What's the science?
A critical goal of neuroscience is to understand the types of cells that make up the brain. Typically, novel cell types have been identified by studying the expression of molecular markers in different cells, and then confirming that the cell appears to have a distinct pattern of morphology of its axon and dendrites (the main processes attached to the cell body of a neuron). Essentially, researchers try to determine the relationship between genotype and phenotype. Some cell types are conserved across species, so a large portion of this type of research is done in rats and also applies to humans. However, not every cell type is conserved across species, so doing research in humans is important too. This week in Nature Neuroscience, Boldog and colleagues study molecular expression of different brain cells and characterize neuron morphology in humans.
How did they do it?
The authors first used single nucleus transcriptomics or RNA sequencing in two healthy post-mortem human brains. This method involves dissecting regions of interest from the cortex, isolating cell nuclei using tissue homogenization, and staining to identify neuronal (NeuN+) and non-neuronal (NeuN-) cells. The region of interest within the cortex was layer 1 of the middle temporal gyrus, which contains mostly inhibitory neurons. The resulting nuclei were then grouped using a clustering method according to the similarity of their transcriptional profiles.
To establish cell morphology, the authors identified interneurons in layer 1 in brain slices prepared from the parietal, temporal, and frontal cortices of 42 patients. Whole-cell recording and light microscopy of the cells was performed. Finally, they authors performed immunohistochemistry on the cells for which morphology was examined to test whether these cells were positive for gene markers indicative of different identified clusters (of gene markers).
What did they find?
Using single nucleus transcriptomics, on average 9937 genes were detected in neurons and 6287 genes were detected in glia (non-neuronal cells). When cells with similar transcriptional profiles were grouped together, different cell types (e.g. oligodendrocytes, microglia, astrocytes, excitatory neurons) were clustered with other cells of the same type as expected. Surprisingly, 11 different clusters of GABAergic or inhibitory neurons were identified within layer 1 of the middle temporal gyrus (however, this doesn’t mean that neurons belonging to these clusters wouldn’t also appear in other layers of the cortex). Different cell types could be identified by different marker genes – for example, GABAergic neurons were identified by the expression of glutamic acid decarboxylase 1 (GAD1).
Using light microscopy of layer 1 interneurons, the authors identified subsets of cells that had been previously described, as well as a novel type of interneuron, newly named the ‘rosehip cell’ for its rosehip-shaped axonal boutons (terminals of an axon). The shape of the dendrites of these neurons were relatively short and bushy. These neurons tended to have the same number of dendrites as basket cells but fewer than neurogliaform cells (two other brain cell types). Dendrites were, however, smaller and less frequent in rosehip cells compared to basket cells. The rosehip cell dendrites were also found to branch more frequently than other cell types, with large boutons. When immunohistochemistry was performed, it was found that the rosehip neurons matched a previously identified cluster of inhibitory neurons with unique transcriptional features. Notably, this cluster was associated with genes involved in axon growth and structure of the synapse, indicating these genes could have contributed to its unique shape. When the electrophysiology of these neurons was examined, they were found to be tuned to beta and gamma frequencies with variable interspike intervals (active and silent periods). The authors also noted which cells partnered with the rosehip cells. Rosehip cells predominantly formed synapses with layer 3 pyramidal cells. Calcium signalling was suppressed upon rosehip cell input to pyramidal cells in some cases, indicating that these cells may be involved in calcium signalling of human pyramidal cells.
What's the impact?
This is the first study to identify transcriptional and morphological characteristics of a unique group of interneuron cells in layer 1 of the human cortex. These new cells are called ‘rosehip cells’ for the shape of their axonal boutons. The identification of this new type of inhibitory cell is groundbreaking because it could lead to significant advances in our understanding of the brain’s circuitry.
Boldog et al., Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type. Nature Neuroscience (2018). Access the original scientific publication here.