Transcriptome Profiling of Layer 5 Intratelencephalic Projection Neurons From the Mature Mouse Motor Cortex

Abstract

The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2−ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2−ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2−ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2−ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2−ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain.

Microglial reduction of colony stimulating factor‐1 receptor expression is sufficient to confer adult onset leukodystrophy

“…The role of CSF-2 as a major mediator of ALSP pathology was recently established by demonstrating that deletion of a single Csf2 allele in ALSP mice prevented development of the behavioral deficits, normalized most pathologies and partially restored the changes in the microglial transcriptome (Chitu et al, 2020). However, since cortical layer V neurons are gradually lost in ALSP mice and a proportion of these neurons express the CSF-1R (Chitu et al, 2015;Clare et al, 2018), we investigated whether decreased CSF-1R expression could affect neuronal survival independently of its effect on microglial functions. While at present it is unclear whether How might Csf1r +/− microglia mediate the selective loss of layer V neurons?…”

“…This could be a consequence of loss of microglial homeostatic functions. However, since previous studies (Luo et al, 2013) reported that CSF-1R signaling mediates neuronal survival cell autonomously and a subpopulation of layer V neurons express Csf1r (Chitu Clare et al, 2018), it is also possible that the reduced survival of layer V neurons is mediated by loss of direct regulation by the CSF-1R. Analysis of cortical neurons showed that MCsf1r het , but not NCsf1r het mice, reproduced the loss in layer V neurons (Figure 4a,b), indicating that these cells are lost due to microglial dysfunction.…”

“…Furthermore, cerebral CSF-1 is elevated in patients and monoallelic Csf2 deletion in Csf1r +/− mice prevents the development of most symptoms of disease in aged mice (Chitu et al, 2020), suggesting that microglial expansion and activation by CSF-2 plays an important role in disease. However, cortical layer V neurons, a subpopulation of which express the CSF-1R (Chitu et al, 2015;Clare et al, 2018), become significantly reduced in Csf1r +/− mice by 21 months of age, following a transient elevation in number at 3 months (Chitu et al, 2015). Monallelic deletion of Csf2 does not improve this phenotype, suggesting that their loss might be due to a failure of direct regulation of neuronal survival via the CSF-1R.…”

Modeling CSF‐1 receptor deficiency diseases – how close are we?

“…In addition to microglia, in the brain, CSF-1R is also expressed by neural progenitor cells, and subpopulations of neuronal cells [21][22][23][24]. While the functional significance of CSF-1R expression in neurons at steady state remains unclear, studies in vitro suggest that CSF-1R signaling in neural progenitor cells suppresses proliferation, enhances their neuronal differentiation and promotes the survival of their early progeny [22].…”

Circuit-Specific Plasticity of Callosal Inputs Underlies Cortical Takeover

“…Efforts to describe these changes are limited to alterations in input or output specificity between interneurons and principal cells (Lee et al, 2016;Lu et al, 2014;Trouche et al, 2013) or laminar specific adaptations (Koester and Johnston, 2005;Sjöström and Häusser, 2006). L5 neurons have been categorized by their electrophysiological properties (Shai et al, 2015), connectivity (Hattox and Nelson, 2007), morphology (Oswald et al, 2013), location (Lefort and Petersen, 2017) and gene expression (Clare et al, 2018;Tasic et al, 2016). These cortical output neurons send long-range projections throughout the brain and are thus optimally situated to integrate columnar information (Baker et al, 2018;Koester and Johnston, 2005;Sjöström and Häusser, 2006) and effect widespread adaptations (Feldmeyer, 2012;Grillner et al, 2005;Petersen, 2019).…”