Synapse-specific retrograde communication revealed by optical quantal analysis
1. Problem
Synapses provide the basis for neuron-to-neuron and neuron-to-muscle communication. From the classical works of Bernard Katz and contemporaries onwards, we have gathered a set of hallmark features that define synaptic transmission, in which presynaptic neurons release neurotransmitters that affect the excitability of postsynaptic neurons (1–5). Endocannabinoids are one of the few contrasting classes of neurotransmitter that allows postsynaptic neurons to retrogradely signal to presynaptic neurons. This retrograde cell-cell communication is based on the release of endocannabinoids from postsynaptic spines, which cross the synaptic cleft and activate the presynaptic receptor CB1, regulating neurotransmitter release. Although the endocannabinoid system has been the center of attention due to its psychoactive properties and its various therapeutic potentials in neurological disorders in the central nervous system – chiefly epilepsy(6) – it is unclear how CB1 is regulated at individual synapses. Are specific synapses prone to CB1 modulation? If so, what are their functional and structural properties? As synapses do not simply conduct signals between neurons, but instead have an active role in coding information(7,8), synapse-specific release properties determine the range of activities neuronal networks can perform(6, 9, 10). A major obstacle to answering these questions is that electrophysiological recording – the gold standard functional essay for synaptic activity – typically measures the aggregate transmission from dozens to thousands of synapses and so blurs the effect of endocannabinoids, missing the diversity present in synapses.
2. Rationale
We overcome this limitation, by capitalizing on recent breakthrough methods in quantal synaptic imaging developed in Volynski’s and Isacoff’s lab(11–13). By combining classical patch-clamp electrophysiology with advanced live fluorescence imaging of glutamate – the main excitatory neurotransmitter in the brain – we can follow transmission at nearly 100 synapses simultaneously with an unprecedented spatiotemporal resolution at the level of single vesicle release, or quanta (Figure 1; example of application shown in Video 1).
Figure 1: Summary of methodological approach: visualizing transmission in multiple synapses with high spatiotemporal resolution. a) Pyramidal neuron expressing the iGluSnFR sensor in a primary mouse cortical neuronal culture is selected for patch-clamp recordings. b) Zoomed axon shown in (a). c,d) Expanded ROI in (b), showing the release profile of individual synapses (#1-17). Note that each bouton displays distinct release properties, even though they belong to the same axonal arbor and are responding to the same 5Hz stimulation (vertical dashed lines indicate AP). Adapted from Mendonca et al., 2022, Nat. Comm.
Video 1: Example of how quantal optical analysis was used to dissect the nature of asynchronous release in a heterogeneous population of synapses. Adapted from Mendonca et al., 2022, Nat. Comm.
3. Details of suggested approach
Using state-of-the-art imaging of glutamate, combined with electrophysiology and pharmacology, I propose to address three key points:
a. How are individual synapses affected by acute CB1 activation?
Background: Presynaptic release sites contain a complex transmitter release machinery based on synaptic vesicles (on the plasma membrane and in the cytoplasm) and various regulatory proteins(14). The amounts and spatial distribution of these components vary greatly between synapses as does the probability of vesicle release in response to an action potential (Pr), and the molecular distributions are correlated to Pr(13, 15–17), providing a molecular logic for how transmission diversity is generated. We will ask whether the manner and degree of modulation of synapses by CB1 are associated with Pr. This would allow us to understand how the endocannabinoid system is differentially regulated between synapses; how endocannabinoids affect the signal processing properties of synapses; and uncover in which conditions it can act as a “circuit breaker” to halt the progress of seizures(18).
Experiment: My previously developed methodology allows to obtain Pr of an entire population of synapses simultaneously, and has already been used to investigate how Pr is associated with the timing of neurotransmitter release(11). Here, we will use a similar approach in primary neuronal cultures from wild-type mice transfected with iGluSnFR – a fluorescence sensor specific to glutamate based on GFP(19). Whole-cell patch clamp recordings of pyramidal neurons are combined with fast imaging of transmission in multiple synapses in the same axon. After obtaining the basal release properties, CB1 agonists (e.g. WIN55) will be added to the bath, allowing us to investigate how CB1 changes the release profile in individual boutons.
b. Is tonic CB1 activation differentially regulated in synapses from the same axonal arbor?
Background: A remarkable property of the endocannabinoid system is that CB1 can be persistently activated in some cells, effectively muting the synaptic outputs of these neurons(6, 7). Tonic CB1 receptor activation has been suggested to mediate the long-lasting effects of cannabinoids in the cortex(7). CB1 antagonists effectively convert these silent synapses into high-fidelity ones, revealing a possible two-state switch in synaptic activity. It is not clear if endocannabinoid release is narrowly regulated at individual postsynaptic sites and acts only at the apposed presynaptic site, or if the effect is more widespread so that multiple neighboring synapses are co-regulated. Moreover, are synapses with specific activity profiles prone to phasic or tonic CB1 activation? Understanding these properties will reveal the fundamentals of how CB1 regulates synaptic activity.
Experiment: Tonic CB1 effects will be studied using CB1 antagonists (e.g. AM251), which will be acutely applied in the bath to uncover synapses silenced by CB1. The well-established iGluSnFR imaging essay will be used as the main readout for the functional synaptic profile. In the second stage, we will investigate how tonic CB1 pathway is homeostatically regulated. Does persistent CB1 activation lead to long-term synaptic plasticity adjustments? This will be achieved by incubating CB1 agonists in primary neuronal cultures (48-72h) and comparing the release profile before and after removal of CB1 activation from the system (using acute CB1 antagonist application).
c. Can synaptic molecular markers predict the effect of CB1 activation?
Background: Most recently, the Isacoff lab has developed advanced imaging methods allowing the detection of neurotransmitter release sites in the fly neuromuscular junction, while mapping the structural properties using super-resolution 3D molecular reconstructions of each release zone(12). We aim to adapt this unique approach to mammalian neurons to understand the molecular underpinnings of Pr diversity and potential CB1 modulation diversity between synapses. These findings may provide mechanistic insights into how CB1 regulates activity in a different population of synapses.
Experiment: After extracting the release profile of individual synapses with the iGluSnFR sensor, the neuronal culture coverslips will be fixed and stained for proteins located in the presynaptic machinery, including synapsin, synaptotagmin 1-7 isoforms, and CB1 receptors. We will use the algorithms established by Isacoff’s lab to align super-resolution reconstructed images to functional images and evaluate how the density of these molecular markers is associated with CB1 effects.
4. How it will affect the broader field
Mammalian presynaptic boutons are small (~1μm) and mediate remarkably fast transmission (from presynaptic action potential to postsynaptic current takes ~4ms). Therefore, associating individual synaptic structures to function has been a tantalizing objective and a constant technical challenge in neurobiology. However, new optical methods now provide faster imaging, better sensors, and improved molecular detection. We aim to take full advantage of these methods to dissect the endocannabinoid system – one of the most remarkable and ubiquitous cell-cell communication systems in the brain – to uncover how it regulates activity at a single synapse level and to reveal the organizing principles of retrograde synaptic transmission. The endocannabinoid system is a source of promising avenues for untreated neurological disorders; therefore, it is crucial to understand how CB1 controls release from synapses coming from the same axonal arbor to help tailor effective therapies in the future.
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