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Rapid Ultrastructural Changes of PSD and Extrasynaptic Axon-spine Interface Membrane during LTP Induced in Single Dendritic Spine

Ye Sun, Michael Smirnov, Naomi Kamasawa, Ryohei Yasuda

Preprint posted on November 13, 2019 https://www.biorxiv.org/content/10.1101/840629v1

Shining a light on the synapse: using targeted glutamate uncaging and electron microscopy to study changing dendritic spines and synaptic strengthening

Selected by Alyson Smith

Why I think this study is interesting

Dendritic spines, small protrusions from the dendrites of neurons, are primary sites of neuron-to-neuron communication. Spines can grow, change shape, or disappear depending on synaptic input or lack thereof, allowing them to mediate neuronal circuit formation and memory storage. Spines are tiny (less than a femtoliter), making them difficult to study in the crowded environment of the brain. This research coupled light and electron microscopy to track individual spine changes over time, providing a clearer picture of how spines respond to synaptic input. The methods used in this preprint open the door to detailed study of the molecular mechanisms behind synapse function.  

Background

Most excitatory neuronal synapses form between an axon terminal or bouton of one neuron and a dendritic spine of another neuron. Each spine forms a postsynaptic density, an electron-dense area containing scaffolding proteins, ion channels, and neurotransmitter receptors that facilitates signal transmission across the synapse (Nimchinsky, et al 2002).

Repeated stimulation (e.g., exposure to the excitatory neurotransmitter glutamate) changes spine structure: the spine and postsynaptic density grow larger, the spine head expands, the postsynaptic density segments, local protein translation increases, and multiple synapses may form at single spines. These changes enable activity-induced strengthening of synaptic transmission, the key process behind the development of neural circuits and memory storage (Sala and Segal 2014). 

To study spine changes, scientists expose large numbers of neurons to glutamate or other stimuli (in culture, brain slices, or live animals) and then measure average spine and/or postsynaptic density attributes by light microscopy (standard or super-resolution) or electron microscopy. Some approaches, such as correlated light and electron microscopy, integrate multiple modalities to measure spine changes at multiple length scales. To directly link spine stimulation with changes in spine attributes, scientists have used 2-photon laser pulses to uncage 4-methoxy-7-nitroindolinyl (MNI)-glutamate within 0.5 micrometers of individual spines and then looked for spine changes by electron microscopy (Bosch et al., 2014; Meyer et al., 2014).

The authors of this preprint used sparse labeling of neurons with GFP, 2-photon laser pulse glutamate uncaging, and an automated tape-collecting ultramicrotome to speed up identification of stimulated spines for imaging by high-resolution electron microscopy (Kamasawa et al., 2015). They applied this method to quantify changes in stimulated spines at multiple time points: 2-3 minutes, 20 minutes, and 2 hours following glutamate uncaging. They compared stimulated spines to control spines nearby on the same dendrites.  

From Figure 1 of the preprint: (A-C) 2-photon glutamate uncaging (at red dot) of a GFP positive CA1 pyramidal neuron causes growth in the target spine. (D) Electron microscopy image of a section from the stimulated spine. (E) 3D reconstruction of stimulated spine (yellow) and postsynaptic density (red). Scale: 1 micrometer for B and C, 0.5 micrometers for D and E.

 

Key findings

Glutamate has rapid effects that fade over time

By the early time point (2-3 minutes post glutamate uncaging), stimulated spines had developed larger volumes, larger surface areas, and larger postsynaptic densities than controls. These differences faded over time and disappeared by the 2 hour time point. 

In all spines, the postsynaptic density area was proportional to the total spine volume. In stimulated spines, the slope of this relationship was lower compared to controls at the early and intermediate timepoints but the slopes became similar by the 2 hour time point. These data show that a single, localized glutamate stimulus induces large changes in spine size and smaller changes in postsynaptic density size that eventually return to pre-stimulus levels.

Glutamate increases postsynaptic density complexity

Synaptic strengthening is associated with segmentation of the postsynaptic density (Harris et al., 1989). The authors of this preprint identified simple, fenestrated, horseshoe, and segmented postsynaptic densities in their sections, as well as densities with irregular shapes. At all time points after glutamate uncaging, 60-70% of stimulated spines had complex postsynaptic density shapes, while only 20-40% of control spines did. Like the previous measurements, postsynaptic density complexity (perimeter2/4𝜋 x area) decreased over time, although the differences remained statistically significant at 2 hours. 

From Figure 3 of preprint: examples of postsynaptic density morphologies. Scale: 200 nanometers.

At the intermediate and late time points, less than one percent of segmented postsynaptic densities contacted more than one presynaptic terminal and thus could receive input from more than one neuron. It is therefore possible that density segmentation allows complex synaptic connections to form. 

Glutamate increases the potential for later synaptic strengthening

By 2-3 minutes after glutamate uncaging, stimulated spines had greater areas of contact with the axon terminal membrane (within 40 nanometers, the typical size of a synaptic cleft). This difference persisted at 2 hours, at which point over 20 percent of the spine membrane was in contact with axon terminal. 

The authors define the extrasynaptic axon-spine interface (eASI) as the spine membrane area that contacts the axon terminal (within 40 nm) but lacks postsynaptic density. This area contains neurotransmitter receptors and may be able to support synaptic transmission. Like the total membrane contact area, the eASI was larger in stimulated spines at all time points the authors tested.  

From Figure 4 of preprint: An image of a spine section highlighting the extrasynaptic axon-spine interface (eASI) in cyan. Scale: 200 nanometers. 

 

By 2 hours after glutamate uncaging, the axon terminals near stimulated spines had more presynaptic neurotransmitter vesicles than those near control spines. Thus, the early increases in spine size, area of contact between pre- and postsynaptic membranes, and complexity of postsynaptic densities following glutamate stimulation likely initiate later phases of synaptic strengthening by recruiting more neurotransmitters and neurotransmitter receptors to the synapse.

Future directions

In this study, the authors bypass normal synaptic transmission by uncaging glutamate one time near a specific dendritic spine. In vivo, spine maturation and synaptic strengthening depend on multiple stimulations and can take hours to days to occur. The methods used in this preprint can be applied to uncage glutamate multiple times and characterize spines and synaptic densities at later time points. These experiments could establish a quantitative relationship between amount of stimulation and the attributes measured in this study.

This study suggests that increases in the extrasynaptic axon-spine interface (eASI) enable later increases in postsynaptic density size and synaptic strength. Immunoelectron microscopy could be used to test this hypothesis by tracking the recruitment of neurotransmitter receptors to the eASI before and after glutamate stimulation.

References

Nimchinsky, E.A., Sabatini, B.L., Svoboda, K., 2002. Structure and Function of Dendritic Spines. Annu. Rev. Physiol. 64, 313–353. 

Sala, C., and Segal, M. (2014). DENDRITIC SPINES: THE LOCUS OF STRUCTURAL AND FUNCTIONAL PLASTICITY. Physiol Rev 94, 48. 

Bosch, M., Castro, J., Saneyoshi, T., Matsuno, H., Sur, M., and Hayashi, Y. (2014). Structural and molecular remodeling of dendritic spine substructures during long-term potentiation. Neuron 82, 444– 459.

Meyer, D., Bonhoeffer, T., and Scheuss, V. (2014). Balance and Stability of Synaptic Structures during Synaptic Plasticity. Neuron 82, 430–443.

Kamasawa, N., Sun, Y., Mikuni, T., Guerrero-Given, D., and Yasuda, R. (2015). Correlative Ultrastructural Analysis of Functionally Modulated Synapses Using Automatic Tape-Collecting Ultramicrotome – SEM Array Tomography. Microsc. Microanal. 21.

Harris, K.M., and Stevens, J.K. (1989). Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982–2997.

Questions for the authors

You describe your imaging method as high throughput. How does the throughput compare to the previous papers (Bosch et al., 2014; Meyer et al., 2014)?

In the introduction, you mention that previous studies found that PSD area and bouton volume increase slowly, over several hours. Did you look at spine ultrastructure at any time points after 2 hours? 

Your study included large numbers (50-80) of control spines and small numbers (10-13) of stimulated spines. Did you account for this sample size discrepancy in your statistical analysis? Also, is it technologically feasible to increase the number of stimulated spines studied? 

Would it be possible to stimulate a single spine with two-photon glutamate uncaging more than once before fixation and imaging? If so, how do you think the number of glutamate uncagings would relate to changes in the parameters you studied?

Tags: 2p microscopy, clem, correlative light and electron microscopy, learning and memory, long term potentiation, ltp, psd

Posted on: 24th November 2019

doi: https://doi.org/10.1242/prelights.15291

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