Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents

Abstract

Fast inhibition in the cortex is gated primarily at GABAergic synapses formed by local interneurons onto postsynaptic targets. Although GABAergic inputs to the somata and axon initial segments of neocortical pyramidal neurons are associated with direct inhibition of action potential generation, the role of GABAergic inputs to distal dendritic segments, including spines, is less well characterized. Because a significant proportion of inhibitory input occurs on distal dendrites and spines, it will be important to determine whether these GABAergic synapses are formed selectively by certain classes of presynaptic cells onto specific postsynaptic elements. By electron microscopic observations of synapses formed by different subtypes of nonpyramidal cells, we found that a surprisingly large fraction (33.4 +/- 9.3%) of terminals formed symmetrical synaptic junctions onto a subset of cortical spines that were mostly coinnervated by an asymmetrical terminal. Using VGLUT1 and VGLUT2 isoform of the glutamate vesicular transporter immunohistochemistry, we found that the double-innervated spines selectively received thalamocortical afferents expressing the VGLUT2 but almost never intracortical inputs expressing the VGLUT1. When comparing the volumes of differentially innervated spines and their synaptic junction areas, we found that spines innervated by VGLUT2-positive terminal were significantly larger than spines innervated by VGLUT1-positive terminal and that these spines had larger, and more often perforated, synapses than those of spines innervated by VGLUT1-positive afferent. These results demonstrate that inhibitory inputs to pyramidal cell spines may preferentially reduce thalamocortical rather than intracortical synaptic transmission and are therefore positioned to selectively gate extracortical information.

Figure 1.

Cortical nonpyramidal cells used in this study. Nine nonpyramidal cells were studied for synaptic target structures using 3D reconstructions of successive EM sections. Red indicates axons, and black indicates soma and dendrites. WA, Wide arbor cell; BS, basket cell; MA, Martinotti cell; RSNP, regular spiking nonpyramidal.

Figure 2.

Synaptic target structures for cortical nonpyramidal cells. A, Successive ultrathin sections showing a symmetrical synapse (white arrow) of the LS NG cell onto a postsynaptic soma. B, Successive ultrathin sections showing a symmetrical synapse (white arrow) from an RSNP DA-CRF cell onto a dendritic shaft with frequent asymmetrical inputs (black arrows). C, Successive ultrathin sections showing a symmetrical synapse (white arrow) from a BSNP DA-CR cell onto a spine head, which is also innervated by an asymmetrical synapse (black arrow). Scale bar in A also applies to B and C.

Figure 3.

3D reconstructed image of the synaptic structure of cortical nonpyramidal cells. A, 3D reconstructed image of the synaptic structure shown in Figure 2A. The axonal terminal of the LS NG cell (red) innervates the soma (green). Right image shows 3D image of targeted soma after removing the bouton. The synaptic junction is shown (pink). B, 3D reconstructed image of synaptic structure shown in Figure 2B. Arrows indicate the same axon terminal boutons shown in Figure 2B. Note that no spines are observed in the target dendrite (green). Red is an axon terminal of RSNP DA-CRF cell. Blue structures are boutons forming asymmetrical synapses. Yellow structures are boutons forming symmetrical synapses. C, 3D reconstructed image of synaptic structure shown in Figure 2C. An axon terminal of BSNP DA-CR cell (red) innervates the spine head (green), which is also innervated by an asymmetrical synaptic terminal (blue). Right image without boutons shows two synaptic junctions (pink and dark blue). Scale bar in A also applies to B and C.

Figure 4.

Summary of target structures of nonpyramidal cell boutons. All types of nonpyramidal cells targeted spine heads, most of which were double-innervated (DI) spines. MA, Martinotti cell.

Figure 5.

VGLUT-immunoreactive fiber distributions in successive neocortical coronal sections. A, VGLUT1-positive fibers were distributed throughout all cortical layers. B, VGLUT2-positive fibers were dense in upper layer I, layer IV, and in the lower half of layer V. Barrel cortex was identifiable in both sections shown in A and B (arrowheads). Cortical areas were divided into frontal cortex area 1 (Fr1), frontal cortex area 2 (Fr2), and parietal cortex area 1 (Par1) according to Paxinos and Watson (1998). Scale bar in A apples to B. C, Higher-magnification photographs showing cell architecture by Nissl staining and VGLUT1- and VGLUT2-immunoreactive fiber distributions in successive neocortical coronal sections. Scale in the Nissl and VGLUT2 micrographs is the same as in the VGLUT1 micrograph. Str, Striatum; WM and W, white matter.

Figure 6.

Cortical spine innervated by a VGLUT1-positive terminal. A–N, Successive ultrathin sections of an entire spine head that was innervated by a VGLUT1-positive axon terminal. A single asymmetrical synapse was observed (arrow in F).

Figure 7.

Cortical spine innervated by a VGLUT2-positive terminal. A–H, Successive ultrathin sections of a spine head innervated by a VGLUT2-positive axon terminal (black arrow in F). Two additional symmetrical synapses contacted this spine head (white arrows in F).

Figure 8.

Synaptic innervations of cortical spines. A, A cortical spine (Sp) was coinnervated by a VGLUT2-positive (T2) asymmetrical synapse (black arrow) and two symmetrical synaptic terminals (white arrows) and was shown in Figure 7F. B, A cortical spine (Sp) was innervated by a VGLUT1-positive (T1) asymmetrical synapse (black arrow) and was shown in Figure 6F. C, A spine head (Sp) was innervated by both a VGLUT2-positive (T2) asymmetrical synapse (black arrow) and a GABA-positive (gold particle labeled) symmetrical synaptic terminal (white arrow). D, A spine head (Sp) innervated by an asymmetrical synapse (black arrow) was also innervated by a symmetrical synaptic terminal (white arrow). Gold particles label GABA_A α1 subunits localized along the synaptic junction of the symmetrical synapse. E, A spine head (Sp) innervated by an asymmetrical synapse (black arrow) was also innervated by a symmetrical synaptic terminal (white arrow). Larger gold particles (15 nm) labeled GABA_A β2/3 subunits localized along the synaptic junction of the symmetrical synapse and smaller particle (10nm) labeled glutamate receptor GluR2/3 subunits of the associated asymmetrical synaptic junction. Scale bars: A applies to B and C; E applies to D. F, 3D reconstructed image of the spine and presynaptic terminals shown in A. Bottom image is the VGLUT2-positive bouton (T2, black) contacting the spine head (Sp, light gray) with a synaptic junction. This spine is also innervated by two symmetrical inputs (Sym, dark gray). Top image shows synaptic junctions (black arrow for VGLUT2 synapse and white arrows for symmetrical synapses) on the spine head. G, 3D reconstructed image of the spine and presynaptic terminal shown in B. The VGLUT1-positive bouton (T1, black) contacts the spine head (SP, light gray) with a synaptic junction. Left image is with the VGLUT1 bouton (black), and right image shows the synaptic junction only (black).

Figure 9.

A, B, Summary of spine head volume (A) and synaptic junction area (B) of VGLUT1- and VGLUT2-innervated spines. Mean and SD of volume of different spines types innervated by VGLUT1 and VGLUT2. *p < 0.0001; ^#p < 0.01. T1, VGLUT1; T2, VGLUT2; SI, single-innervated spine; DI, double-innervated spine; PE, spine innervated by perforated synapse; NP, spine innervated by nonperforated synapse. C, 3D reconstructed images of VGLUT2-innervated spines. Spines with nonperforated (NP), perforated (PE), U-shape (U), or twin-junctional (TJ) synapse.

Figure 10.

Electron micrograph showing a perforate synapse (Sp1) corresponding with 3D image “T2 PE” shown in Figure 9C. A–L, Successive ultrathin sections of an entire spine head (Sp1) that was innervated by a VGLUT2-positive axon terminal. An asymmetrical synapse was observed, and the middle of the successive ultrathin sections postsynaptic junction was split in two parts (E–G) and again merged together, indicating a typical perforated synapse. A second synapse (Sp2) formed by the VGLUT2-positive axon terminal (onto different postsynaptic target) is also a perforated synapse because the postsynaptic density is split into two parts in J–L.

Figure 11.

Schematic summary of the GABAergic input to dendritic spines. Most VGLUT1-positive axon terminals originate from cortical cells (purple) and innervate spines of cortical pyramidal neurons (gray) that receive no secondary synaptic input. VGLUT2-positive axon terminals (green) originate from the thalamus and innervate larger spine heads of pyramidal cells (gray) that exhibit a second, GABAergic synaptic input (orange) in ∼10% of cases.