Glutamatergic and GABAergic synapses in the human spinal dorsal horn revealed with immunohistochemistry

Abstract

Primary afferent neurons detect sensory stimuli in the periphery and transmit this information to the dorsal horn of the spinal cord, where it is extensively processed by excitatory and inhibitory spinal controls before being sent to higher brain centres. Immunohistochemical analysis of the synaptic architecture of these spinal circuits has been notoriously difficult in formaldehyde-fixed tissue and often requires antigen retrieval techniques to reveal antigen binding epitopes. To avoid the damage this harsh treatment can induce, studies have used antibodies raised against scaffolding proteins Homer1 and gephyrin, which anchor glutamate and GABA receptors to the membrane, respectively, to identify synaptic associations in rodent spinal cord underlying pain and itch transmission. In contrast, few studies have attempted to visualise spinal cord synapses in the human, partly due to a lack of high-quality tissue with low postmortem intervals. In this study, we reveal both excitatory and inhibitory synapses at a high resolution in human lumbar spinal cord tissue using antibodies to Homer1 and gephyrin and show that the basic organisation of these proteins within the dorsal horn is similar to that seen in the rodent. We show these postsynaptic markers are highly colocalised with glutamate and GABA receptor subunits and are in close apposition to presynaptic markers, confirming their specificity. Finally, we use Homer1-immunolabelling to demonstrate that primary afferents can form complex synaptic arrangements in human. We conclude that these antibodies can be used as reliable tools for the study of the human CNS and we have used them to reveal insight into the microanatomy of somatosensory connections in the human spinal cord that can be expanded upon in future studies.