Laminar Differences in the Targeting of Dendritic Spines by Cortical Pyramidal Neurons and Interneurons in Human Dorsolateral Prefrontal Cortex

Abstract

Activation of specific neural circuits in different layers of the primate dorsolateral prefrontal cortex (DLPFC) is essential for working memory, a core cognitive function. Recurrent excitation between pyramidal neurons in middle and deep layers of the DLPFC contributes to the laminar-specific activity associated with different working memory subprocesses. Excitation between cortical pyramidal neurons is mediated by glutamatergic synapses on dendritic spines, but whether the relative abundance of spines receiving cortical inputs differs between middle and deep cortical layers in human DLPFC is unknown. Additionally, GABAergic inputs to spines sculpt pyramidal neuron activity. Whether dendritic spines that receive a glutamatergic input from a cortical pyramidal neuron are targeted by GABAergic interneurons in the human DLPFC is unknown. Using triple-label fluorescence confocal microscopy, we found that 1) the density of spines receiving an input from a cortical pyramidal neuron is greater in the middle than in the deep laminar zone, 2) dendritic spines dually innervated by a cortical pyramidal neuron and an interneuron are present in the human DLPFC, and 3) the density of spines dually innervated by a cortical pyramidal neuron and an interneuron is also greater in the middle than in the deep laminar zone. Ultrastructural analyses support the presence of spines that receive a cortical pyramidal neuron synapse and an interneuron synapse in human and monkey DLPFC. These data support the notion that the DLPFC middle laminar zone is particularly endowed with a microcircuit structure that supports the gating, integrating and fine-tuning of synaptic information in recurrent excitatory microcircuits.

Keywords: GABA; axospinous; glutamate; layer 3; layer 5; ultrastructure.

Figure 1.. Representative image of human DLPFC triple-labeled for spinophilin, Vglut1 and gephyrin.

Single channel images of (A) spinophilin, (B) Vglut1, (C) gephyrin and (D) lipofuscin autofluorescence within the neuropil. (E) Merged image of A-D. (F) Object masks generated from immunoreactive puncta. All object masks overlapping the lipofuscin mask (arrow) are removed from analysis. Scale bar is 10 μm.

Figure 2.. Identification of dendritic spine subtypes in human DLPFC.

(A1–A5) Pyramidal neuron innervated spine (arrowhead) defined as a spinophilin object mask that is adjacent to and overlaps with a Vglut1 object mask (spinophilin+/Vglut1+). (B1–B5) Pyramidal neuron and GABA interneuron dually innervated spine (arrowhead) defined as a spinophilin object mask that is adjacent to and overlaps with a Vglut1 object mask and overlaps a gephyrin object mask (spinophilin+/Vglut1+/gephyrin+). Dually innervated spines (B) represent a subset of pyramidal neuron innervated spines (A). Scale bar is 1 μm.

Figure 3.. Density of dendritic spine subtypes in middle and deep laminar zones of the DLPFC.

(A) Mean density of spinophilin+/Vglut1+ objects in each laminar zone. (B) Correlation between age and the mean density of spinophilin+/Vglut1+ objects in each laminar zone. (C) Mean density of spinophilin+/Vglut1+/gephyrin+ objects in each laminar zone. Each marker represents the mean value for an individual subject. In panels A and C, markers above the diagonal unity line indicate subjects with greater density of objects in the middle laminar zone than in the deep laminar zone.

Figure 4.. Ultrastructural identification of dendritic spines dually innervated by a cortical pyramidal neuron and an interneuron in human and monkey DLPFC.

(A1–A4) Successive ultrathin serial sections showing a dually innervated spine receiving a Type II synapse (arrowhead) from a PV-IR interneuron axon terminal (PV+AT) and a glutamatergic Type I synapse (arrow) from a non-PV-IR axon terminal (PV-AT) in human DLPFC middle laminar zone. (B) Serial section 3D reconstructed image of the synaptic structure identified in A1–A4. (C) Electron micrograph of a BDA–labeled axon terminal originating from a local DLPFC pyramidal neuron in layer 3 of monkey DLPFC area 9. This BDA-labeled axon terminal (BDA AT) forms a Type I glutamatergic synapse (arrow) onto a dendritic spine that also receives a Type II, likely GABAergic, synapse (arrowhead) from an unlabeled axon terminal (u AT). Scale bar is 500nm in A-B and 400nm is C.