Science: Technological innovation!

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With the rapid development of single-cell technology, more and more different molecular types of neurons can be defined, which is of great significance for deciphering the function of neural circuits
.

But cell types defined by single-cell sequencing lack spatial location information
.

In 2018, the main inventor of optogenetics, Professor Karl Deisseroth of Stanford University, developed a three-dimensional brain in situ single-cell sequencing technology to obtain the spatial distribution and gene expression map of cell types in a certain tissue, solving the above problems
.

Linking a specific type of neuron to its function during a specific behavior requires monitoring the activity of that cell type in vivo
.

In general, functional analysis can be achieved by labeling specific cell types with mouse tools, but the number of labeled cell types is limited (1 to 3 molecular markers)
.

On January 7, 2022, the research team of Jerry L.
Chen of Boston University combined two-photon calcium imaging technology and multiple rounds of in situ hybridization experiments to form CRACK technology, which realized the analysis of the correlation between multiple cell subtypes and behavioral functions
.

Figure 1: CRACK technology process In a nutshell, first, during the behavioral process, two-photon microscopy is used to image calcium at different depths of tissue, and the changes in neuronal calcium ions (the activity of all neuronal groups in the imaging area) are recorded; second, through Clear brain technology combined with mRNA probe in situ hybridization to achieve cell type localization in three-dimensional space, define specific molecular type of neurons (neuronal activity of specific molecular cell type in the imaging area); finally, use barcode reading scheme to decode and behavioral associated with new cell types (Figure 1)
.

Figure 2: Whiskers tactile spatial memory task flow The researchers utilized a whisker-based tactile spatial memory task, which used a rotating rotor to deflect mouse whiskers forward or backward (Figure 2)
.

During the experiment, the "sample" stimulus was given first, followed by a 2-second delay period, and finally the "test" stimulus was given
.

If the "sample" stimulus and the "test" stimulus caused the whiskers to deflect in the same direction, licking the water tube was rewarded with water; on the contrary, if the above two stimuli caused different directions, the water tube was not licked
.

Based on previous single-cell sequencing results, they subdivided the excitatory neurons in the layer 2/3 (S1 L2/3) region of the primary somatosensory cortex into three subtypes, Adamts2, Baz1a, and Agmat, and subdivided the inhibitory neurons into three subtypes.
are Lhx6-positive neurons, vasoactive intestinal peptide receptor 2 (Vipr2)-positive parvalbuminergic neurons, cartilage agglutinin (Chodl)-positive somatostatinergic neurons, and parathyroid-like hormone (Pthlh)-positive vasoactive intestinal peptidergic neurons
.

By correlating changes in calcium ion in the excitatory and inhibitory neurons of the above subtypes with the experimental variables of the above tasks (stimulus type, direction, selection process, etc.
), it was found that Baz1a excitatory neurons and Pthlh positive vasoactive intestinal Peptidergic neurons primarily encode information related to whisker movement (i.
e.
, touch)
.

Figure 3: Virus tracing experiments reveal that the output of Baz1a excitatory neurons above-mentioned various subtypes form a complex network functional connection with the experimental variables of the tactile spatial memory task, and it is found that Baz1a excitatory neurons play the most important role in coordinating the above overall network activities main role
.

Further virus tracing experiments found that Baz1a excitatory neurons in the S1 L2/3 region mainly projected to somatostatin neurons in this region
.

In summary, this paper uses CRACK technology to discover that Baz1a excitatory neurons serve as key nodes in neural circuit networks of multiple molecular types, processing sensory information
.

The emergence of CRACK technology can solve the pain point that single-cell sequencing technology defines molecular types but cannot define functions, and greatly promotes the development of neuroscience
.

When this technology is combined with the free-moving micro two-photon microscope technology developed by academician Cheng Heping, it can also be extended to more behavioral functional studies and collide with larger "calcium sparks"
.

[References] 1.
https://doi.
org/10.
1126/science.
abl5981 The pictures in the text are from the references