Characterization of the three-dimensional synaptic and mitochondrial nanoarchitecture within glutamatergic synaptic complexes in postmortem human brain via focused ion beam-scanning electron microscopy.

Glutamatergic synapses are the primary site of excitatory synaptic signaling and neural communication in the cerebral cortex. Electron microscopy (EM) studies in non-human model organisms have demonstrated that glutamate synaptic activity and functioning are directly reflected in quantifiable ultrastructural features. Thus, quantitative EM analysis of glutamate synapses in ex vivo preserved human brain tissue has the potential to provide novel insight into in vivo synaptic functioning. However, factors associated with the acquisition and preservation of human brain tissue have resulted in persistent concerns regarding the potential confounding effects of antemortem and postmortem biological processes on synaptic and sub-synaptic ultrastructural features. Thus, we sought to determine how well glutamate synaptic relationships and nanoarchitecture are preserved in postmortem human dorsolateral prefrontal cortex (DLPFC), a region that substantially differs in size and architecture from model systems. Focused ion beam-scanning electron microscopy (FIB-SEM), a powerful volume EM (VEM) approach, was employed to generate high-fidelity, fine-resolution, three-dimensional (3D) micrographic datasets appropriate for quantitative analyses. Using postmortem human DLPFC with a 6-hour postmortem interval, we optimized a tissue preservation and staining workflow that generated samples of excellent ultrastructural preservation and the high-contrast staining intensity required for FIB-SEM imaging. Quantitative analysis of sub-cellular, sub-synaptic and organelle components within glutamate axo-spinous synapses revealed that ultrastructural features of synaptic function and activity were well-preserved within and across individual synapses in postmortem human brain tissue. The synaptic, sub-synaptic and organelle measures were highly consistent with findings from experimental models that are free from antemortem or postmortem effects. Further, dense reconstruction of neuropil revealed a unique, ultrastructurally-complex, spiny dendritic shaft that exhibited features characteristic of neuronal processes with heightened synaptic communication, integration and plasticity. Altogether, our findings provide a critical proof-of-concept that ex vivo VEM analysis provides a valuable and informative means to infer in vivo functioning of human brain.

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

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.

Selective alterations in postsynaptic markers of chandelier cell inputs to cortical pyramidal neurons in subjects with schizophrenia.

Markers of GABA neurotransmission between chandelier neurons and their synaptic targets, the axon initial segment (AIS) of pyramidal neurons, are altered in the dorsolateral prefrontal cortex (dlPFC) of subjects with schizophrenia. For example, immunoreactivity for the GABA membrane transporter (GAT1) is decreased in presynaptic chandelier neuron axon terminals, whereas immunoreactivity for the GABA(A) receptor alpha2 subunit is increased in postsynaptic AIS. To understand the nature and functional significance of these alterations, we determined the density, laminar distribution, and length of AIS immunoreactive (IR) for ankryin-G and betaIV spectrin, two proteins involved in the regulation of synapse structure and ion channel clustering at AIS, in dlPFC area 46 from 14 matched triads of subjects with schizophrenia or major depressive disorder (MDD) and normal comparison participants. The density of ankyrin-G-IR AIS in the superficial, but not in the deep, cortical layers was significantly decreased by 15-19% in the subjects with schizophrenia relative to the other participant groups. In contrast, no group differences were present in the density of betaIV spectrin-IR AIS. The length of labeled AIS did not differ across participant groups for either ankyrin-G or betaIV spectrin. The density of ankyrin-G-IR AIS was not altered in the dlPFC of macaque monkeys chronically exposed to antipsychotic medications. Given the important role of ankyrin-G in the recruitment and stabilization of sodium channels and other integral membrane proteins to AIS, our findings suggest that these processes are selectively altered in superficial layer pyramidal neurons in subjects with schizophrenia.

Dendritic-targeting GABA neurons in monkey prefrontal cortex: comparison of somatostatin- and calretinin-immunoreactive axon terminals.

Different subclasses of gamma-aminobutyric acid (GABA) cortical neurons can be distinguished by their content of neuropeptides such as somatostatin (SST), or calcium-binding proteins such as calretinin (CR). SST, but not CR, neurons have been reported to be altered in the prefrontal cortex (PFC) of subjects with schizophrenia. Understanding the functional significance of the SST neuron disturbances in schizophrenia requires knowledge of the specialized synaptic circuitry of these neurons relative to that of CR neurons. Consequently, we used immuno-electron microscopy to examine the synaptic type and postsynaptic targets of SST-immunoreactive (IR) axon terminals in monkey PFC and compared these findings with similar data for CR-IR axon terminals. SST-IR axon terminals formed exclusively symmetric synapses and contacted only dendritic shafts (86%) and dendritic spines (14%), whereas CR-IR terminals also formed synapses with cell bodies. The postsynaptic targets of SST-IR axon terminals also differed across layers with synapses onto dendritic spines more frequent in the superficial (20%) than in the deep (8%) layers. Dual-label immunoelectron microscopy revealed that CR-IR axon terminals targeted GABA-IR dendritic shafts with a greater frequency (60%) than did SST-IR axon terminals (21.5%). Conversely, SST-IR axon terminals contacted unlabeled dendritic shafts, presumably belonging to pyramidal neurons, more frequently than did CR-IR axon terminals (57% vs. 19%, respectively). This specialized synaptic circuitry of SST neurons in the primate PFC suggests that the alterations of these neurons in schizophrenia is likely to have distinct functional consequences.

The human precentral sulcus: chemoarchitecture of a region corresponding to the frontal eye fields.

Human functional neuroimaging studies have consistently shown that the superior element of the precentral sulcus (sPCS), near the caudal end of the superior frontal sulcus (cSFS), is activated during oculomotor tasks, and refer to this area as the frontal eye field (FEF). However, the anatomy of this area in humans has not been examined systematically, nor has the correspondence between traditional cytoarchitectonic maps and recent studies of the functional neuroanatomy of the FEF been determined. To identify the chemoarchitectonic features of this fMRI-defined area of the human sPCS, we labeled tissue sections of postmortem human brains containing the sPCS and the cSFS with antibodies against the neuronal nuclear protein (NeuN), the nonphosphorylated neurofilament triplet protein (NNFP), and the calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV). Distinctive chemoarchitectural features of this area of the sPCS compared with rostral and caudal regions were consistently found across subjects and consisted in clusters of large, intensely immunoreactive (IR) pyramidal cells in deep layer V and the presence of layer IV in both NNFP and NeuN-labeled material. In sections labeled for calcium binding proteins, the two walls of the sPCS were characterized by: (1) a higher density of CR-IR neurons; (2) a relative lack of CB-IR pyramidal cells; and (3) higher density of layers II-III CB-IR neurons and more large PV-IR interneurons in deep layer III. These specific chemoarchitectural features may differentiate the location of the human FEF from rostral cortical regions and support the functions for which it is specialized.

Pyramidal neuron local axon terminals in monkey prefrontal cortex: differential targeting of subclasses of GABA neurons.

In monkey prefrontal cortex (PFC), approximately 50% of the local axon terminals of pyramidal neurons form synapses with the dendritic shafts of GABA neurons. Subclasses of GABA neurons can be distinguished by the presence of different calcium-binding proteins. For example, in monkey PFC, parvalbumin (PV)-containing cells comprise approximately 25% of GABA neurons and are predominantly located in layers 3b-4, whereas calretinin (CR)-containing cells, which are present in greatest density in layers 2-3a, constitute 50% of GABA neurons. Consequently, in order to determine the cell class and laminar specificity of the dendritic targets of pyramidal neuron local axon collaterals in monkey PFC area 9, we conducted ultrastructural analyses of local axon terminals labeled with the anterograde tracer, biotinylated dextran amine, and dendrites immunoreactive (IR) for PV or CR. In layer 3b, the majority of the local axon terminals targeted PV-IR dendritic shafts, whereas CR-IR dendritic shafts were targeted infrequently. This differential targeting was also present in layers 2-3a, although it was less pronounced. In addition, PV-IR dendrites had a significantly greater density of excitatory inputs than did CR-IR dendrites. These findings indicate that PV-containing interneurons, which have a potent inhibitory effect on pyramidal neurons, are selectively targeted by the excitatory local axon terminals of supragranular pyramidal neurons in monkey PFC. These connections may provide the anatomical substrate for the coordinated activity of pyramidal neurons and fast-spiking GABA neurons during working memory.

Specificity in the functional architecture of primate prefrontal cortex.

Multiple lines of evidence indicate that the performance of complex cognitive processes, such as those involving working memory, depend upon the functional properties of the circuitry of the prefrontal cortex (PFC). In primates, working memory has been proposed to be dependent upon the sustained activity of specific populations of PFC pyramidal cells, with this activity regulated by certain types of GABAergic interneurons. Thus, knowledge of the connectivity between PFC pyramidal cells and interneurons is crucial to the understanding the neural mechanisms that subserve working memory. This paper reviews recent findings that reveal specificity in the spatial organization, synaptic targets and postnatal development of pyramidal cells and interneurons in the primate prefrontal cortex, and considers the relevance of these findings for the neural circuitry that subserves working memory.