Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons

Abstract

The generation of emotional responses by the basolateral amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons, the pyramidal cells. The activity of these neurons is tightly controlled by gamma-aminobutyric acid (GABA)-ergic interneurons, especially a parvalbumin-positive (PV(+)) subpopulation that constitutes almost half of all interneurons in the basolateral amygdala. In the present semiquantitative investigation, we studied the incidence of synaptic inputs of PV(+) axon terminals onto pyramidal neurons in the rat basolateral nucleus (BLa). Pyramidal cells were identified by using calcium/calmodulin-dependent protein kinase II (CaMK) immunoreactivity as a marker. To appreciate the relative abundance of PV(+) inputs compared with excitatory inputs and other non-PV(+) inhibitory inputs, we also analyzed the proportions of asymmetrical (presumed excitatory) synapses and symmetrical (presumed inhibitory) synapses formed by unlabeled axon terminals targeting pyramidal neurons. The results indicate that the perisomatic region of pyramidal cells is innervated almost entirely by symmetrical synapses, whereas the density of asymmetrical synapses increases as one proceeds from thicker proximal dendritic shafts to thinner distal dendritic shafts. The great majority of synapses with dendritic spines are asymmetrical. PV(+) axon terminals form mainly symmetrical synapses. These PV(+) synapses constitute slightly more than half of the symmetrical synapses formed with each postsynaptic compartment of BLa pyramidal cells. These data indicate that the synaptology of basolateral amygdalar pyramidal cells is remarkably similar to that of cortical pyramidal cells and that PV(+) interneurons provide a robust inhibition of both the perisomatic and the distal dendritic domains of these principal neurons.

Figure 1

Light micrograph illustrating the innervation of CaMK+ pyramidal cell perikarya and proximal dendrites labeled by the V-VIP peroxidase technique (purple), by PV+ axon terminals labeled by the DAB peroxidase technique (brown). Scale bar = 20 μm.

Figure 2

Electron micrographic composite of a CaMK+ pyramidal cell perikaryon (Cpk) innervated by PV+ axon terminals (arrows). PV+ terminals are clearly marked with intense DAB reaction product; all 13 synapses indicated were confirmed through serial sections. The inset shows a magnified view of one PV+ terminal (asterisk) and its synaptic contact. The moderately dense, particulate V-VIP reaction product for CaMK immunoreactivity is more obvious, at this low magnification, in the neighboring CaMK+ dendrites (Cd) than in the perikaryon (Cpk), where it is somewhat obscured by cytosolic features such as polysomes (see Fig. 3). An oblique view of a PV+ myelinated axon (Pma) is also indicated, with an unlabeled myelinated axon profile (Uma) shown for comparison. Scale bars = 1 μm, 0.2 μm for inset.

Figure 3

Electron micrographs of CaMK+ pyramidal cell perikarya receiving synaptic inputs from PV+ terminals (Pt). A: A CaMK+ perikaryon (Cpk) receives input (arrows) from two PV+ terminals (Pt). The particulate V-VIP reaction product for CaMK is clearly distinguishable from other perikaryal features at this magnification (compared to Fig. 2). The axosomatic synapse of the upper PV+ terminal was seen more clearly in adjacent serial sections; this terminal also synapses with a thin unlabeled dendrite located below the terminal. B: A PV+ terminal (Pt) makes synaptic contact (arrow) onto a CaMK+ perikaryon (Cpk) labeled by the IGS technique, with 4 silver grains apparent in this view. Scale bars = 0.5 μm.

Figure 4

A CaMK+ axon initial segment (Cais), labeled by the V-VIP method, receives symmetrical synaptic inputs from PV+ axon terminals (arrows) and unlabeled terminals (arrowheads). Five of the PV+ terminals shown are adjacent to one another (top five arrows). One of these PV+ terminals (asterisk) is shown magnified in the inset. This CAMK+ axon initial segment was followed through four additional serial sections (from the upper right of the field shown). The synaptic nature of each of these axoaxonic contacts was ascertained in these serial sections. Scale bars = 1 μm, 0.25 μm for inset.

Figure 5

Large caliber CaMK+ dendrites (Cd) receive synaptic input from PV+ terminals (Pt, arrows). A: A CaMK+ dendrite labeled by the V-VIP method receives synaptic input from a PV+ terminal. B: A CaMK+ proximal dendrite labeled by the IGS method receives input from a PV+ terminal. An unlabeled terminal, making symmetrical synaptic contact with the CaMK+ dendrite is also indicated (Ut, arrowhead). Scale bars = 0.5 μm.

Figure 6

Small caliber CaMK+ dendrites (Cd) receive synaptic input from PV+ terminals (Pt, arrows). A: A bifurcating CaMK+ dendrite, labeled by the IGS method, receives synaptic input from PV+ terminals on each of its branches. Each branch also receives symmetrical synaptic contact from unlabeled terminals (Ut, arrowheads). The PV+ terminal contacting the upper branch of the CaMK+ dendrite makes synaptic contact with a neighboring spine (sp, short arrow), which also receives additional synaptic input from two unlabeled terminals (asterisks). B: Two neighboring CaMK+ dendrites, labeled by the V-VIP method, receive synaptic input from the same PV+ terminal. The CaMK+ dendrite on the left also receives a symmetrical synaptic contact from an unlabeled terminal (Ut). The CaMK+ dendrite on the right gives rise to a spine (Csp), which receives an apparent asymmetrical contact from an unlabeled terminal (asterisk). Scale bars = 0.5 μm.

Figure 7

A, B: Two serial sections of a CaMK+ dendrite (Cd) and emerging spine (Csp) receiving synaptic input from a PV+ terminal (Pt, arrow). The CaMK+ dendrite is labeled by the V-VIP method. A neighboring spine (sp) receiving an asymmetrical synaptic contact from an unlabeled terminal (Ut, arrowhead) and a PV+ myelinated axon profile (Pma) are also indicated. Scale bar = 0.5 μm.

Figure 8

Synaptic inputs to CaMK+ spines. Three CaMK+ spine heads (Csp), labeled with the V-VIP method, receive perforated asymmetrical synaptic input (pairs of arrowheads). Two of the terminals contacting the CaMK+ spines were also found, in serial sections, to be CaMK+ (Ct). Other terminals, with no apparent immunoreactivity (small asterisks), also make asymmetrical contact with spines. Two spines receive synaptic contacts from PV+ terminals (Pt). One of these spines (lower left) emerges from a CaMK+ dendrite (Cd), as shown in serial section in the inset. Large asterisks mark a common feature (a mitochondrion), for orientation. As seen in the inset, the emerging spine receives synaptic input from a PV+ terminal (arrow) as well as a small apparently unlabeled terminal (small asterisk). The other PV+ terminal (left of center) makes synaptic contact with a spine (sp, short arrow) and a CaMK+ perikaryon (Cpk, arrow). The synaptic nature of the axosomatic contact was seen more clearly in adjacent serial sections. Scale bar = 0.5 μm.

Figure 9

Synaptic input to spines from PV+ terminals. A, B: Serial electron micrographs show synaptic inputs to spines (sp) from PV+ terminals (Pt, short arrows). A: One of the spines receiving input from a PV+ terminal (left center) also receives an asymmetrical synaptic contact from an unlabeled terminal (arrowhead, asterisk). Another spine (upper left corner) is shown to receive an asymmetrical contact only from an unlabeled terminal. A lightly labeled CaMK+ terminal (Ct) is shown in serial section (panel B) to make a perforated asymmetrical synaptic contact (two arrowheads) with a spine (sp). B: The PV+ terminal (Pt), central in the field, makes synaptic contact onto two emerging spines (sp, two short arrows), confirmed by additional serial sections. Scale bar = 0.5 μm.