Periodic Reporting for period 2 - OPTONEUROCHEM (Genetically encoded sensors for imaging neurochemical dynamics in vivo)
Berichtszeitraum: 2022-08-01 bis 2024-01-31
Our brain has a language of its own. At the core of its complex functions is chemical communication occurring between and among neurons and astrocytes, which use an alphabet composed of a myriad secreted signaling molecules, including neurotransmitters, neuropeptides, hormones etc. Understanding the chemical language of the brain is a goal of fundamental importance, as many of these signaling molecules or the cellular receptors that relay their signals, are involved in diseases of the nervous system and are potential targets of pharmaceuticals that could restore physiological brain functions. Towards this goal, an important first step is to decipher the associations between animal behavior, neural activity, and the precise spatial and temporal dynamics of these secreted chemical neuromodulators.
To aid our understanding of neural communication, along with continuous improvements in neuroimaging technologies, a range of new molecular tools needs to be developed and deployed. Genetically encoded fluorescent sensors, such as the widely utilized calcium sensors GCaMPs, occupy the center stage, due to their ideal properties for in vivo imaging and their flexible combination with the most advanced imaging modalities.
Standing on the shoulders of these giants, recent developments by us and others led to the first genetically encoded indicators for dopamine (dLight1), a key neuromodulator best known for its roles in reward and motivation.
These ultrasensitive probes are built by engineering a single fluorescent protein (circularly permutated Green Fluorescent Protein) into G-protein coupled receptors (GPCRs).
Our team is interested in continuing the optimization and expansion of this neurotechnology toolbox, to shine a new light on the in vivo dynamics of diverse neuromodulatory molecules involved in neural communication. Our goal is to reveal previously hidden aspects of neural communication in intact living animals. Ultimately, the biological findings enabled by the new tools we propose to develop are expected to translate into new and improved therapeutic approaches for several neurological and neuropsychiatric disorders.
Within this context, we are pursuing the following overall objectives:
1) Development of novel high-quality sensors for chemical neuromodulators (i.e. monoamines, neuropeptides, neurohormones), optimized for in vitro and in vivo imaging with high sensitivity;
2) Deployment of these sensors in brain slices and awake behaving animals to investigate the spatial and temporal scales of neuromodulatory signals, as well as their relationship with neural circuit activity and animal behavior.
To aid our understanding of neural communication, along with continuous improvements in neuroimaging technologies, a range of new molecular tools needs to be developed and deployed. Genetically encoded fluorescent sensors, such as the widely utilized calcium sensors GCaMPs, occupy the center stage, due to their ideal properties for in vivo imaging and their flexible combination with the most advanced imaging modalities.
Standing on the shoulders of these giants, recent developments by us and others led to the first genetically encoded indicators for dopamine (dLight1), a key neuromodulator best known for its roles in reward and motivation.
These ultrasensitive probes are built by engineering a single fluorescent protein (circularly permutated Green Fluorescent Protein) into G-protein coupled receptors (GPCRs).
Our team is interested in continuing the optimization and expansion of this neurotechnology toolbox, to shine a new light on the in vivo dynamics of diverse neuromodulatory molecules involved in neural communication. Our goal is to reveal previously hidden aspects of neural communication in intact living animals. Ultimately, the biological findings enabled by the new tools we propose to develop are expected to translate into new and improved therapeutic approaches for several neurological and neuropsychiatric disorders.
Within this context, we are pursuing the following overall objectives:
1) Development of novel high-quality sensors for chemical neuromodulators (i.e. monoamines, neuropeptides, neurohormones), optimized for in vitro and in vivo imaging with high sensitivity;
2) Deployment of these sensors in brain slices and awake behaving animals to investigate the spatial and temporal scales of neuromodulatory signals, as well as their relationship with neural circuit activity and animal behavior.
Orexin biosensor:
Orexins/hypocretins are neuropeptides carrying out critical neuromodulatory functions in the brain. Normally, they regulate arousal, wakefulness, motivation and appetite. Defects in the release or sensing of orexin neuropeptides cause, both in humans and in animals, a sleep disorder called narcolepsy. Affected individuals suffer from overwhelming daytime drowsiness and often exhibit cataplexic states, in which they remain conscious but are unable to control body movement.
Through our work in the context of this ERC-funded research, we developed a genetically encoded fluorescent biosensor that enables us for the first time to study orexin action and release mechanisms directly in the brain of living animals with high spatial and temporal resolution.
The new orexin biosensor named "OxLight1" is based on a specially designed green fluorescent protein integrated into one of the human orexin receptors. Engineering the receptor with this fluorescent protein makes it visible under the microscope. When the neuropeptide binds to this sensor it makes it "light up". OxLight1 thus offers practically a real-time outlook on orexin release.
We used this new biosensor to investigate the relationship between neuronal activity and neuropeptide release in living animals (mice), one of the most pressing and long-sought questions in neurophysiology that remained elusive until now.
By combining photometry imaging of orexin dynamics and neuronal activity recordings to score the sleep status of the animals, we observed for the first time that a rapid drop in orexin levels occurs during REM sleep of the mice. Further work with colleagues from the Istituto Italiano di Tecnologia in Italy, experts in two-photon microscopy, revealed another so far unknown process: spatially-localized orexin fluctuations occurring in the somatosensory cortex upon awakening from anesthesia.
Photocaged orexin-B:
We introduced the first photocaged orexin-B derivative (photo-OXB). Since OXB lacks functionalizable amino acids in its biologically relevant C-terminal region, we developed an approach for C-terminal photocaging of peptides, which should be directly applicable to other peptides that activate their cognate receptor via their C terminus. We demonstrated the compatibility between optical uncaging of OXB and biosensor imaging and used all-optical assays to functionally characterize photo-OXB light sensitivity and bioactivity. Finally, we showed that photo-OXB can be used for light-controlled activation of endogenous orexin receptors ex vivo by demonstrating the effect of its uncaging on the membrane potential of medium spiny neurons in acute brain slices. We expect that the photo-OXB tool described here will be rapidly adopted to investigate the multifaceted functions of orexin signaling in physiological or disease contexts. In addition, the caging strategy we developed, in combination with optical biosensor assays, will be a valuable resource for the broader chemical biology community as it allows for rapid development of photocaged peptides and their optical characterization. Read the article here: https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(22)00413-5
Orexins/hypocretins are neuropeptides carrying out critical neuromodulatory functions in the brain. Normally, they regulate arousal, wakefulness, motivation and appetite. Defects in the release or sensing of orexin neuropeptides cause, both in humans and in animals, a sleep disorder called narcolepsy. Affected individuals suffer from overwhelming daytime drowsiness and often exhibit cataplexic states, in which they remain conscious but are unable to control body movement.
Through our work in the context of this ERC-funded research, we developed a genetically encoded fluorescent biosensor that enables us for the first time to study orexin action and release mechanisms directly in the brain of living animals with high spatial and temporal resolution.
The new orexin biosensor named "OxLight1" is based on a specially designed green fluorescent protein integrated into one of the human orexin receptors. Engineering the receptor with this fluorescent protein makes it visible under the microscope. When the neuropeptide binds to this sensor it makes it "light up". OxLight1 thus offers practically a real-time outlook on orexin release.
We used this new biosensor to investigate the relationship between neuronal activity and neuropeptide release in living animals (mice), one of the most pressing and long-sought questions in neurophysiology that remained elusive until now.
By combining photometry imaging of orexin dynamics and neuronal activity recordings to score the sleep status of the animals, we observed for the first time that a rapid drop in orexin levels occurs during REM sleep of the mice. Further work with colleagues from the Istituto Italiano di Tecnologia in Italy, experts in two-photon microscopy, revealed another so far unknown process: spatially-localized orexin fluctuations occurring in the somatosensory cortex upon awakening from anesthesia.
Photocaged orexin-B:
We introduced the first photocaged orexin-B derivative (photo-OXB). Since OXB lacks functionalizable amino acids in its biologically relevant C-terminal region, we developed an approach for C-terminal photocaging of peptides, which should be directly applicable to other peptides that activate their cognate receptor via their C terminus. We demonstrated the compatibility between optical uncaging of OXB and biosensor imaging and used all-optical assays to functionally characterize photo-OXB light sensitivity and bioactivity. Finally, we showed that photo-OXB can be used for light-controlled activation of endogenous orexin receptors ex vivo by demonstrating the effect of its uncaging on the membrane potential of medium spiny neurons in acute brain slices. We expect that the photo-OXB tool described here will be rapidly adopted to investigate the multifaceted functions of orexin signaling in physiological or disease contexts. In addition, the caging strategy we developed, in combination with optical biosensor assays, will be a valuable resource for the broader chemical biology community as it allows for rapid development of photocaged peptides and their optical characterization. Read the article here: https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(22)00413-5
The sensors and photocaged peptide that we developed throughout the course of this ERC-funded research greatly advance our ability to investigate neurmodulator dynamics at high resolution in intact behaving animals. These technological advances make it possible to ask questions that we previously unaddressable, such as what is the precise relationship between neuronal activity and the release of specific neuropeptide molecules, or what are the real-time dynamics of these molecules in different brain areas. The orexin biosensor that we developed as part of this project is now being used to investigate physiological functions of orexins neuropeptides at high-resolution in laboratories around the world. The photocaged peptide (photo-OXB) that we developed is currently being used to investigate the causal link between orexin signaling and feeding/reward behaviors in living animals with unprecedented resolution.
This molecular toolbox also benefits drug discovery and preclinical validation efforts targeting orexin receptors, by making it possible to establish high-throughput screening assays for the identification of novel orexin agonists/antagonists, and to investigate the precise pharmacokinetic profiles of these drugs in vivo.
This molecular toolbox also benefits drug discovery and preclinical validation efforts targeting orexin receptors, by making it possible to establish high-throughput screening assays for the identification of novel orexin agonists/antagonists, and to investigate the precise pharmacokinetic profiles of these drugs in vivo.