Gap junctions among dendrites of cortical GABAergic neurons establish a dense and widespread intercolumnar network

Abstract

Gap junctions are common between cortical GABAergic interneurons but little is known about their quantitative distribution along dendritic profiles. Here, we provide direct morphological evidence that parvalbumin-containing GABAergic neurons in layer 2/3 of the cat visual cortex form dense and far-ranging networks through dendritic gap junctions. Gap junction-coupled networks of parvalbumin neurons were visualized using connexin36 immunohistochemistry and confocal laser-scanning microscopy (CLSM). The direct correspondence of connexin36-immunopositve puncta and gap junctions was confirmed by examining the same structures in both CLSM and electron microscopy. Single parvalbumin neurons with large somata (> or =200 microm2) formed 60.3 +/- 12.2 (mean +/- SD) gap junctions with other cells whereby these contacts were not restricted to proximal dendrites but occurred at distances of up to 380 microm from the soma. In a Sholl analysis of large-type parvalbumin neurons, 21.9 +/- 7.9 gap junctions were within 50 microm of the soma, 21.7 +/- 7.6 gap junctions in a segment between 50 and 100 microm, 11.2 +/- 4.7 junctions between 100 and 150 microm, and 5.6 +/- 3.6 junctions were in more distal segments. Serially interconnected neurons could be traced laterally in a boundless manner through multiple gap junctions. Comparison to the orientation-preference columns revealed that parvalbumin-immunoreactive cells distribute randomly whereby their large dendritic fields overlap considerably and cover different orientation columns. It is proposed that this dense and homogeneous electrical coupling of interneurons supports the precise synchronization of neuronal populations with differing feature preferences thereby providing a temporal frame for the generation of distributed representations.

Correlated CLSM-EM images of Cx36-labeled gap junctions. A, PV-IR neuron in layer 5 of area 18, shown in a projected image from a stack file, gives rise to a descending dendrite that forms a dendrodendritic contact at a distal site (arrow). B, Enlargement of the contact site in A, visualized by dual CLSM for PV (green) and Cx36 (red). Two Cx36-IR punctate structures (1, 2) are located between the contacting dendrites (asterisks). C, Immunoelectron micrograph of the same structures shown in B. The contacting dendrites (asterisks) demarcated by open squares and circles, as well as another neighboring profile (arrowhead), can be identified with DAB labeling for PV. Enlarged views of the contact sites (1, 2) clearly indicate ultrastructural profiles of gap junctions at the positions corresponding to Cx36-IR puncta in B. Scale bars: A, 50 μm; B, C, 1 μm; right panels, 0.1 μm.

Figure 2.

Direct confirmation of Cx36-labeled gap junction in dual labeled CLSM-EM. A, B, A Cx36-IR spot (red) is located at the contact site between PV-IR dendrites (green). C, Electron micrograph of the contact site in A with DAB labeling for PV and silver-enhanced Fluoronanogold labeling for Cx36. D, Enlargement of the contact site showing gap junction and silver grains on both sides. Scale bars: A, B, 1 μm; C, D, 0.1 μm.

Figure 3.

Size distributions of heterogeneous PV neurons in layer 2/3 of cat area 18. A, Cross-sectional areas of somata of Cx36-negative (filled columns) and the entire (open columns) populations of PV neurons. B, Somatic areas of CB/PV double-positive (filled) and the entire population of PV neurons. C, CLSM image, shown in a projected image from a stack file, demonstrates CB (green) and PV (red) immunoreactivity. One small cell (arrow) is double-labeled. Scale bar, 50 μm.

Figure 4.

Distal gap junctions. A–C, PV neuron (arrowhead) in the deepest layer 3 (layers are represented as roman numerals) of area 18, shown in a projected image from a stack file, gives rise to an ascending dendrite, along which Cx36-IR puncta (insets, red) are observed at the contact sites (asterisks) between PV-IR dendrites (insets, green). The most distal contact (site 1), located 380 μm away from the soma at the most superficial part of layer 2, shows the ultrastructure of the gap junction in C. Continuity of this ascending dendrite was ascertained in serial ultrathin sections in EM. D, E, PV neuron (cyan arrowhead) in layer 6 of area 18, shown in a projected image from a stack file, has many Cx36-labeled contact sites (asterisks), both proximally (sites 6–12) and distally (sites 4, 5, 13). Part of the contact sites are shown in colors (insets). Scale bars: A, B, D, E, 100 μm; insets, 1 μm; C, 0.1 μm.

Figure 5.

Single-cell analysis of the distribution of Cx36-IR contact sites. A, Reconstruction of a PV neuron in layer 2/3 of area 18. Small circles along dendrites indicate 59 Cx36-IR contact sites. Shells with different radii (50–200 μm) are used for analyzing the distribution by the Sholl method. B, Comparison of the number of Cx36-IR contact sites (top), dendritic length (middle), and density of contact sites per unit of dendritic length (1 mm; bottom) for 50-μm-wide segments. Data are averaged values from nine cells; error bars indicate SD.

Figure 6.

Reconstruction of a dendritic network of PV neurons linked by gap junctions in layer 2/3 of area 18. Cells shown are linked to one another through Cx36-IR contact sites, with the interconnectivity and number of mutual contacts depicted in the inset. Different symbols along dendrites indicate contact sites belonging to the traced cells. Note that all of these sites have contacting partners, only a small part of which were traced and included in the graph. Scale bar, 100 μm.

Figure 7.

Spatial relationship between an orientation preference map and the two-dimensional distribution of PV neurons in layer 2/3 of area 18. All PV neurons residing in layer 2/3 with large (≥200 μm²; open circles) and small (<200 μm²; closed circles) somata are superimposed on the orientation map.

Figure 8.

Random distribution of PV neurons. A, A rectangle centered on an orientation singularity in Figure 7, 400 × 400 μm in size, contains 344 PV neurons including 63 L-type cells. The rank of u₁ (see supplemental Fig. 1, available at www.jneurosci.org as supplemental material) among 99 Monte Carlo tests for L-type cells and the entire population is 53 and 22, respectively, both indicating a random distribution pattern. B, The empirical distribution function for observed cells (the entire population) in A (blue line) is within the envelope between U(y) and L(y), also suggesting a random distribution (see supplemental Fig. 2, available at www.jneurosci.org as supplemental material). C, A square located between two singularities in Figure 7, 400 × 400 μm in size, contains 330 PV neurons, including 55 L-type cells. The rank of u₁ among 99 Monte Carlo tests for L-type cells and the entire population is 71 and 50, respectively, indicating random distribution. D, The empirical distribution function for observed cells (the entire population) in B (blue line) is within the envelope.

Figure 9.

Spatial relationship between an orientation preference map and dendritic fields of L-type PV neurons. A, Dendrites arising from five PV neurons are reconstructed and shown in arbitrary colors. Note the substantial overlap of their large dendritic fields that extend across different orientation columns. Scale bar, 150 μm. B, Circles with radii of 150 μm are centered on L-type PV neurons. Each circle is colored according to the position of the parent soma. Note the dense overlap between the dendritic arbors of neurons located in different orientation compartments.