Abstract

Somatostatin fluorescent in situ hybridization in the frontal pole cortex (ba10): application for psychiatric disorders

Krolewski, DM, Kumar V, Medina A, Foltz M, Myers R, Lee FS, Barchas JD, Schatzberg AF, Bunney B, Bunney WE, Akil H, Watson SJ
51st Annual Meeting of the Society for Neuroscience. 2022.

Background: Somatostatin (SST) peptide is produced by a specific subpopulation of inhibitory GABA-ergic interneurons which exhibit pronounced synaptic connectivity with distal dendrites of pyramidal neurons. Previous studies indicate SST participates in cognitive processes as SST mRNA is one of the more downregulated interneuron markers the dorsolateral prefrontal cortex in schizophrenia (SZ). Our laboratory recently employed qPCR to investigate SST gene expression in the frontal pole cortex (BA10); a region linked to multiple functions including those related to emotion, nociception, and pain. The latter study also revealed frontal pole SST mRNA to be amongst the most reduced transcripts in SZ. (Medina et al., 2022). To potentially uncover a more specific neuroanatomical foundation to these findings, we look to perform a BA10 cortical layer-based SST+ neuron analysis in SZ and bipolar disorder (BPD) subjects using fluorescent in situ hybridization. Methods: Frontal pole cortical tissue was obtained from the Brain Donor Program, University of California, Irvine. All samples had zero agonal factors and met stringent criteria for pH and PMI. Hybridization chain-reaction (HCR) fluorescent in situ hybridization (FISH) was performed on 30µm and 10µm-thick fresh-frozen cryosection. Images were captured on an Olympus Fluoview 3000 and SST+ cells quantified in stitched z-plane stacks with Amira and ImageJ software. Results: Using control subjects, we were able to achieve FISH methods that demonstrate clear visualization and quantification of frontal pole layer-specific SST+ neurons while eliminating lipofuscin autofluorescence. During testing, as expected, 30µm-thick sections displayed more SST+ cells versus 10µm, but neurons also appeared more fully labeled superficially and visually more uniform morphologically. SST+ neurons were widely present in layers II-VI with larger neurons in layers II/III and V versus those smaller in layer IV. Given this successful methodology, we are currently processing HCR-FISH for BA10 control, SZ, and BPD subjects for comparison (n=12/group). Conclusion: We have completed visual and quantitative methods for frontal pole cortex SST+ interneuron analyses in thicker cryosections. These results will allow for determining possible BA10 cortical layer-based neuropeptide differences in our ongoing study investigating psychiatric disorders.