Distinct GABAergic targets of feedforward and feedback connections between lower and higher areas of rat visual cortex

Abstract

Processing of visual information is performed in different cortical areas that are interconnected by feedforward (FF) and feedback (FB) pathways. Although FF and FB inputs are excitatory, their influences on pyramidal neurons also depend on the outputs of GABAergic neurons, which receive FF and FB inputs. Rat visual cortex contains at least three different families of GABAergic neurons that express parvalbumin (PV), calretinin (CR), and somatostatin (SOM) (Gonchar and Burkhalter, 1997). To examine whether pathway-specific inhibition (Shao and Burkhalter, 1996) is attributable to distinct connections with GABAergic neurons, we traced FF and FB inputs to PV, CR, and SOM neurons in layers 1-2/3 of area 17 and the secondary lateromedial area in rat visual cortex. We found that in layer 2/3 maximally 2% of FF and FB inputs go to CR and SOM neurons. This contrasts with 12-13% of FF and FB inputs onto layer 2/3 PV neurons. Unlike inputs to layer 2/3, connections to layer 1, which contains CR but lacks SOM and PV somata, are pathway-specific: 21% of FB inputs go to CR neurons, whereas FF inputs to layer 1 and its CR neurons are absent. These findings suggest that FF and FB influences on layer 2/3 pyramidal neurons mainly involve disynaptic connections via PV neurons that control the spike outputs to axons and proximal dendrites. Unlike FF input, FB input in addition makes a disynaptic link via CR neurons, which may influence the excitability of distal pyramidal cell dendrites in layer 1.

Figure 1.

CR- and SOM-immunolabeled neurons in rat visual cortex. A, B, Laminar distribution of CR neurons in area 17 and the secondary visual area, LM. Most CR neurons are located in superficial layers. Layer 1 contains CR somata, but lacks SOM (D, E) and PV (Fig. 2C) cell bodies. C, CR stains cell bodies including the entire dendritic tree. Dendritic spines (arrows) are rare. The neuropil contains numerous labeled axons and boutons (arrowhead). D, E, Laminar distribution of SOM neurons in areas 17 and LM. F, SOM immunolabeling reveals soma, primary, and secondary dendrites. The neuropil contains labeled axons and boutons (arrowheads). Scale bars: (in E) A, B, D, E, 100 μm; (in F), C, F, 20 μm.

Figure 7.

SOM and CR neurons in layer 2/3 of rat visual cortex are GABAergic and receive inputs from GABAergic and non-GABAergic synapses. A, Coexpression of SOM (dark amorphous DAB reaction product) and GABA immunogold particles in cell body, which receives inputs from symmetric GABAergic (AT2, arrow) and asymmetric, GABA-negative synapse (AT1) synapses. B, Coexpression of CR and GABA in cell body that receives asymmetric synapse from GABA-negative, type 1 axon terminal (AT1). C, Large SOM/GABA double-labeled axon terminal (SOM AT) which forms symmetric synapse (arrow) on SOM/GABA double-labeled cell body. D, SOM-labeled GABAergic axon terminals (SOM AT) forming symmetric synapses (arrows) with SOM-negative/GABA-negative cell body. E, Anterogradely biocytin-labeled (dark TMB/tungstate crystal) FF axon terminal in close association with SOM-immunoreactive dendrite (SOM) forming asymmetric synapse (AT1) with nearby unlabeled dendrite (D). Scale bars: A-E, 0.5 μm.

Figure 3.

PV, CR, and SOM expression in different types of GABAergic neurons that are differentially innervated by FF and FB axons. A, Triple labeling with antibodies against PV (red), CR (green), and SOM (blue) in layer 2/3 of rat area 17 showing that each cell is labeled by only a single marker. Scale bar, 25 μm. B, C, Confocal images of anterogradely biocytin-labeled FF and FB axons (green) forming multiple putative synaptic contacts (arrows) with PV neurons (red) in layer 2/3 of areas LM (B) and 17 (C). Scale bar: (in C) B, C, 10 μm. D, Biocytin-labeled FF axons (green) making infrequent contacts (arrow) with SOM (red) neuron in layer 2/3 of area LM. E, Biocytin-labeled FB axons (green) making occasional contacts (arrows) with CR (red) neuron in layer 2/3 of area 17. F, FB axons (green) in layer 1 of area 17 strongly innervate CR (red) neurons. Arrows indicate putative synaptic contacts. Scale bar: (in F) D-F, 10 μm.

Figure 2.

Confocal microscopic images of putative synaptic FF and FB inputs to CR and SOM expressing GABAergic neurons in rat visual cortex. A, Anterogradely labeled terminal field of FF axons (red) in area LM after injection of biocytin into area 17. CR-labeled somata and dendrites are shown in green. The boxed area is shown at higher magnification in B. Scale bar, 100 μm. B, CR-immunolabeled neuron (green) located in the terminal field of FF axons (red) in layer 2/3 of area LM. Insets (bottom right) show two high-power images taken of boxed area in the center of the picture. Arrows indicate putative contacts (see movies at http://thalamus.wustl.edu/burkhalterimages/) of FF boutons (red) with CR dendrites (green). The colocalization of SV2 (blue, arrows) in axon terminals (red, arrows) indicates that they contain vesicle proteins that are used in synapses. Optical thickness of section: 5 μm. Scale bars: B, 20 μm; inset, 2 μm. C, Anterogradely labeled FB axons (red) in layers 1 and 2/3 of area 17 after injection of biocytin into area LM. PV (blue) and SOM (green) immunolabeled neurons. Boxed area contains SOM and PV neurons (arrows) that are shown at higher magnification in D. Scale bar, 100 μm. D, High-magnification image of boxed area shown in C, indicating putative synaptic FB (red) inputs to SOM (green, arrows) and PV (blue, arrowheads) neurons. Optical thickness of section: 5 μm. Scale bar, 10 μm.

Figure 6.

Asymmetric and symmetric synaptic inputs to SOM neurons in layer 2/3 of rat area 17. A, Densely innervated SOM-immunoreactive (dark amorphous DAB reaction product) dendrite with multiple inputs from asymmetric (arrows) and symmetric Gray type 2 synapses (type 2 axon terminal, AT2). B, Small SOM dendrite with inputs from three asymmetric synapses. Scale bar, 0.25 μm. C, GABA immunogold-labeled (black dots), SOM-immunoreactive dendrite with input from asymmetric synapse (arrow). Nearby GABA-negative dendrite (D) receives inputs from asymmetric, Gray type 1 synapse (type 1 axon terminal, AT1). Scale bar, 0.5 μm.

Figure 4.

Relative strengths of FF and FB inputs to PV, CR, and SOM neurons in rat visual cortex, assessed by confocal and electron microscopy (EM). A, Percentage of all FF and FB connections providing inputs to PV, CR, and SOM neurons in layer 2/3. Total number of boutons and synapses is indicated in parentheses. Asterisks indicate EM data from Gonchar and Burkhalter (1999a). B, Percentage of all FB connections making inputs to PV, CR, and SOM cell bodies and dendrites in layer 1.

Figure 5.

Synaptic targets of FF and FB connections in rat visual cortex. A, Anterogradely biocytin-labeled (needle-shaped TMB/tungstate reaction product indicated by arrow) FF axon terminal in layer 2/3 of area LM. The large axon terminal contains round vesicles and forms asymmetric synapse with dendritic spine (Sp), which contains a prominent spine apparatus. B, Large biocytin-labeled (see dark TMB/tungstate crystal on lower left) FF terminal forming asymmetric synapse with dendritic shaft (D). C, Biocytin-labeled (see dark TMB/tungstate crystal) FB axon terminal forming asymmetric synapse with small dendrite (D). Nearby spine shows prominent spine apparatus (Sp). D, Asymmetric synapse of SOM-immunoreactive soma (SOM, indicated by dark DAB reaction product). Scale bars: A-D, 0.25 μm.

Figure 8.

FB inputs to CR and SOM neurons in rat area 17. Anterogradely biocytin-labeled (dark TMB/tungstate crystal) FB axon terminals forming asymmetric synapse with CR dendrite in upper layer 2/3 (A) and with CR immunoreactive cell body in layer 1 (B, arrows). Nu indicates cell nucleus. C, FF input to SOM neuron in rat area LM. Two anterogradely biocytin-labeled (dark TMB/tungstate crystals) FF axon terminals in layer 2/3 of area LM which both form asymmetric synapses. The FF terminal marked with an asterisk makes synaptic contact (arrows) with SOM-immunoreactive dendrite. The terminal marked AT forms symmetric synapse onto unlabeled dendrite. Scale bars: A, 0.2 μm; B, C, 0.5 μm.