Periodic Reporting for period 4 - NovelNMDA (Novel NMDA receptor signaling in cortical synaptic depression)
Período documentado: 2023-08-01 hasta 2025-01-31
In the mammalian brain, glutamate is the principle excitatory neurotransmitter. The NMDA receptor, a glutamate-gated ion channel, is essential for many forms of synaptic plasticity, including long-term depression. Synaptic plasticity is essential for experience-dependent changes in neuronal connectivity, and underlies the formation of receptive fields. Understanding how the plastic properties of synapses arise can help us understand how the developmental refinement of cortical representations of sensory information occurs, and whether it can be manipulated.
The relevance of NMDARs in long-term plasticity has been attributed to the flux of calcium through the NMDAR ion channel. However, recent work has shown that NMDARs may signal through a non-ionic mechanism in long-term depression. Understanding the functional role of NMDARs in synaptic plasticity is the goal of this research project.
To achieve this, we first aim to understand how long-term depression changes the synapses between cortical layer 4 and layer 2/3 neurons, and how NMDAR activity triggers those changes. We are applying functional imaging using 2-photon laser scanning microscopy, coupled with electrophysiological measurements to observe how single, identified synapses function and change after plasticity induction. As we develop approaches to study the activity of NMDARs in this cortical synapse, we are testing whether NMDARs in other synapse types behave in a similar manner, or whether there are unique properties in these synapses that are important for cortical experience dependent plasticity.
At the conclusion of this project, we found that non-ionic NMDAR signaling appears to be a general plasticity signaling mechanism that is present in multiple glutamatergic synapse types, and can be activated by multiple plasticity induction paradigms. In the cortical layer 4 to layer 2/3 synapses, long-term depression depends on non-ionic NMDAR signaling and alters the properties of presynaptic release. We found the role of cannabinoid signaling in this process does not induce further glutamate release, in contrast to previous models of long-term depression at this synapse type. We also found that short-term plasticity at these cortical synapses arises from a combination of two distinct release mechanisms, which may be independently target by modulatory and plasticity signaling. Altogether, we discovered new aspects of glutamatergic synaptic signaling at a cortical synapse and a previously overlooked role of NMDARs in synaptic plasticity.
The relevance of NMDARs in long-term plasticity has been attributed to the flux of calcium through the NMDAR ion channel. However, recent work has shown that NMDARs may signal through a non-ionic mechanism in long-term depression. Understanding the functional role of NMDARs in synaptic plasticity is the goal of this research project.
To achieve this, we first aim to understand how long-term depression changes the synapses between cortical layer 4 and layer 2/3 neurons, and how NMDAR activity triggers those changes. We are applying functional imaging using 2-photon laser scanning microscopy, coupled with electrophysiological measurements to observe how single, identified synapses function and change after plasticity induction. As we develop approaches to study the activity of NMDARs in this cortical synapse, we are testing whether NMDARs in other synapse types behave in a similar manner, or whether there are unique properties in these synapses that are important for cortical experience dependent plasticity.
At the conclusion of this project, we found that non-ionic NMDAR signaling appears to be a general plasticity signaling mechanism that is present in multiple glutamatergic synapse types, and can be activated by multiple plasticity induction paradigms. In the cortical layer 4 to layer 2/3 synapses, long-term depression depends on non-ionic NMDAR signaling and alters the properties of presynaptic release. We found the role of cannabinoid signaling in this process does not induce further glutamate release, in contrast to previous models of long-term depression at this synapse type. We also found that short-term plasticity at these cortical synapses arises from a combination of two distinct release mechanisms, which may be independently target by modulatory and plasticity signaling. Altogether, we discovered new aspects of glutamatergic synaptic signaling at a cortical synapse and a previously overlooked role of NMDARs in synaptic plasticity.
This ERC starting grant provided an opportunity to establish my laboratory at the European Neuroscience Institute Göttingen, and acquire the necessary equipment to complete this research project.
Objectives 1 and 2 focused on synaptic plasticity at the cortical layer 4 to layer 2/3 synapse. We tested the functional role of NMDA receptors in both spike-timing dependent plasticity (Objective 1) and low-frequency stimulation induced long-term depression (Objective 2), and found that glutamate signaling through NMDA receptors required for long-term depression is independent of ion flux through the NMDA receptor ion channel. This raised the question of how plasticity is expressed, either as postsynaptic or presynaptic changes. We found evidence for presynaptic changes occurring after plasticity induction, in contrast to long-term depression in hippocampal synapses, where plasticity arises through postsynaptic changes. This work formed the basis of a Master Thesis titled “Presynaptic expression of non-ionotropic NMDA receptor dependent LTD in the barrel cortex,” is being drafted into a manuscript, and has been presented at multiple scientific meetings and invited talks.
In addition to long-term plasticity, we observed a peculiar short-term plasticity time course at the cortical synapses that shows depression developing over time. To understand how this unique time course arises, and whether novel synaptic mechanisms exist in these synapses, we examined this more closely. We found that two release mechanisms coexist in individual synapses between layer 4 and layer 2/3 neurons, one with an initial high release probability that depresses after a synaptic event, and a separate release mechanism that requires the calcium sensor Synaptotagmin 7 which enhances release probability for a short while after an event. This was published in a manuscript titled “Synaptotagmin 7 sculpts short-term plasticity at a high probability synapse,” and has also been presented at conferences and invited talks.
Objective 3 uses optical methods to investigate NMDAR function at single synapses. We noticed that we were able to detect synaptic activity at voltages near the neuronal resting potential. This was surprising because the calcium was due to NMDAR activity, which is thought to be minimal at these negative voltages. We found that this NMDAR calcium signal did not require coincident depolarization; it was still present when AMPARs were blocked. This work was published in a manuscript titled, “Synaptic NMDA receptor activity at resting membrane potentials,” and the results have been presented at several scientific meetings.
Objective 4 addressed the potential role of non-ionic NMDAR signaling at other synapse types. We tested plasticity induction in hippocampal synapses, and whether NMDAR pharmacology can bias plasticity towards long-term depression. We also assessed NMDAR function during long-term depression in cerebellar parallel fiber to Purkinje cell synapses. This work formed the basis for a Master’s Thesis titled, “Synaptic plasticity modulation by NMDA receptor antagonists,” and has been presented at multiple meetings.
Objectives 1 and 2 focused on synaptic plasticity at the cortical layer 4 to layer 2/3 synapse. We tested the functional role of NMDA receptors in both spike-timing dependent plasticity (Objective 1) and low-frequency stimulation induced long-term depression (Objective 2), and found that glutamate signaling through NMDA receptors required for long-term depression is independent of ion flux through the NMDA receptor ion channel. This raised the question of how plasticity is expressed, either as postsynaptic or presynaptic changes. We found evidence for presynaptic changes occurring after plasticity induction, in contrast to long-term depression in hippocampal synapses, where plasticity arises through postsynaptic changes. This work formed the basis of a Master Thesis titled “Presynaptic expression of non-ionotropic NMDA receptor dependent LTD in the barrel cortex,” is being drafted into a manuscript, and has been presented at multiple scientific meetings and invited talks.
In addition to long-term plasticity, we observed a peculiar short-term plasticity time course at the cortical synapses that shows depression developing over time. To understand how this unique time course arises, and whether novel synaptic mechanisms exist in these synapses, we examined this more closely. We found that two release mechanisms coexist in individual synapses between layer 4 and layer 2/3 neurons, one with an initial high release probability that depresses after a synaptic event, and a separate release mechanism that requires the calcium sensor Synaptotagmin 7 which enhances release probability for a short while after an event. This was published in a manuscript titled “Synaptotagmin 7 sculpts short-term plasticity at a high probability synapse,” and has also been presented at conferences and invited talks.
Objective 3 uses optical methods to investigate NMDAR function at single synapses. We noticed that we were able to detect synaptic activity at voltages near the neuronal resting potential. This was surprising because the calcium was due to NMDAR activity, which is thought to be minimal at these negative voltages. We found that this NMDAR calcium signal did not require coincident depolarization; it was still present when AMPARs were blocked. This work was published in a manuscript titled, “Synaptic NMDA receptor activity at resting membrane potentials,” and the results have been presented at several scientific meetings.
Objective 4 addressed the potential role of non-ionic NMDAR signaling at other synapse types. We tested plasticity induction in hippocampal synapses, and whether NMDAR pharmacology can bias plasticity towards long-term depression. We also assessed NMDAR function during long-term depression in cerebellar parallel fiber to Purkinje cell synapses. This work formed the basis for a Master’s Thesis titled, “Synaptic plasticity modulation by NMDA receptor antagonists,” and has been presented at multiple meetings.
During the work towards this project, we have noticed several phenomena that went beyond our expectations.
First, during our synaptic imaging experiments, we noticed NMDAR calcium signals, even at potentials near the neuronal resting potential. This is counter to the traditional idea NMDARs pass calcium only if there is simultaneous depolarization. We found this was a robust phenomenon at cortical synapses, and also present in hippocampal synapses. These results have been published in a manuscript titled “Synaptic NMDA receptor activity at resting membrane potentials.”
A second unexpected observation was the peculiar short-term plasticity phenomenon at cortical synapses that arises from a combination of both facilitation and depression processes. This was beyond our expectations, as the cortical layer 4 to 2/3 synapse has been traditionally thought of as a high release probability, depressing synapse. We further found that the short-term dynamics were due to independent release properties which arose from single synaptic sites, and we were able to dissociate these release mechanisms by manipulating Synaptotagmin7. The short-term synaptic plasticity that we describe may be an important function for sensory cortical processing. These results have been published in a manuscript titled “Synaptotagmin 7 sculpts short-term plasticity at a high probability synapse.”
A third important outcome was to adapt a method of measuring extracellular glutamate concentration using NMDARs as sensitive, endogenous glutamate detectors. We found that this method could resolve nanomolar changes in glutamate concentration. This direct approach to measure glutamate can resolve questions about glutamate release that have been difficult to establish with previous indirect methods.
First, during our synaptic imaging experiments, we noticed NMDAR calcium signals, even at potentials near the neuronal resting potential. This is counter to the traditional idea NMDARs pass calcium only if there is simultaneous depolarization. We found this was a robust phenomenon at cortical synapses, and also present in hippocampal synapses. These results have been published in a manuscript titled “Synaptic NMDA receptor activity at resting membrane potentials.”
A second unexpected observation was the peculiar short-term plasticity phenomenon at cortical synapses that arises from a combination of both facilitation and depression processes. This was beyond our expectations, as the cortical layer 4 to 2/3 synapse has been traditionally thought of as a high release probability, depressing synapse. We further found that the short-term dynamics were due to independent release properties which arose from single synaptic sites, and we were able to dissociate these release mechanisms by manipulating Synaptotagmin7. The short-term synaptic plasticity that we describe may be an important function for sensory cortical processing. These results have been published in a manuscript titled “Synaptotagmin 7 sculpts short-term plasticity at a high probability synapse.”
A third important outcome was to adapt a method of measuring extracellular glutamate concentration using NMDARs as sensitive, endogenous glutamate detectors. We found that this method could resolve nanomolar changes in glutamate concentration. This direct approach to measure glutamate can resolve questions about glutamate release that have been difficult to establish with previous indirect methods.