Nat Commun-Liu's team was the first to comprehensively map single-cell cells in the primate visual cortex and found differences in gene expression across species

Written by Wei Jiaru, Hao Zhaozhe

Responsible editor—Wang Sizhen, Fang Yiyi

Editor—Wang Ruhua

The visual system is the most important sensory system of human beings, perceiving 80% of the sensory information
of the outside world.
The analysis and processing of external information by the visual system is the basis of various intellectual activities of the brain, and it is also an important window for studying and understanding the working principle of the brain [1-3].

The primary visual cortex is a brain area
in the cerebral cortex that specializes in processing visual information.
Although existing studies have established detailed primary cortical cell profiles on mice, primary cortical cell profiles for primates remain unknown
.
Primates have a more advanced and complex visual system
than rodents.
Rodents are mainly active in the dark, poor visual sharpness, most of them only have dichromatic vision, while primates have clearer three-dimensional vision and trichromatic vision, with a unique retinal foveal structure, which is extremely important for the production of fine vision [4].

Correspondingly, the primate visual cortex has a unique primary visual cortex structure, stronger visual information processing ability and computing ability [3].

Has the primate visual cortex evolved new cell types, is its neural cell type composition different, and is the gene expression of cells different? Studying the cellular and molecular mechanisms of primary visual cortex evolution in non-human primates is not only of great significance for recognizing and understanding the formation of higher and complex visual functions in humans, but also laying an important foundation
for studying the abnormalities of visual functions of amblyopia and strabismus.
The analysis of the composition of cell types and circuit formation in the primate visual primary cortex also provides important inspiration
for the development of artificial vision.

Recently, the team of Liu Sheng, a researcher at the Zhongshan Ophthalmology Center of Sun Yat-sen University, together with the research team of Miao Zhichao in Guangzhou Laboratory and the team of Professor Teichmann of the Wellcome Sanger Institute, published an online report in the journal Nature Communications "Identification of visual cortex cell types and species differences using single-cell RNA sequencing.
"
This work is the first comprehensive mapping of primate optic cortex cells, revealing the conservation and differences of different cell types across species, and identifies novel marker genes and cell types by combining whole-cell electrophysiological patch-clamp combined with single-cell sequencing (Patch-seq) and fluorescence in situ hybridization.

The comprehensive mapping of the optic cortex cell map will facilitate the pathogenesis and clinical treatment of amblyopia and strabismus
.

Researchers in our group have achieved a technological breakthrough by developing a whole-cell isolation technology for primate brain tissues to obtain high-quality single-cell suspension (cell activity > 90%) to achieve whole-cell single-cell sequencing
of adult primate brains 。 In order to delineate the cellular diversity of the primate visual cortex, the researchers applied this isolation technology to the visual cortex of cynomolgus monkeys for large-scale single-cell transcriptome sequencing to obtain 133454 high-quality single-cell data and a high proportion of neurons
.
Combining cell layer distribution, classical/novel marker genes, and data integration methods, the researchers identified 67 different cell types, including 25 excitatory neurons, 37 suppressor neurons, and methods 5 glial cell types
.
Next, the researchers used fluorescence in situ hybridization to verify candidate marker genes
for excitatory and inhibitory neurons.
They identified two novel layer-specific marker genes HPCAL1 and NXPH4 (Figure 1).
, mainly in the visual cortex L2/3 and L6 expression
.

Figure 1 Cell atlases of the visual cortex of cynomolgus monkeys

(Source: Wei JR, et al.
, Nat Commun, 2022).

Neuropeptide Y (NPY) is one of the most abundant and widely distributed neuropeptides in the central nervous system, and it is involved in a wide range of physiological functions, such as food intake, circadian rhythm, and memory
.
In previous studies, NPY+ neurons in the cortex were inhibitory neurons [5].

In this study, the researchers reported a class of novel excitatory neurons Exc NPY (Exc L1-3 HPCAL1 NPY) expressing NPY and SLC17A7, and these neurons did not express the marker gene GAD1 of suppressor neurons (Figure 2).

Such neurons specifically express DRD3, which encodes a subtype of dopamine receptor associated with addiction, indicating that Exc NPY has a unique physiological function
.
In addition, Exc NPY highly expresses NNAT and NOV; NNAT regulates ion channels during development and maintains nervous system structure during adulthood; NOV encodes extracellular matrix-associated signaling proteins
.
KEGG enrichment analysis found that genes upregulated by Exc NPY are mainly related to the biology of reward and addiction, and that such neurons may play an important role
in visual learning and memory.

Figure 2 Exc-NPY novel excitatory neurons

(Source: Wei JR, et al.
, Nat Commun, 2022).

In order to investigate whether Exc NPY cells have unique electrophysiological, morphological and transcriptome multimodal information, the researchers used the recently developed whole-cell electrophysiological patch-clamp combined with single-cell sequencing technology (Patch-seq) to simultaneously obtain the precise location, morphological characteristics, electrophysiological characteristics and gene expression characteristics of individual neurons
。 The researchers obtained multimodal information of suppressor neurons, HPCAL1 L2/3 excitatory neurons and Exc NPY neurons, and the Patch-seq experimental results showed that Exc NPY neurons were expressed SLC17A7, HPCAL1, NPY, NOV, NNAT, and CRYAB (Figure 2) are consistent with high-throughput single-cell transcriptome sequencing results
。 The researchers further compared the electrophysiological properties of HPCAL1 L2/3 and Exc NPY neurons and found that Exc NPY neurons had high input resistance ), the width of the action potential, the rising time of the action potential and the decay time
.

Previous studies have found that visual experience drives high expression of OSTN in the primate primary visual cortex, thereby affecting dendritic branches and neural network circuits of neurons [6].

In this study, Exc L4-6 RORB OSTN, a class of primate-specific neurons with high expression of LGI2 (Figure 3), was identified in the primary visual cortex LGI2 participates in synaptic maturation and inhibits the growth
of dendrites.
GO and KEGG analysis found that genes upregulated by Exc L4-6 RORB OSTN were involved in oxidative phosphorylation and ATP metabolic processes and synaptic signaling, suggesting that these neurons are in an activated state; The downregulated genes are related to neuronal projection development and synaptic composition, suggesting that the morphogenesis of these neurons is inhibited, consistent with previous in vitro studies, where OSTN limits dendritic growth
.
These results reveal mechanisms of visual plasticity unique to primates
.

Figure 3 Neurons induced by neural activity in RORB OSTN

(Source: Wei JR, et al.
, Nat Commun, 2022).

In order to systematically study the conserved and different gene expression across species, and whether there are cross-species gene expression differences in different cell types, the researchers studied the gene expression patterns
of the selected gene sets through cross-species correlation analysis.
First, the researchers used 461 genes in the developmental stage of the nervous system and 54 genes in the long-term enhancement gene set for correlation analysis
.
The study found that the correlation between monkey and human neurons was higher than between mice and humans, suggesting that the expression of these genes was conserved between primates, but there was a large difference between primates and mice; Suppressor neurons have a greater correlation than excitatory neurons, suggesting that expression of these genes is more conserved
in suppressor neurons.
Next, the researchers analyzed the correlation of 15 gene sets, including neural structure, neural modulation, cell signaling, cell adhesion molecules, and plasticity, and found that 14 gene sets were more strongly correlated in monkeys and humans than between mice and humans, and the differences were mainly manifested in gene sets
related to neural modulation, synaptic connectivity and plasticity.
In addition, these gene sets differ significantly more across species in excitatory neurons than in suppressor neurons
.
Finally, the researchers performed the same analysis on the gene sets of the GO and HGNC (Human Genome nomencature Committee) gene families, and found that monkeys and humans correlated more strongly than mice and humans, and suppressor neurons correlated stronger than excitatory neurons.
Glial cells are more strongly correlated than excitatory neurons
.
These results suggest that excitatory neurons are the source
of neurological differences between primates and rodents.