagon released from alpha cells 35 . We propose that alpha-beta interactions are specifically 226 regulated by pancreatic innervation since peri-islet neural ablation led to a similar decrease in 227 alpha-beta coupling and the alpha-active/beta-active state. However, given that pan-neural 228 inhibition is required to induce changes in nearest delta-beta interactions, it is possible that 229 central nervous control of other organs could in part be driving these changes. Importantly, our 230 data further support a role for neurons in modulating delta cell activity, since delta cells that 231 display neural activity connection exhibit a significant increase in their coupling to other delta 232 cells. Future studies will determine whether the observed changes in delta cell activity directly 233 reflect alterations in somatostatin release.

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Selective targeting of subsets of neurons will advance our understanding of the pancreatic 235 islet-neural interplay in health and disease, including diabetes pathophysiology. Through our in 236 vivo studies of homotypic and heterotypic activity coupling, we have illustrated how studying 237 functional connectivity can be achieved for the endocrine pancreas and discovered a critical role 238 for neurons in mediating these connections. Given the cellular heterogeneity of organ 239 composition, simultaneously evaluating the function of the different cell types that make up an 240 organ provides insights that can be missed by simply investigating one cell type at a time.

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Interrogating the neural regulation of other organs, such as the liver, intestine, and kidney, can be 242 achieved through the extended application of these tools. In combination with the ability to 243 monitor organ development, function, and regeneration in vivo, this approach will allow one to 244 address complex questions pertaining to the autonomic nervous system and its role in organ 245 maintenance and dysfunction. 28.5°C. Zebrafish embryos and larvae were grown in egg water at 28.5°C. Transgenic and 257 mutant lines used were on the mitfa w2/w2 background and as described in Table 1.  The Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) line was generated by Tol2 transgenesis of a 8.7  levels, pools of 40 larvae swimming in 9 cm diameter petri-dishes filled with 35 mL egg water 313 were exposed to 0.3 mW blue LED light for 30 min prior to sample collection; control animals 314 were not exposed to blue light and were randomly selected siblings from the same clutch.  Figure 5A). Induction of a stress response likely led to 317 this increase in glucose levels in wild-type larvae, as chronic 14 hr exposure to blue LED light 318 for 3 consecutive days kills zebrafish larvae 43 . To assess calcium changes, larvae were exposed 319 to a 3 min pulse of blue light (2.1 mW 470 nm LED, Colibri) and the GCaMP6s signal was 320 measured pre-and post-blue light exposure. While blinding was not possible, we randomized 321 the order in which animals on a given day were imaged.         n=24-32 batches of 5 larvae per replicate, p-value from t-test; see Figure 3 source data 1. C.

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Quantification of primary islet mass; n=21-29, p-value from t-test; see Figure 3 source data 2.  Figure 3D. Mean distance of pancreatic islet cells to islet core.        LG HG HG -post light ΔF/F