Differentially interconnected networks of GABAergic interneurons in the visual cortex of the cat

Abstract

Networks of GABAergic neurons have been implicated in neuronal population synchronization. To define the extent of cellular interconnections, we determined the effect, number, and subcellular distribution of synapses between putative GABAergic neurons in layers II-IV of the cat visual cortex using paired intracellular recordings in vitro followed by correlated light and electron microscopy. All neurons having interneuronal electrophysiological properties were classified by their postsynaptic target profile and were identified as basket (BC; n = 6), dendrite-targeting (DTC; n = 1), and double bouquet (DBC; n = 2) cells. In four out of five anatomically fully recovered and reconstructed cell pairs, synaptic connections were found to be reciprocal. Generally BCs established synaptic junctions closer (21 +/- 20 micron) to postsynaptic somata than did DBCs (43 +/- 19 micron; p < 0.01). The unitary number of synapses (n values, 10, 7, and 20) in each of three BC-to-BC pairs was higher than that in three BC-to-DBC (n values, 1, 2, and 2) and three DBC-to-BC (n values, 1, 4, and 4) connections (p < 0.05). A BC innervated a DTC through two synaptic junctions. Unitary postsynaptic effects mediated by five BCs could be recorded in two BCs, two DBCs, and a DTC. The BCs elicited short-duration fast IPSPs, similar to those mediated by GABAA receptors. At a membrane potential of -55.0 +/- 6.4 mV, unitary IPSPs (n = 5) had a mean amplitude of 919 +/- 863 microV. Postsynaptic response failures were absent when an IPSP was mediated by several release sites. Thus, distinct GABAergic interneurons form reciprocally interconnected networks. The strength of innervation and the proximal placement of synapses suggest a prominent role for BCs in governing the activity of intracortical GABAergic networks in layers II-IV.

Fig. 1.

Reconstruction and synaptic effect of a BC-to-BC pair in layers II–III of area 18. A, B, Axonal (A) and dendritic (B) arborization of the presynaptic (black) and the postsynaptic (gray) BCs is shown. The axon of the postsynaptic cell was only partially recovered. C, The route of the presynaptic axon (black) to synaptic boutons on the postsynaptic cell (gray) is shown. Branch points are indicated by asterisks. D, The locations of electron microscopically verified synaptic junctions between the two BCs are indicated. Four synapses were found on the soma, and six synapses were on proximal dendrites. Three boutons established two distinct synaptic release sites each (grouped numbers). Ea, In response to (Figure legend continues).

Fig. 2.

Physiologically and anatomically identified BC-to-DTC connection in area 17. The sketch andnumbers on the top of the figure represent the synaptic and autaptic connectivity (straight arrow, synaptic connection; round arrows, autaptic connections) in the cell pair. A, Axonal pattern of the presynaptic BC (black) and the dendritic arbor of the postsynaptic DTC (gray) that was located at the margin of the presynaptic axonal arbor are shown.B, The axonal arbor of the postsynaptic DTC (gray) did not overlap with the dendritic tree of the presynaptic BC (black). C, (Figure legend continues).

Fig. 3.

Reciprocally connected BC-to-DBC pair in area 17. The sketch and numbers on thetop of the figure represent the synaptic and autaptic connectivity (arrows) in the cell pair.A, B, Axonal (A) and dendritic (B) arborization of the presynaptic BC (blue) and the postsynaptic DBC (red) is shown. C, The route of axonal branches of the BC and DBC to boutons establishing synaptic and autaptic junctions, all of which were verified by electron microscopy, is shown. The BC innervated the DBC through a single synapse (b-db) and formed eight autaptic junctions (blue; a1–a8). One of the autaptic terminals formed two separate synaptic junctions (a2,3). The DBC established one synaptic junction on the BC (db-b) and innervated itself via three autapses (red; a1–a3). Note that when the release sites formed by the BC are compared with those established by the DBC, the former targeted proximal parts, whereas the latter innervated more distal regions of the cell. D–F, Synaptic coupling could be tested electrophysiologically from the BC to the DBC.D, Action potentials (∼1 Hz) of the BC (Da) resulted in a short-latency IPSP (Db) with a mean amplitude of −0.13 ± 0.53 mV with the DBC being depolarized to a membrane potential of −57 mV.E, Amplitude distributions of IPSPs and baseline noise in the DBC show a slight shift of the IPSP distribution relative to the noise. F, Brief trains of presynaptic APs (Fa) elicited a summated response in the DBC (Fb) at a membrane potential of −57 mV.

Fig. 4.

Electron microscopic evidence of synaptic junctions between GABAergic neurons shown in Figures 1-3.A, Synapses between the two BCs illustrated in Figure 1.Aa, One of the two synaptic junctions (arrow) established by a bouton (b2,3) of the presynaptic BC on the soma (BCs) of the postsynaptic BC is shown. Ab, An axonal terminal of the presynaptic BC (b10) forms a synapse (arrow) on a dendrite (BCd) of the postsynaptic BC. An unlabeled terminal (t) also establishes a synaptic junction (arrowhead). B, One of the two synaptic junctions (arrow) formed by the same bouton (b1,2) of the presynaptic BC on the soma of theDTC illustrated in Figure 2. C, The two synaptic junctions from the reciprocally connected BC-to-DBC pair shown in Figure 3. Ca, A bouton (bc) of the BC establishes a synapse (arrow) on a dendrite of theDBC. Cb, In the reverse direction, a bouton (dbc) of the DBC formed a synaptic junction (arrow) on a dendrite of the BC. A neighboring unlabeled terminal (t) also established a synapse (arrowhead) on the postsynaptic dendrite. The numbering of presynaptic boutons corresponds to that in Figures 1D, 2D, and 3C, respectively. Scale bars, 0.2 μm, withB and C at the same magnification.

Fig. 5.

Reciprocally interconnected network of two BCs and a DBC in the medial bank of area 17. A, Light microscopic reconstructions of the three interneurons [basket cell 1 (BC1), white soma and dendrites, greenaxon; basket cell 2 (BC2), blue soma and dendrites,pink axon; double bouquet cell, red soma and dendrites, yellow axon] are shown.Arrows indicate the identified directions of connections (for details, see Figs. 6, 7); the numbers of synapses are given in circles. Each cell was in reciprocal connection with the other two, and the two BCs also innervated themselves. For the display of overlapping axonal and dendritic arborizations of the cells, layer III is shown expanded, maintaining the position of layer I relative to the DBC and that of layers IV–VI to the BCs. B, The topographically correct position of dendritic trees is illustrated. C, D, Functional synaptic coupling could be tested electrophysiologically in only two directions. Action potentials (1–2 Hz) of BC1 (Ca) and BC2 (Da) resulted in short-latency, fast IPSPs (Cb, Db) with mean amplitudes of −0.59 and −0.70 mV in BC2 and DBC, respectively. The latter were held at membrane potentials of −61 and −60 mV.

Fig. 6.

Route of axons to synaptic and autaptic boutons and subcellular placement of synapses and autapses within the trineuronal network established by two BCs and a DBC presented in Figure 5. A–C, Within the network. a total of 70 synapses and autapses were identified with correlated light and electron microscopy. Each panel shows a single presynaptic axon and all dendritic branches innervated by the cell.A, Synaptic and autaptic output of BC1.Aa, The route of the axon to its identified targets is shown (green). Ab, BC1 established one somatic (white; a6) and seven dendritic (a1–a5; a7,8) autapses, innervated BC2 via seven synaptic junctions (blue; two on the soma, b6,7; five on dendrites,b1–b5), (Figure legend continues).

Fig. 7.

Examples of electron microscopically verified synaptic and autaptic junctions within the trineuronal network of two BCs and a DBC presented in Figures 5 and 6. A, Synaptic and autaptic output of BC1. Aa, An axon terminal (a7,8) of BC1 establishes two separate autaptic junctions (arrows) on its own dendrite (BC1). Ab, Two separate synaptic junctions (arrows) between a bouton (b6,7) of BC1 and the soma (BC2) of BC2 are shown. Ac, A bouton (bd2) of BC1 forms a synaptic junction (arrow) on a dendrite (DBC) of the DBC. The convention of labeling presynaptic boutons corresponds to that of Figure 6Ab.B, Synaptic and autaptic output of BC2.Ba, An axon terminal (a19) of BC2 establishes an autapse(Figure legend continues).