RESUMEN
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.
RESUMEN
Age-related dopamine (DA) neuron loss is a primary feature of Parkinson's disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.