Periodic Reporting for period 1 - P-appetite (Dissecting how the Drosophila brain regulates behavioral sequences of feeding to ensure protein homeostasis)
Período documentado: 2019-11-03 hasta 2021-11-02
Balanced intake of nutrients is an important determinant of lifespan and reproductive success across animal phyla. Lack or imbalances in the intake of nutrients (e.g. proteins, carbohydrates, fats etc.) lead to malnutrition which in humans is the major cause of dietary diseases. The overconsumption of foods, on the other hand, is linked to pathologies like diabetes and obesity. To attain nutrient homeostasis, animals have evolved nutrient-specific appetites where the deficiency of (or high demand for) a nutrient class leads to compensatory consumption of that nutrient. A comprehensive understanding of physiological, behavioural and neural mechanisms underlying nutrient-specific appetites will therefore help develop strategies against malnutrition-related pathologies and eating disorders. Fruit flies, like humans, develop protein-specific appetite in response to protein deprivation. In this project, we made use of the unprecedented neurogenetic toolbox and single synapse-level connectomics data for Drosophila to identify neural mechanisms underlying protein-specific appetites. Since the behavioural strategies to control food intake underlying nutrient-specific appetites are highly conserved, the neural mechanisms we identify will shed light on the mechanisms underlying the perturbations to nutrient homeostasis in humans. At the end of this project, we have identified a neural circuit at synaptic resolution that control the length of the feeding bursts in protein-specific appetite. This is one of the first examples of an entire sensorimotor pathway that controls a specific motor pattern in a nutrient specific way in Drosophila. Our findings demonstrate that brains evolve multiple, overlapping strategies to attain nutrient homeostasis.
In this project, we performed a behavioral screen of by genetically silencing different subsets of central neuronal populations in the subesophageal zone (SEZ), the feeding Drosophila centre. We hypothesised that high order neurons controlling feeding microstructure in a nutrient-specific way should be located here since this neuropil contains both gustatory sensory projections as well as the motor neurons controlling the feeding motor program.
By using the flyPAD assay which allows us to monitor the feeding microstructure at single-sip resolution and screening different Gal4 driver lines labeling different sets of (SEZ) neurons, we identified a subset of neurons that are important for regulating the length of the feeding bursts in protein-specific appetite. By using genetic intersections, we narrowed down the population of neurons to a single pair of neurons in the SEZ. We further show that this single pair of neurons (sustain neurons) are necessary for sustaining the feeding burst in protein deprived flies. In addition, optogenetic activation of these neurons in a closed-loop feeding assay (optoPAD) is sufficient to increase the number of sips upon initiation of feeding bursts. However, silencing these neurons did not affect the probability of initiating feeding bursts suggesting that these two parameters are controlled by different circuit elements. Calcium imaging experiments suggest that these neurons respond to yeast (a protein-rich food source) in a protein state-dependent way. In order to map the neural circuit the sustain neuron involved in we used the electron microscopy reconstruction of the synaptic partners of this neuron in a female fruit fly brain (ngl.flywire.ai). Connectomic analysis showed that sustain neuron is a high order neuron dowsntream of taste peg gustatory receptor neurons. It connects to specific proboscis (feeding organ of Drosophila) motor neurons via a two neurons, one of which is excitatory and the other one is inhibitory. Interestingly, sustain neurons are excitatory neurons that connect to the contralateral sustain neurons potentially creating a sustained activity underlying the sustaining of feeding bursts. To our knowledge, this is one of the first examples of an entire sensorimotor pathway from sensory neurons to motor neurons which control a specific feeding motor parameter in a nutrient specific way. This work demonstrates that the nervous system can control different feeding motor parameters via dedicated sensorimotor loops and it is important to know which of these parameters are affected and specifically target them in different eating disorders.
A brief overview of the results:
(1) Using a combination of Drosophila neurogenetics and connectomics, we have identified a neuron that is crucial for sustaining feeding bursts on proteinaceous food.
(2) We performed optogenetic gain-of-function experiments and calcium imaging to show that this neuron respond to proteinaceous food and is sufficient to induce feeding behavior.
(3) By using Drosophila connectomics, we showed that this neuron is a 3rd order neuron downstream of taste peg gustatory receptor neurons and project to pharyngeal motor neurons that control ingestion.
(4) By silencing pharyngeal motor neurons we verified that pharyngeal pumping is necessary to sustain yeast feeding
I presented the findings (1), (2), (3) and (4) in regular lab meetings, institute seminars and international conferences.
By using the flyPAD assay which allows us to monitor the feeding microstructure at single-sip resolution and screening different Gal4 driver lines labeling different sets of (SEZ) neurons, we identified a subset of neurons that are important for regulating the length of the feeding bursts in protein-specific appetite. By using genetic intersections, we narrowed down the population of neurons to a single pair of neurons in the SEZ. We further show that this single pair of neurons (sustain neurons) are necessary for sustaining the feeding burst in protein deprived flies. In addition, optogenetic activation of these neurons in a closed-loop feeding assay (optoPAD) is sufficient to increase the number of sips upon initiation of feeding bursts. However, silencing these neurons did not affect the probability of initiating feeding bursts suggesting that these two parameters are controlled by different circuit elements. Calcium imaging experiments suggest that these neurons respond to yeast (a protein-rich food source) in a protein state-dependent way. In order to map the neural circuit the sustain neuron involved in we used the electron microscopy reconstruction of the synaptic partners of this neuron in a female fruit fly brain (ngl.flywire.ai). Connectomic analysis showed that sustain neuron is a high order neuron dowsntream of taste peg gustatory receptor neurons. It connects to specific proboscis (feeding organ of Drosophila) motor neurons via a two neurons, one of which is excitatory and the other one is inhibitory. Interestingly, sustain neurons are excitatory neurons that connect to the contralateral sustain neurons potentially creating a sustained activity underlying the sustaining of feeding bursts. To our knowledge, this is one of the first examples of an entire sensorimotor pathway from sensory neurons to motor neurons which control a specific feeding motor parameter in a nutrient specific way. This work demonstrates that the nervous system can control different feeding motor parameters via dedicated sensorimotor loops and it is important to know which of these parameters are affected and specifically target them in different eating disorders.
A brief overview of the results:
(1) Using a combination of Drosophila neurogenetics and connectomics, we have identified a neuron that is crucial for sustaining feeding bursts on proteinaceous food.
(2) We performed optogenetic gain-of-function experiments and calcium imaging to show that this neuron respond to proteinaceous food and is sufficient to induce feeding behavior.
(3) By using Drosophila connectomics, we showed that this neuron is a 3rd order neuron downstream of taste peg gustatory receptor neurons and project to pharyngeal motor neurons that control ingestion.
(4) By silencing pharyngeal motor neurons we verified that pharyngeal pumping is necessary to sustain yeast feeding
I presented the findings (1), (2), (3) and (4) in regular lab meetings, institute seminars and international conferences.
Our work demonstrates that it is crucial to study feeding motor program at high resolution as different aspects of the feeding microstructure are controlled by different neuronal elements in a nutrient specific way. We used state of the art techniques such as optogenetics, two photon calcium imaging and connectomics analysis to map the sensorimotor circuit underlying the control of the feeding burst lengths. The neuronal computations performed by these circuit are likely to be conserved in other animals including humans which control the feeding motor program in a similar way. Understanding these computational mechanisms will be crucial to understand and treat human eating disorders in the future.