Massive autaptic self-innervation of GABAergic neurons in cat visual cortex

Abstract

Autapses are transmitter release sites made by the axon of a neuron on its own dendrites. We determined the numbers and precise subcellular position of autapses on different spiny and smooth dendritic cell types using intracellular biocytin filling in slices of adult neocortex. Potential self-innervation was light microscopically assessed on 10 pyramidal cells, 7 spiny stellate cells, and 41 smooth dendritic neurons from cortical layers II-V. Putative autapses occurred on each smooth dendritic neuron and on seven pyramids, but not on spiny stellate cells. However, electron microscopic examination of all light microscopically predicted sites on pyramids (n = 28) showed only one case of self-innervation with two autapses on dendritic spines. Interneurons were classified by postsynaptic target distribution () and all putative autapses of seven basket, three dendrite-targeting, and three double bouquet cells were scrutinized. All basket and dendrite-targeting cells established self-innervation, the number of autapses being 12 +/- 7 and 22 +/- 12 (mean +/- SD), respectively; only one of the double bouquet cells formed autapses (n = 3). Basket cell autapses (n = 74) were closer to the soma (12.2 +/- 22.3 microm) than autapses established by dendrite-targeting cells (51.8 +/- 49.9 microm; n = 66). The degree of self-innervation is cell type-specific. Unlike on spiny cells, autapses are abundant on GABAergic basket and dendrite-targeting interneurons, with subcellular location similar to that of synapses formed by the parent cell on other neurons. The extensive self-innervation may modulate integrative properties and/or the firing rhythm of the neuron in a manner temporally correlated with its own activity.

Fig. 2.

Electron microscopic demonstration of autaptic connections established by BCs. A, Two self-innervating boutons (a1, a2) on the soma (s) of the BC shown in Figure1A–C. Ai, Aii, The autaptic junctions were identified by the rigid membrane apposition and the widening of extracellular space (between arrows) and the clustering of vesicles. Numbering is the same as in Figure 1C. B, Correlated LM (Bi) and EM of a self-innervating bouton (a2) targeting the soma (s) of the parent BC 1102941. The autaptic junction is shown betweenarrows (Bii). C, A bouton (b) of BC 0812942 forms a membrane apposition (star) with its own axon initial segment (ais) without autaptic specialization. In a serial section (Ci), the same bouton establishes a synaptic junction on an unlabeled soma (s). Scale bars:A, Ai, Aii, Bii, C, Ci, 0.2 μm; Ai, Aii, Bii, same magnification; C, Ci, same magnification; B, 2 μm; Bi, 10 μm.

Fig. 3.

Exclusively dendritic self-innervation established by a DTC in area 18. A, Dendritic (red) and axonal (black) arborization of the DTC.B, Location of electron microscopically verified (see Fig. 4) autaptic innervation by boutons. All autapses were on dendrites at various distances from the soma. One bouton established two separate autaptic junctions (10, 11). The cell showed a synaptic target preference toward dendritic shafts (72.7%) and also innervated spines (27.3%) of other neurons. Asterisks indicate axonal branching points.

Fig. 4.

Correlated LM and EM of autaptic junctions established by the DTC shown in Figure 3. A, A myelinated axonal branch (ax) gives rise to six en terminaux boutons (a1–a5 and unlabeled arrows) apposed to a distal dendrite (d) of the parent cell. Unmarked arrows indicate autaptic boutons not shown by EM on this figure. B, Three of the autaptic boutons shown in A are seen emerging from the myelinated axonal trunk (ax) and target successive dendritic beads.Bi–Biii, The three autaptic junctions ofB are illustrated at higher magnification.C, A self-innervating bouton targeting the interbead segment of the parent dendrite is surrounded by unlabeled terminals (t) establishing synapses (betweenarrowheads). The numbering is identical to Figure3B. Autaptic clefts are indicated betweenarrows. Scale bars: A, 10 μm;B, 1 μm; Bi–Biii (same magnification),C, 0.3 μm.

Fig. 6.

Examples of autaptic junctions established by the DTCs shown in Figure 5. A, B, Autapses on the DTC of Figure 5A. A, An autaptic terminal (a 15, 16,17) targeting the soma (s) established three separate autaptic junctions, two of which (underlined numbers) are illustrated here.B, A bouton (a2) of the DTC forms an autapse on the dendrite of the cell. C, One (underlined number) of the two autapses established by a terminal (a 6,7) of the DTC shown in Figure 5B. A synapse (between arrowheads) formed by an unlabeled bouton (t) and the dendrite (d) of the DTC are indicated. Numberingis identical to Figure 5. Autaptic clefts are indicated betweenarrows. Scale bar for all panels, 0.2 μm.

Fig. 8.

The autaptic junctions established by the pyramidal cell shown in Figure 7. A, B, The axon of the pyramidal cell (ax) formed two closely located en terminaux autaptic boutons (a1, a2), which innervated two neighboring dendritic spines (s). Autaptic junctions are indicated between arrows. Scale bar for both panels, 0.2 μm.

Fig. 1.

Self-innervation by BCs. A–C, Light microscopic reconstructions of a large BC in layer III of area 17. The soma and dendrites are illustrated in red; axons are shown in black. Although the axonal cloud is relatively sparse in the neighborhood of the soma, the parent cell body is heavily innervated by high-order axonal branches. B, Route of the axon back to the parent soma. C, All electron microscopically verified autaptic junctions were located on, or very close to, the cell body. Three boutons established more than one autaptic junction, as indicated by groupednumbers. D, Autaptic junctions of a different type of BC in layer III. The dendritic arborization is shown in red; the axonal branches forming autapses are presented in black. The complete axonal arborization is illustrated by Buhl et al. (1997, their Fig. 3). E, Subcellular distribution of autaptic junctions. Both cells have higher autaptic target preference toward their soma than in the overall synaptic target distribution on other neurons. Asterisksindicate axonal branching points.

Fig. 5.

Comparison of synaptic and autaptic connections established by individual interneurons. A, Synaptic and autaptic connections of a layer IV DTC (soma and dendrites,red; axon, black) and a spiny stellate cell (soma and dendrites, green; axon not shown). The complete axonal and dendritic arborizations are shown by Tamás et al., (1997, their Fig. 6). The DTC innervated the spiny cell through three dendritic synapses (s1–s3) and established 32 autapses (a1–a32). Grouped numbersrepresent multiple autaptic junctions formed by the same bouton. Ten autapses were found on the soma, a higher proportion than the somatic targets (10.7%) in the overall synaptic target distribution.B, Synaptic and autaptic relationships between a layer IV DTC (soma and dendrites, red; axon,black) and a layer III–IV border pyramidal cell (soma and dendrites, green; axon, blue). The complete axonal and dendritic arborizations are shown by Buhl et al., (1997, their Fig. 1). The cell pair was in reciprocal synaptic connection, but only the effect of the pyramidal cell could be recorded. The DTC established nine autaptic junctions (a1–a9), innervated the pyramidal cell via 17 synapses (i1–i17), and received five synapses from the pyramid (p1–p5). Note that the subcellular distribution of synaptic and autaptic junctions established by the interneuron was similar on both cells.

Fig. 7.

Reconstruction of the only self-innervating pyramidal cell of 10 tested. A, Dendritic and axonal arborization of the pyramidal cell from area 17. B, Location of the two electron microscopically verified autaptic junctions on the pyramidal cell. Autapses were formed by two neighboring boutons of a seventh order axon collateral on two adjacent dendritic spines on a fourth order dendrite. For correlated EM of the connection, see Figure 8. Asterisks indicate axonal branching points.