Periodic Reporting for period 4 - nanoAXON (Nano-physiology of small glutamatergic axon terminals)
Reporting period: 2022-10-01 to 2024-03-31
Background: Our understanding of neuronal networks relies on knowledge about how single neuron activities encode information. However, the principles governing how single neurons extract information from the activity of single upstream neurons remained elusive, partly because investigating how individual axons contribute to this process was challenging.
Approach: In this project we investigated the principles of axonal signaling in the major glutamatergic pathways to and from the dentate gyrus region of the hippocampus. To achieve this, we employed cutting edge techniques, including direct patch clamp recordings from small axons, which were meticulously identified anatomically, computational simulations, and voltage- and Ca2+ imaging techniques. The unprecedented direct functional access to small axon terminals allowed us to investigate previously unknown general mechanisms.
Conclusions: As the detailed results below demonstrate, this project demonstrated several examples that axons are not simply devices that only relay neuronal information from the neuronal soma to synapses, but axonal signaling actively contributes to neuronal information processing.
Approach: In this project we investigated the principles of axonal signaling in the major glutamatergic pathways to and from the dentate gyrus region of the hippocampus. To achieve this, we employed cutting edge techniques, including direct patch clamp recordings from small axons, which were meticulously identified anatomically, computational simulations, and voltage- and Ca2+ imaging techniques. The unprecedented direct functional access to small axon terminals allowed us to investigate previously unknown general mechanisms.
Conclusions: As the detailed results below demonstrate, this project demonstrated several examples that axons are not simply devices that only relay neuronal information from the neuronal soma to synapses, but axonal signaling actively contributes to neuronal information processing.
The results were built into seven major works.
The first work has yielded a groundbreaking finding: axons employ uniform action potentials (APs). We examined axonal functions in novel ways by direct recording and voltage imaging from various axon types. Our results bridge a critical gap between two key aspects of basic neuroscience: the biophysics of AP signaling and the triggering of synaptic release. It was predicted that morphology imposes limitations on axonal voltage signals, resulting in substantial filtering in smaller elements, leading to unpredictability due to the large variability in diameter within individual axons. Consequently, if passive membrane principles are uncompensated, APs would vary, meaning that the presynaptic “digital” trigger would be different in every synapse depending on its size. It was not possible to measure these properties directly and resolve this contradiction. Surprisingly, we found that within individual fibers, APs do not depend on axon size. In fact, axons actively compensate for the biophysical limitations of size. Therefore, AP uniformity emerges as a fundamental rule essential for digital output functions of axons, and as such, it is involved in essentially all neuron-to-neuron communications.
We also measured AP-Ca2+ current coupling (AP-ICa coupling) directly in mossy fiber axons. This simpler work was published before the first work. There were examples in the literature showing that distinct neuron types use identical ion channels or synaptic proteins in various combinations to achieve cell type-specific excitability or synaptic properties. But what about complete modules, in which mechanisms cooperate for fundamental functions, such as the coupling between axonal APs and Ca2+ currents that translates presynaptic activity to Ca2+ influx that triggers synaptic release. We demonstrated that distinct postsynaptic cell types can utilize identical presynaptic axonal modules.
To address the correlation between axonal morphology and axonal signaling from a different perspective, and to better understand how glutamatergic pathways contribute to the activity in the dentate gyrus, in the next work we examined two types of perforant pathway axons, which are morphologically similar but contribute to different physiological functions. Here we revealed intriguing differences between lateral and medial perforant pathway axons (LPP and MPP) within the dentate gyrus region of the hippocampus. Within this work, first, using direct patch clamp recordings, we showed that axonal membrane properties and APs from LPP and MPP in the dentate gyrus region of the hippocampus are different. Next, we verified these observations with a new approach using axonal voltage imaging with the sensor, Voltron2. This results also suggested that LPP activity evokes large Ca2+ influx and reliable synaptic release. Finally, we identified the underlying molecular mechanisms of the differential signaling in LPP and MPP axons, which interestingly is also know to play crucial role in some forms of epilepsy and ataxias. This is a fundamental discovery because we demonstrated axonal properties significantly contribute to distinct physiological functions, allowing LPP and MPP to convey different information about the environment and objects, ultimately shaping distinct representations in the dentate gyrus. Thus, these results sheds light on the intricate interplay between axonal characteristics and functional diversity within the hippocampus.
Ensuring precise reporting of biological signals collected through direct axonal recordings was essential. The quantitative descriptions of signaling properties of small axons are scarce not only because of their limited visual and physical accessibility, but also because the comparable physical size and electrical contribution of the recording instrument and the biological structures substantially affects recorded signals. These technical obstacles prevented understanding of axonal signaling mechanisms. Therefore, we built a complex electrical model of recording instruments together with the biologically properties of axons, that allowed us to separate instrumental interference from biologically relevant signals. Thus, we could see how axonal APs would look (i.e. native APs) if the recording pipette was not there.
Mapping functional synaptic connectivity and understanding the contribution of individual glutamatergic traditionally rely on using a combination of patch clamp recordings and correlated anatomical identification (see above). However, this labor-intensive method has limitations. It can measure only a limited number of synaptic connections in detail that is not sufficient to map the contribution of individual pathways within a larger network capable of complex neuronal computations. Therefore, we evaluated the applicability of a voltage imaging technique in hippocampal slices to map unitary synaptic connections by using a new voltage sensor, which can detect both APs and small subthreshold events, such as unitary EPSPs. With this approach we can map the functional properties of connections between 10-40 neurons simultaneously. We draw the fundamental connectivity matrix of excitatory cells of hippocampus and resolved one of the major controversies about hippocampal connectivity. This innovative method not only opens new avenues for understanding synaptic connectivity but also shapes the future experimental direction of our research group.
In 6th work, we elucidated the contribution of specific channels to the excitability of hippocampal specific neurons. This work has important implications for the definition of the boundaries between cell types based on their activities and for understanding how target cells integrate glutamatergic inputs.
In a collaboration that is related to the topic of the project we helped characterize a protein that induces axon growth and synapse formation in the mossy cell to dentate granule cell-pathway that may explain the mechanisms of connectivity changes associated with temporal lobe epilepsy.
The first work has yielded a groundbreaking finding: axons employ uniform action potentials (APs). We examined axonal functions in novel ways by direct recording and voltage imaging from various axon types. Our results bridge a critical gap between two key aspects of basic neuroscience: the biophysics of AP signaling and the triggering of synaptic release. It was predicted that morphology imposes limitations on axonal voltage signals, resulting in substantial filtering in smaller elements, leading to unpredictability due to the large variability in diameter within individual axons. Consequently, if passive membrane principles are uncompensated, APs would vary, meaning that the presynaptic “digital” trigger would be different in every synapse depending on its size. It was not possible to measure these properties directly and resolve this contradiction. Surprisingly, we found that within individual fibers, APs do not depend on axon size. In fact, axons actively compensate for the biophysical limitations of size. Therefore, AP uniformity emerges as a fundamental rule essential for digital output functions of axons, and as such, it is involved in essentially all neuron-to-neuron communications.
We also measured AP-Ca2+ current coupling (AP-ICa coupling) directly in mossy fiber axons. This simpler work was published before the first work. There were examples in the literature showing that distinct neuron types use identical ion channels or synaptic proteins in various combinations to achieve cell type-specific excitability or synaptic properties. But what about complete modules, in which mechanisms cooperate for fundamental functions, such as the coupling between axonal APs and Ca2+ currents that translates presynaptic activity to Ca2+ influx that triggers synaptic release. We demonstrated that distinct postsynaptic cell types can utilize identical presynaptic axonal modules.
To address the correlation between axonal morphology and axonal signaling from a different perspective, and to better understand how glutamatergic pathways contribute to the activity in the dentate gyrus, in the next work we examined two types of perforant pathway axons, which are morphologically similar but contribute to different physiological functions. Here we revealed intriguing differences between lateral and medial perforant pathway axons (LPP and MPP) within the dentate gyrus region of the hippocampus. Within this work, first, using direct patch clamp recordings, we showed that axonal membrane properties and APs from LPP and MPP in the dentate gyrus region of the hippocampus are different. Next, we verified these observations with a new approach using axonal voltage imaging with the sensor, Voltron2. This results also suggested that LPP activity evokes large Ca2+ influx and reliable synaptic release. Finally, we identified the underlying molecular mechanisms of the differential signaling in LPP and MPP axons, which interestingly is also know to play crucial role in some forms of epilepsy and ataxias. This is a fundamental discovery because we demonstrated axonal properties significantly contribute to distinct physiological functions, allowing LPP and MPP to convey different information about the environment and objects, ultimately shaping distinct representations in the dentate gyrus. Thus, these results sheds light on the intricate interplay between axonal characteristics and functional diversity within the hippocampus.
Ensuring precise reporting of biological signals collected through direct axonal recordings was essential. The quantitative descriptions of signaling properties of small axons are scarce not only because of their limited visual and physical accessibility, but also because the comparable physical size and electrical contribution of the recording instrument and the biological structures substantially affects recorded signals. These technical obstacles prevented understanding of axonal signaling mechanisms. Therefore, we built a complex electrical model of recording instruments together with the biologically properties of axons, that allowed us to separate instrumental interference from biologically relevant signals. Thus, we could see how axonal APs would look (i.e. native APs) if the recording pipette was not there.
Mapping functional synaptic connectivity and understanding the contribution of individual glutamatergic traditionally rely on using a combination of patch clamp recordings and correlated anatomical identification (see above). However, this labor-intensive method has limitations. It can measure only a limited number of synaptic connections in detail that is not sufficient to map the contribution of individual pathways within a larger network capable of complex neuronal computations. Therefore, we evaluated the applicability of a voltage imaging technique in hippocampal slices to map unitary synaptic connections by using a new voltage sensor, which can detect both APs and small subthreshold events, such as unitary EPSPs. With this approach we can map the functional properties of connections between 10-40 neurons simultaneously. We draw the fundamental connectivity matrix of excitatory cells of hippocampus and resolved one of the major controversies about hippocampal connectivity. This innovative method not only opens new avenues for understanding synaptic connectivity but also shapes the future experimental direction of our research group.
In 6th work, we elucidated the contribution of specific channels to the excitability of hippocampal specific neurons. This work has important implications for the definition of the boundaries between cell types based on their activities and for understanding how target cells integrate glutamatergic inputs.
In a collaboration that is related to the topic of the project we helped characterize a protein that induces axon growth and synapse formation in the mossy cell to dentate granule cell-pathway that may explain the mechanisms of connectivity changes associated with temporal lobe epilepsy.
As explained above, we published 4 papers during the project and 3 additional papers will be published after the end of the project.