Periodic Reporting for period 1 - TANYFEEDNEURONS (Gliotransmission and shuttling of metabolic signals to feeding neuronal circuits by tanycytes) Reporting period: 2015-10-01 to 2017-09-30 Summary of the context and overall objectives of the project Tanycytes are specialized hypothalamic glial cells located at the interface between blood, cerebrospinal fluid and brain areas that control food intake. Little is known about their physiological roles. Recent studies indicate that tanycytes sense the circulating nutrient glucose, release the neuroactive factor adenosine triphosphate and the energy substrate lactate, both of which can potentially modulate neuronal activity. Tanycytes also permit the access to the brain of the blood-borne peptide leptin produced by fat to inhibit hunger. Understanding how this glia might interact with neurons that control food intake is critical to identify new cellular targets for the development of therapeutic strategies for treating metabolic disorders such as obesity, leptin resistance and type 2 diabetes. Therefore, the overall objective of the project was to determine how tanycytes could influence electrical activity of ARH neurons. More specifically, the project was based on answering the following questions: i) Does adenosine triphosphate released by tanycytes modulate activity of neurons that control food intake? ii) Do tanycytes interact with these neurons through the release of lactate? iii) How do tanycytes convey leptin to them? Towards this end, I used electrophysiological techniques in acute mouse brain slices and newly-developed genetic tools that allow selective manipulations of tanycyte signaling. The TANYFEEDNEURONS project provide direct evidence for tanycyte-neuron communication in the context of energy homeostasis and identify tanycytes as potential therapy for metabolic disorders, which are age-related diseases and a leading cause of morbidity and mortality in Europe. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The project has achieved most of its objectives and milestones for the period, with relatively minor deviations.Given the strong electrophysiological components of the project, my work in the first months of the funding period focused on setting up an electrophysiology station in the host laboratory to appropriately execute the initially planned experiments. In the first aim, by combining electrophysiology in living brain slices with DREADDs and Cre/LoxP strategies, I demonstrated that activation of calcium signaling in tanycytes reduced the activity of neurons that control food intake, likely through the release of neuroactive substances. These data suggest that an active communication between tanycytes and neurons may exist. My current work carried out during the third year thanks to funding from an ANR Grant acquired by the host group aims to i) identify ATP as the neuroactive substance, ii) determine the release mechanisms of ATP and iii) evaluate the consequences of obesity on ATP release.In the second aim, I tested whether the intercellular channels gap junctions in tanycytes allow the trafficking of the energy substrates glucose and its derived metabolite lactate towards neurons that control food intake. Immunohistochemistry showed that tanycyte cell bodies highly expressed the connexin 43 (Cx43) gap junction proteins. Using patch-clamp technique and intracellular filling in living brain slices, I found that tanycytes communicated to each other through gap junctions and this permitted the passage of a fluorescent glucose analog. Interestingly, diffusion of glucose was completely blocked when I selectively knocked out Cx43 gene in tanycytes and this led to perturbations in energy balance towards an increase in food intake and a decrease in energy expenditure. Together, these results reveal that tanycytes form a functional network of highly interconnected cells made up of Cx43 gap junctions through which energy substrates can diffuse to regulate energy balance. I also found that the electrical activity of the neurons that inhibit hunger relied on the entry of the lactate into the cells, suggesting that these neurons could use glucose-derived lactate shuttled from the tanycyte networks as energy fuel to sustain their activity and drive satiety. My current work carried out during the third year thanks to funding from an ANR Grant acquired by the host group intends to determine i) whether tanycytes and neurons that control food intake are metabolically coupled through the lactate shuttle and ii) whether obesity influences trafficking of energy substrates through tanycyte networks.In the third aim, I tested whether tanycytes can shuttle the blood-born leptin from the cerebrospinal fluid to neurons that control food intake. In the early stage of this study, my work focused on setting up a protocol in living brain slices to visualize a fluorescent bioactive leptin transported by tanycytes towards neurons that control food intake. The protocol consisted in applying a fluorescent bioactive leptin locally at the apical surface of the cell body of tanycytes. However, after adjusting multiple parameters to optimize the local application, it was not possible to clearly visualize the uptake of the fluorescent bioactive leptin. Consequently, this method was not reliable enough to pursue the third aim of this project. Instead, I tested the new hypothesis that tanycytes could sense leptin from the cerebrospinal fluid and respond to it by releasing neuroactive substances to mediate the inhibitory effect of leptin on hunger. Interestingly, I found that local application of leptin at the apical surface of the cell body of tanycytes stimulated intracellular calcium signaling which was blocked by the mutated recombinant leptin antagonist, suggesting that the effect was mediated by functional leptin receptors. These compelling results led us to define a new aim in which a series of experiments will be designed to determine i) whether leptin-induced calcium increases in tanycytes trigger the release of ATP, ii) whether this ATP release influences the activity of nearby neurons that control food intake and iii) whether this signaling is impacted by obesity. The full completion of these experiments will be carried out by myself during the third year thanks to funding from an ANR Grant acquired by the host group. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The research work that I implemented during the reporting period has led to progress towards the achievement of the major scientific goals of this project. So far, no study has explored the active communication and metabolic cooperation between tanycytes and neurons that control food intake. I believe that the research questions addressed in this project provide new insights into the cellular mechanisms involving tanycytes in the regulation of energy balance. Moreover, the new questions proposed during the last part of the fellowship will reinforce that tanycytes are active signaling cells in the brain that directly modulate activity of neurons that control food intake. Thus, the knowledge obtained here is likely to provide a much better understanding of human obesity and leptin resistance, and consequently pave the way for the development of new therapeutic strategies to overcome metabolic disorders, a leading cause of morbidity and mortality in Europe.