Periodic Reporting for period 4 - LIFE synapses (Long-term Investigation of Functional Excitatory Synapses: Linking Plasticity, Network Wiring andMemory Storage)
Berichtszeitraum: 2021-10-01 bis 2022-09-30
The nature of the physical substrate of memory – or engram – is probably one of the longest studied mysteries in neuroscience, and yet it still remains elusive. In recent years, the search for the engram has gained new momentum due to the possibilities of selective activation and silencing of specific neurons in the brain. Recent studies suggest that the engram could be defined as the subset of neurons that is necessary and sufficient to cause recall of a specific memory when activated. But where is the engram when the neurons are not active? Most likely, ‘lasting alterations’ during memory formation are encoded in the connections between nerve cells, termed synapses. The connection pattern and strength of these synapses determine how information is flowing between nerve cells and thereby underlie memory recall.
The central aim of this project was to identify synapses participating in the engram and to study their morphological stability and functional properties, but also to identify general rules that determine the lifetime of synapses and their participation in information processing. We develop novel approaches to precisely control synaptic circuits and neuronal networks with light termed "optogenetics". We use these techniques to investigate functional synapses and neuronal populations in their native circuit over the time scale of weeks, relevant for memory encoding and long-term storage. Thus, by connecting functional long-term analysis of single synapses and neuronal circuits with morphological observations we filled a gap in our understanding of how synapses contribute to formation and storage of memories.
To solve the fundamental question, how synaptic connections contribute to information storage in the brain is essential if want to better understand how neurodegenerative diseases affect our cognitive capacities. A number of neuropathies are associated with loss or alterations of synapses. However, the causal relationship between synapse loss and cognitive decline is often not well understood. For example, it remains unclear whether loss of synapses is causative for cognitive disabilities or whether this is an epiphenomenon. Thus, to solve the question whether synapses are sites of memory storage is of high general relevance.
The central aim of this project was to identify synapses participating in the engram and to study their morphological stability and functional properties, but also to identify general rules that determine the lifetime of synapses and their participation in information processing. We develop novel approaches to precisely control synaptic circuits and neuronal networks with light termed "optogenetics". We use these techniques to investigate functional synapses and neuronal populations in their native circuit over the time scale of weeks, relevant for memory encoding and long-term storage. Thus, by connecting functional long-term analysis of single synapses and neuronal circuits with morphological observations we filled a gap in our understanding of how synapses contribute to formation and storage of memories.
To solve the fundamental question, how synaptic connections contribute to information storage in the brain is essential if want to better understand how neurodegenerative diseases affect our cognitive capacities. A number of neuropathies are associated with loss or alterations of synapses. However, the causal relationship between synapse loss and cognitive decline is often not well understood. For example, it remains unclear whether loss of synapses is causative for cognitive disabilities or whether this is an epiphenomenon. Thus, to solve the question whether synapses are sites of memory storage is of high general relevance.
During the first half of the project, we worked on technologies to control neurons with light (termed "optogenetics") at a wide optical spectrum and at different temporal scales. In parallel, we worked on improved optical methods to investigate synaptic transmission. Using fast imaging at single synapses together with patch-clamp electrophysiology and biophysical modeling, we measured novel, genetically encoded sensors of fast synaptic transmission. In another collaborative effort, we developed a new, genetically encoded optical sensor termed SynTagMA that enables identification of thousands of active synapses at the same time. We demonstrated its applicability in vivo as tool to map active synapses in the living mouse brain.
Moreover, we investigated the long-term consequences of synaptic plasticity on the stability of single synapses during the following days. This work demonstrates that strengthening synapses reduces their risk for elimination from the circuit. We further showed that sequential synaptic potentiation and depression interact over days to influence synapse stability, with the last plasticity event fully determining synaptic survival probability. The long-term consequence is that initial functional adaptations of synapses, seen as potentiation or depression of synaptic pathways are transformed into changes in circuit wiring, which, from a macroscopic point-of-view, have the same effect on synaptic pathways.
During the third funding period, we published a novel optogenetic tool termed BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing), for bidirectional control of neuronal activity. To better understand brain function and dysfunction, including neurological disorders, one must be able to manipulate nerve cells with high precision and specificity in the living organism. In principle, optogenetic manipulations allow the activation and inhibition of the same population of neurons to test their sufficiency and necessity for a particular brain function. However, existing optogenetic tools do not easily allow such bidirectional manipulations with light. BiPOLES overcomes these limits and enables multiple new applications including (1) potent excitation and inhibition of the same neurons, (2) exclusive dual-color excitation of two distinct neuronal populations, and (3) precise optical tuning of the membrane voltage. Due to its applicability in numerous model systems - such as worms, flies, mice and ferrets - BiPOLES fills an important gap in the optogenetic toolbox and might become the tool of choice to address a number of yet inaccessible problems in neuroscience.
Finally, we investigated how different anesthetics alter synaptic connections, neuronal activity and memories. Understanding how different anesthetics affect the brain, particularly the hippocampus, is important for both clinicians with human patients and experimental scientists who work with animals. We recorded brain activity from the hippocampus while mice were anesthetized using one of three common combinations of general anesthetics: isoflurane, ketamine/xylazine (Keta/Xyl), and medetomidine/midazolam/fentanyl (MMF). We found a number of differences in how the specific anesthetics affected the brain, synaptic connections and memory stabilization. Knowing these varying effects on the hippocampus and memory formation should be useful for doctors or experimenters when considering which method to use.
Moreover, we investigated the long-term consequences of synaptic plasticity on the stability of single synapses during the following days. This work demonstrates that strengthening synapses reduces their risk for elimination from the circuit. We further showed that sequential synaptic potentiation and depression interact over days to influence synapse stability, with the last plasticity event fully determining synaptic survival probability. The long-term consequence is that initial functional adaptations of synapses, seen as potentiation or depression of synaptic pathways are transformed into changes in circuit wiring, which, from a macroscopic point-of-view, have the same effect on synaptic pathways.
During the third funding period, we published a novel optogenetic tool termed BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing), for bidirectional control of neuronal activity. To better understand brain function and dysfunction, including neurological disorders, one must be able to manipulate nerve cells with high precision and specificity in the living organism. In principle, optogenetic manipulations allow the activation and inhibition of the same population of neurons to test their sufficiency and necessity for a particular brain function. However, existing optogenetic tools do not easily allow such bidirectional manipulations with light. BiPOLES overcomes these limits and enables multiple new applications including (1) potent excitation and inhibition of the same neurons, (2) exclusive dual-color excitation of two distinct neuronal populations, and (3) precise optical tuning of the membrane voltage. Due to its applicability in numerous model systems - such as worms, flies, mice and ferrets - BiPOLES fills an important gap in the optogenetic toolbox and might become the tool of choice to address a number of yet inaccessible problems in neuroscience.
Finally, we investigated how different anesthetics alter synaptic connections, neuronal activity and memories. Understanding how different anesthetics affect the brain, particularly the hippocampus, is important for both clinicians with human patients and experimental scientists who work with animals. We recorded brain activity from the hippocampus while mice were anesthetized using one of three common combinations of general anesthetics: isoflurane, ketamine/xylazine (Keta/Xyl), and medetomidine/midazolam/fentanyl (MMF). We found a number of differences in how the specific anesthetics affected the brain, synaptic connections and memory stabilization. Knowing these varying effects on the hippocampus and memory formation should be useful for doctors or experimenters when considering which method to use.
With our ability to chronically follow individual synapses with a known history both functionally and structurally over many days, we were able to investigate the structure-function relationship at single synapses over time scales that were not accessible until now. Having established an all-optical way to control synaptic plasticity allowed us to directly test the effects of plasticity on synapse survival. Usually, investigations of synaptic plasticity with single-synapse resolution are limited to only a few hours due to the invasive nature of commonly used recording techniques. Imaging methods that permit long-term investigations of synapses usually are limited to structural/morphological aspects, thus ignoring the functional state of the synapse. Our work showed that modifications of synaptic strength are not maintained over long time periods, but rather are converted to a "survival signal" instructing synapses whether to remain part of the circuit or to get removed. A second highlight is the development of a new optogenetic sensor to label thousands of active synapses at once, termed SynTagMA. This new probe overcomes limitations of commonly used technologies, such as acute two-photon calcium imaging, expanding the number of synapses accessible for analysis by orders of magnitude. Finally, the development of BiPOLES will enable many scientists to perform experiments that were not possible before. Inhibiting and activating the same neuronal populations enables them to test their sufficiency and necessity for a particular brain function. Aside from this, other manipulations become possible, such as all-optical voltage and excitability tuning of neurons either with single- or two-photon light.