Aminergic and peptidergic modulation of Insulin-Producing Cells in Drosophila

Reviewer #1 (Public review):

Summary:

Insulin is crucial for maintaining metabolic homeostasis, and its release is regulated by various pathways, including blood glucose levels and neuromodulatory systems. The authors investigated the role of neuromodulators in regulating the dynamics of the adult Drosophila IPC population. They showed that IPCs express various receptors for monoaminergic and peptidergic neuromodulators, as well as synaptic neurotransmitters with highly heterogeneous profiles across the IPC population. Activating specific modulatory inputs, e.g. dopaminergic, octopaminergic or peptidergic (Leucokinin) using an optogenetic approach coupled with in vivo electrophysiology unveiled heterogeneous responses of individual IPCs resulting in excitatory, inhibitory or no responses. Interestingly, calcium imaging of the entire IPC population with or without simultaneous electrophysiological recording of individual cells showed highly specific and stable responses of individual IPCs suggesting their intrinsic properties are determined by the expressed receptor repertoire. Using the adult fly connectome they further corroborate the synaptic input of excitatory and inhibitory neuronal subsets of IPCs. The authors conclude that the heterogeneous modulation of individual IPC activity is more likely to allow for flexible control of insulin release to adapt to changes in metabolic demand and environmental cues.

Strengths:

This study provides a comprehensive, multi-level analysis of IPC properties utilizing single-nucleus RNA sequencing, anatomical receptor expression mapping, connectomics, electrophysiological recordings, calcium-imaging and an optogenetics-based 'intrinsic pharmacology' approach. It highlights the heterogeneous receptor profiles of IPCs, demonstrating complex and differential modulation within the IPC population. The authors convincingly showed that different neuromodulatory inputs exhibit varied effects on IPC activity and simultaneous occurrence of heterogeneous responses in IPCs with some populations exciting a subset of IPCs while inhibiting others, showcasing the intricate nature of IPC modulation and diverse roles of IPC subgroups. The temporal dynamic of IPC modulation showed that polysynaptic and neuromodulatory connections play a major role in IPC response. The authors demonstrated that certain neuromodulatory inputs, e.g. dopamine, can shift the overall IPC population activity towards either an excited or inhibited state. The study thus provides a fundamental entry point to understanding the complex influence of neuromodulatory inputs on the insulinergic system of Drosophila.

Weakness:

GPCRs are typically expressed at low levels and while the transcriptomic and reporter expression analysis was comprehensive, both approaches have the caveat that they do not allow validating protein level expression. Thus, some receptors might have been missed while others might be false positives. The authors acknowledged the challenges in accurately accessing receptor expression in complex modulatory systems indicating there are limitations in full understanding of the receptor profiles of IPCs.

While this study provides valuable insights into the heterogeneity of IPC responses and receptor expression, it will require future studies to elucidate how these modulatory inputs affect insulin release and transcriptional long-term changes.
The authors further analyzed male and female snRNAseq data and claimed that the differences in receptor expression were minimal. The experimental analyses used mated females only and while the study is very complete in this respect, it would have been extremely interesting to compare male flies in terms of their response profiles.
Lastly as also pointed out by the authors, their approach of using optogenetically driven excitation of modulatory neuronal subsets limits the interpretation of the results due to the possibly confounding direct or indirect effect of fast synaptic transmission on IPC excitation/inhibition, and the broad expression of some neuromodulatory lines used in this analysis.

Overall, however, the conclusions of this study are well supported by the data provided by the authors. Moreover, their detailed and thorough analysis of IPC modulation will have a significant impact on the field of metabolic regulation to understand the complex regulatory mechanism of insulin release, which can now be studied further to provide insight about metabolic homeostasis and neural control of metabolic processes.

Reviewer #2 (Public review):

Summary:

Held et al. investigated the distinct activities of Insulin-Producing Cells (IPCs) by electrophysiological recordings and calcium imaging. In the brain of the fruit fly Drosophila melanogaster, there are approximately 14 IPCs that are analogous to mammalian pancreatic beta cells and provide a good model system for monitoring their activities in vivo. The authors performed single-nucleus RNA sequencing analysis to examine what types of neuromodulatory inputs are received by IPCs. A variety of neuromodulatory receptors are expressed heterogeneously in IPCs, which would explain the distinct activities of IPCs in response to the activations of neuromodulatory neurons. The authors also conducted the connectome analysis and G-protein prediction analysis to strengthen their hypothesis that the heterogeneity of IPCs may underlie the flexible insulin release in response to various environmental conditions.

Strengths:

The authors succeeded patch-clamp recordings and calcium imaging of individual IPCs in living animals at a single-cell resolution, which allows them to show the heterogeneity of IPCs precisely. They measured IPC activities in response to 9 types of neurons in patch-clamp recordings and 5 types of neurons in calcium imaging, comparing the similarities and differences in activities between two methods. These results support the idea that the neuromodulatory system affects individual IPC activities differently in a receptor-dependent manner.

Weaknesses:

One concern is how much extent the heterogeneity of IPC activities in a short time scale is relevant to the net output, a release of insulin-like peptides in response to metabolic demands in a relatively longer time scale. The authors can test their hypothesis by manipulating the heterogeneous expressions of receptor genes in IPCs and examining IPC activities on a longer time scale. Moreover, while the authors focus on IPC activities, they did not show the activation of the neuromodulatory inputs and the net output of insulin levels in the data. The readers might want to know which neurons are indeed activated to send signals to IPCs and how IPC activities result in the secretion of insulin peptides.