Network architecture of gap junction-coupled neuronal linkage in the striatum

Abstract

Previous studies have revealed the existence of gap junctions between GABAergic interneurons of a particular type in the striatum. Because of the technical difficulties, however, there is no information about their positions within the striatal circuitry. We have developed a method to detect neuronal gap junctions reliably at the light microscopic level and thereby explored the network architecture of the gap junctional linkage. Gap junction-coupled networks among parvalbumin-containing GABAergic interneurons extended nonuniformly in the feline striatum. They were located predominantly in the methionine-enkephalin-poor matrix. Moreover, the density of gap junctional coupling showed a marked regional difference along the anterior-posterior axis of the striatum. The densest interconnectivity was found in the posterior part of both caudate nucleus and putamen that corresponds to the sensory-recipient area of the feline striatum. Electron microscopic observations provided clear evidence of internalization of neuronal gap junction, indicating the dynamic nature of gap junctional linkage between neurons in vivo. The nonuniform organization of gap junction networks suggests differential modes of information processing in heterogeneous subregions of the striatum.

Figure 1.

Identification of neuronal gap junctions in the striatum. A, Two PV-immunoreactive dendrites (d) make direct contact with each other. B, Enlargement of the contact site in A, demonstrating a typical ultrastructure of gap junction. C, Dual CLSM image showing Cx36 (red)-immunoreactive punctuate structure located at the contact site (arrow) between two PV (green)-immunoreactive dendrites (asterisks), one running horizontally while the other perpendicular to the image. D, The same contact site shown in C was re-examined in EM by converting fluorescent signals for PV to DAB reaction products. High-power view (inset) indicates strict correspondence of Cx36 labeling in CLSM to gap junction in EM. E–L, Functional and dynamic aspects of gap junctional coupling. After taking dual CLSM image (PV green; Cx36 red), the same contacting dendrites (d) were identified by EM (G), reconstructed from serial ultrathin sections (F), and confirmed to form gap junction between them (H). Blue dots on the surface of reconstructed dendrites in F indicate sites of synaptic contacts. Red arrowheads in G and H demarcate gap junction. Serial ultrathin sections (I–L) next to H demonstrate a presumed internalization of gap junction (blue arrows). Images in J and K are taken from the same specimen with different tilt angles for EM observations. Coated pits are visible around the internalized membranes in K. M, Enlargement of the framed area (bottom) in L. Note hexagonal array of spot-like reaction products. N, Enlargement of the framed area (top) in L, showing an alternating pattern of reaction products along the central line of the gap junction. Scale bars: A, C, G, 1 μm; B, 50 nm; D, 0.5 μm; inset, 100 nm; E, F, 10 μm; H-L, 100 nm; M, N, 10 nm.

Figure 2.

Gap junctions establish a dense network of PV neurons in the posterior putamen. Images are taken from the area shown in supplemental Figure 1B, available at www.jneurosci.org as supplemental material, at +11 in Horsely–Clarke coordinate. A–G, Dendrites arising from three somata (No. 1–3) come close to one another (triangles), making direct contacts between cells 1 and 2 (B, C) as well as between cells 2 and 3 (E, F). Exactly at these contact sites are Cx36-positive gap junctions (D, G). H, Computer-assisted reconstruction of 11 PV neurons including the three in A. All these neurons are directly connected to one another through gap junctions. Different symbols along dendrites indicate sites of gap junctions. Detailed interconnectivity is depicted in supplemental Figure 3, available at www.jneurosci.org as supplemental material, and the actual CLSM image of a part of this tracing is shown in supplemental Figure 2, available at www.jneurosci.org as supplemental material. I, Enlargement of the central part of H. Note that not only dendrites belonging to the reconstructed 11 neurons but also all contacting pairs that were traced from all Cx36-positive contact sites (different symbols) are included in the drawing here, although many of the paired dendrites were traced only in short segments. Scale bars: A, H, 100 μm; B–G, 10 μm; I, 50 μm.

Figure 3.

PV neuron networks avoid mENK-rich patches. A, B, PV immunoreactive structures (green) are nonuniformly distributed in the caudate nucleus with their somata and dendrites located predominantly outside the patches that are recognizable as mENK (red)-rich zones. Images are taken at +13 in Horsely–Clarke coordinate. C–F, High-power view confirms preferential localization of PV immunoreactivity in the mENK-poor matrix. Although the patch contains a few thinner dendrites, the greater part of dendritic plexus is confined to the matrix. Note the course of thick dendrites arising from somata located near the patch/matrix border. Scale bars, 100 μm.

Figure 4.

The distribution of PV (green) and mENK (red) immunoreactivities in the striatum at +16 in Horsely–Clarke coordinate. A, C, PV immunoreactivity has a gradient along the dorsolateral-ventromedial axis of the caudate nucleus (Cd). CC, Corpus callosum; IC, internal capsule; LV, lateral ventricle; Pu, putamen; S, septum; VP, ventral pallidum. B, mENK-rich patches are rather indistinct and irregularly shaped in both the ventromedial sector of the caudate nucleus and the ventral striatum (nucleus accumbens, NAc). mENK-rich patches are also indistinct in the putamen. D, E, Enlargement of the framed area in A. Scale bars, 1 mm.

Figure 5.

Quantitative analyses on the nonuniform organization of PV neuron networks in the caudate nucleus. Abscissa indicates Horsely–Clarke coordinates. A, The numerical density of PV neuron somata in the mENK-poor matrix (M) is higher than that in the mENK-rich patches (P) at most of the anterior–posterior locations. Asterisks indicate significant difference (p < 0.05, Mann–Whitney test). B, Total dendritic length contained per unit volume in the mENK-poor matrix is also higher than that in the mENK-rich patches at all anterior–posterior levels (p < 0.05, Mann–Whitney test). C, Density of gap junctions in the mENK-poor matrix shows marked regional difference along the anterior–posterior axis (p < 0.05, Friedman test). Dotted lines indicate data from three animals, whereas thick line their average.

Figure 6.

Dense gap junctional network in the matrix of the posterior caudate nucleus (+13 in Horsely–Clarke coordinate). Five reconstructed cells shown here are connected through gap junctions that are indicated by different symbols. In contrast, on the left side of red dotted line is the patch where some PV-immunoreactive dendrites are seen but devoid of gap junctional linkage. Scale bar, 100 μm.