Ultrastructural, Molecular and Functional Mapping of GABAergic Synapses on Dendritic Spines and Shafts of Neocortical Pyramidal Neurons - PubMed (original) (raw)

Ultrastructural, Molecular and Functional Mapping of GABAergic Synapses on Dendritic Spines and Shafts of Neocortical Pyramidal Neurons

Taekyung Kwon et al. Cereb Cortex. 2019.

Abstract

The location of GABAergic synapses on dendrites is likely key for neuronal integration. In particular, inhibitory inputs on dendritic spines could serve to selectively veto or modulate individual excitatory inputs, greatly expanding the computational power of individual neurons. To investigate this, we have undertaken a combined functional, molecular, and ultrastructural mapping of the location of GABAergic inputs onto dendrites of pyramidal neurons from upper layers of juvenile mouse somatosensory cortex. Using two-photon uncaging of GABA, intracellular labeling with gerphyrin intrabodies, and focused ion beam milling with scanning electron microscopy, we find that most (96-98%) spines lack GABAergic synapses, although they still display GABAergic responses, potentially due to extrasynaptic GABA receptors. We conclude that GABAergic inputs, in practice, contact dendritic shafts and likely control clusters of excitatory inputs, defining functional zones on dendrites.

Keywords: 3D electron microscopy; focused ion beam milling and scanning electron microscopy; gerphyrin; intrabodies; two-photon; uncaging.

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Figures

Figure 1.

Figure 1.

Two-photon RuBi-GABA uncaging. (a) Experimental setup for two-photon RuBi-GABA uncaging (b) Typical two-photon uncaging experiment with RuBi-GABA, showing a cortical layer 2/3 pyramid loaded with Alexa Fluor 594. The red dot indicates site of two-photon uncaging. Scale bar: 10 μm. (c) (top panel) Average (black) and standard deviation (gray) of 20 successive two-photon uncaging responses. (bottom panel) Average response induced by two-photon uncaging with application of RuBi-GABA (left), after washing RuBi-GABA (middle), and recovery of the response in reapplication of RuBi-GABA (right). (d) Normalized amplitude of responses induced by two-photon uncaging at different excitation wavelengths. Inset shows an example of responses from the same cell at 800 nm (top, brown), 750 nm (middle, magenta), and 725 nm (bottom, orange). (e) Peak two-photon uncaging response amplitude before, during (gray bar), and after the application of GABA antagonists (gabazine and phaclofen). Insets show the average trace for each condition. Black bars under traces indicate the uncaging laser pulse. (f) (top panel) _XY_-axis resolution of two-photon uncaging: initial response with perisomatic uncaging point (left), response after 1 μm lateral displacement of the uncaging point away from the cell (middle), and response after 2 μm lateral displacement (right). (bottom panel) _XY_-axis resolution of one-photon uncaging: initial response with perisomatic uncaging point (left), response after 5 μm lateral displacement of the uncaging point away from the cell (middle), and response after 15 μm lateral displacement (right). One-photon RuBi-GABA uncaging was performed as described in Rial Verde et al. (2008). (g) Normalized amplitude of responses induced by two-photon (filled circles) and one-photon (empty triangles) uncaging at different distances from the cell soma. Scale bars: 5 pA, 50 ms. Black bars under traces indicate the uncaging laser pulse.

Figure 2.

Figure 2.

Functional GABA receptors mapping in soma and dendrites with two-photon RuBi-GABA uncaging. (a) Somatic mapping. This example shows eight different uncaging sites on the same cell. Points 5 and 6 are at the same XY coordinates, but at different Z planes. Scale bars: 5 pA, 50 ms, 10 μm. Black bars under traces indicate uncaging laser pulse. RuBi-GABA was applied through a glass pipette. (b) Dendritic mapping. Red traces are uncaging responses from stimulation points at red dots, which are on same level and side from dendrite. Green traces are uncaging responses from control stimulations at green dots, ~1 μm away from respective red dots. These reduced control responses reveal a spatial resolution of two-photon RuBi-GABA uncaging is ~1 μm. We observed RuBi-GABA uncaging response everywhere in dendrite with variability in response amplitude. Scale bar: 2 μm.

Figure 3.

Figure 3.

RuBi-GABA uncaging reveals GABAergic hotspots. (a) Two-photon RuBi-GABA uncaging grid map of dendritic GABAergic responses. 153 uncaging pulses were done with 2 s intervals. (b) Representative uncaging responses with stimulations indicated in (a). (c) Pseudocolor map of peak uncaging response peak amplitudes. (d_–_e) Examples of functional GABA receptor mapping in dendrite. Note functional hotspots of GABA receptors on dendritic shafts. Spine with a white arrow in (d) had clear uncaging response. Scale bar: 2 μm.

Figure 4.

Figure 4.

GABAergic hotspots are correlated with gerphyrin hotspots. (a) Schematic drawing of DNA construct and protein structure of gephyrin intrabody. (b) (left) Experimental time line for molecular mapping of GABA receptors by performing in utero electroporation of gephyrin intrabody. (right) Schematic image how in utero electroporation was performed. (c) (left) Molecular labeling of gephyrin intrabody. A gephyrin punctum (or a molecular hotspot for a putative inhibitory synapse) is shown in the right dendritic branch of image. (middle) Functional GABA receptor mapping was done and a functional hotspot is shown on the right side of image. (right) Overlaid images of (left) and (middle) panels show molecular hotspot and functional hotspot are at the same location. Scale bar: 2 μm. (d) another example overlaying molecular hotspot and function hotspot (e) spatial correlation between molecular and functional hotspots in (c) shown as uncaging response as a function of distance from center of mass spatial coordinates of molecular hotspot.

Figure 5.

Figure 5.

Gerphyrin intrabodies mapping of inhibitory synapses. (a_–_d) Molecular hotspots are mainly in dendritic shafts. Occasionally, molecular hotspots are found in dendritic spines or in both dendritic spines and shaft (see middle spot in d). Scale bars: 2 μm.

Figure 6.

Figure 6.

Ultrastructural mapping of synapses by FIB/SEM. (a_–_c) Three nonconsecutive serial sections of the neuropil of the mouse somatosensory cortex (layer 2/3) showing examples of asymmetric (excitatory) and symmetric (inhibitory) synapses. The numbers at the right bottom corners indicate the order of each photomicrograph in the series. Individual section thickness was 20 nm, so the examples shown here are separated by 60 nm. An asymmetric synapse (AS, green arrowheads) is established on a dendritic spine (Sp). A symmetric synapse (SS, red arrowheads) is established on the shaft of the parent dendrite. (d) Snapshot of Espina, the software tool that has been used for the automatic segmentation of synaptic junctions based on the gray level of synaptic densities. Each individual synaptic junction is given a unique number (left panel) and is color-coded as asymmetric (green) or symmetric (red)(central panel). The stack of images can be navigated in 3D to identify the postsynaptic target of each individual synapse as either a dendritic spine or shaft. To help with this task, the program also performs interactive digital reslicing through orthogonal planes (right middle and bottom panels) and three-dimensional reconstructions of synaptic junctions (upper right panel). (e) Three-dimensional reconstruction of the synaptic junctions present inside a stack of neuropil. Asymetric synaptic junctions are represented in red and symmetric synaptic junctions in green. See also Supplemental videos 1 and 2. Scale bar in (a), 1000 nm for (a_–_c).

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