Pathways for Emotions: Specializations in the Amygdalar, Mediodorsal Thalamic, and Posterior Orbitofrontal Network

In MDmc, neurons projecting to pOFC overlapped with amygdalar axons. A–C, Neurons in MDmc projecting to pOFC (left), overlapped with patches of labeled amygdalar axons (middle), as seen in the merged images (right). Scale bar in A is 100 μm and applies to A–C. D, E, Laser scanning confocal microscopy showing close appositions (white arrowheads) between pOFC projection neurons (green) and amygdalar axons (red) in MDmc. Scale bar in D is 20 μm and applies to D and E.

Neurons in MD projecting to pOFC colocalized with CB or PV. A, Thalamic MD neuron projecting to pOFC (top, white arrowhead) is also positive for CB (middle), as seen in the merged images (bottom). B, Thalamic MD neuron projecting to pOFC (top, white arrowhead) is also positive for PV (middle), as seen in the merged images (bottom). Scale bar in A is 10 μm and applies to A and B. C, Thalamic neurons projecting to pOFC more frequently colocalized with CB than PV, in all cases analyzed. Vertical lines indicate SEM. D, Schematic depicting the termination pattern of calbindin thalamocortical neurons, which target robustly the upper cortical layers, and parvalbumin thalamocortical neurons, which terminate focally in the middle cortical layers.

Amygdalar axon terminals form synapses with CB and PV thalamocortical neurons in MD. A, Amygdalar axon terminal forming a synapse (white arrowheads) with a CB dendrite labeled with TMB (black asterisk),and also forming synapses with inhibitory dendrites (black arrowheads). B, Amygdalar axon terminal forming a synapse (white arrowheads) with a CB dendrite labeled with TMB (black asterisk). C, Amygdalar axon terminal forming a synapse (white arrowheads) with a PV dendrite labeled with gold (white asterisk). D, Amygdalar axons formed synapses more frequently with labeled CB dendrites in MD. E, The majority of amygdalar axons formed a synapse with at least one excitatory thalamic dendrite. Amy At, amygdalar axon terminal; CB den, calbindin dendrite; inhib den, inhibitory dendrite; PV den, parvalbumin dendrite.

Amygdalar neurons that project to MD and to pOFC are distinct. A, Amygdalar neuron is labeled with tracer injected in pOFC (left, blue, white arrowhead), but is not labeled with tracer from MD (right). Scale bar in A is 10 μm and applies to A–C. B, Amygdalar neuron is labeled with tracer injected in MD (right, red, white arrowhead), but is not labeled with tracer from pOFC (left). C, Neurons in the same field show that different neurons in the amygdala were labeled retrogradely with tracer from pOFC (left, bottom arrowhead) or from MD (right, top arrowhead); neither neuron is double-labeled. D–F, Representative coronal sections from fresh tissue through the amygdala (anterior → posterior) show spatial overlap of projection neurons directed to MD (red dots) or pOFC (blue dots). Scale bar in D is 1 mm and applies to D–F. G, Mean area of labeled amygdalar projection neurons directed to MD was significantly larger than for projection neurons directed to pOFC. Vertical lines indicate SEM.

Amygdalar axons in MDmc and pOFC express different glutamate transporters. A, B, Amygdalar axons (top, white arrowheads) in the upper layers of pOFC colocalized with VGLUT1 (middle), but not with VGLUT2 (middle, silhouette arrowheads), as seen in the merged images (bottom). Scale bar in A is 10 μm and applies to all panels. C, D, Amygdalar axons in MDmc (top, white arrowheads) colocalized with VGLUT2 (middle, white arrowheads), but not with VGLUT1 (middle, silhouette arrowheads), as seen in the merged images (bottom). E, Scatterplot depicts the proportion of amygdalar boutons in each case which colocalized with VGLUT1 or VGLUT2 in MDmc or pOFC. F, Overall proportion of amygdalar boutons that colocalized with VGLUT1 or VGLUT2 in MDmc or pOFC.