Neural-circuit

• Q1: What are the advantages and disadvantages of Cre-loxP system gene recombination in transgenic animals?
  A: The genetic recombination that transgenic animals rely on can achieve efficient and specific genetic recombination. However, transgenic animals are difficult to obtain, animal mating requires a lot of time (at least 2 generations of mating to obtain gene knockout animals), and the regional or tissue specificity is not High (relies on promoter regulation only).
• Q2: What issues need to be paid attention to when analyzing the results of the horseradish peroxidase (HRP) tracing method?

  A：

  (1) Effective injection range: The injection range of HRP spreads from the injection center to the surroundings, and the concentration decreases. The effective range of the injection area is difficult to determine, which affects the analysis of the results. It is generally believed that the area in the center of the injection that is deeply stained and whose structure cannot be discerned is the effective area, while the area around it where labeled cells can be discerned is the ineffective area.

  (2) Passing fiber problem: Fibers passing through the injection area can also take up HRP and be transported in both forward and reverse directions. Therefore, neurons or terminals present at the labeled site may not originate or terminate at the injection site. This is a problem often encountered when analyzing results.

  (3) Side branch labeling: After the HRP label is taken up by an axon terminal, during its retrograde transport, some of it can be transported along the side branches of the axon to the terminals of the side branches. The terminal portion creates a terminal mark. Therefore, the presence of terminal markers at one site does not necessarily mean that the cell bodies from which these fibers emanate are located within the injection zone.

  (4) Trans-synaptic labeling: After HRP is transported to the terminal, it may be released, taken in by the next-level neuron, and even transported to the terminal of the next-level neuron. This phenomenon is rare, mainly seen when the more sensitive WGA-HRP is used as a tracking agent, and it occurs when the first-level terminals are densely packed and closely related to the second-level neurons. For example, HRP within retinal ganglion cell terminals may transsynaptically label lateral geniculate neurons.

• Q3: How to judge and eliminate labeling artifacts of horseradish peroxidase method?

  A：

  (1) Other cells other than neurons: glial cells, vascular endothelial cells, pericytes, macrophages, etc. at and near the injection site can take in HRP and react with it to color. This has less impact on tracing long-distance fiber connections, but hampers the study of short-distance connections. Generally, they are easy to distinguish from nerve cells based on their cell shape, especially the shape of the nucleus and its relationship with the lumen of blood vessels. Moreover, the reaction granules in these cells are often thick and uneven in size, and the cytoplasm is often evenly colored. However, identification can sometimes be difficult, especially when there are only single cells.

  (2) Endogenous substances of neurons: Some neurons contain lipofuscin, which increases with age and may be confused with DAB-reactive granules. On slices counterstained with tar violet, its dark field effect may also be strengthen. In mice and monkeys, it has been found that some neurons may contain some endogenous enzymes or other substances, which can also react with DAB-H2O2 to produce brown reactants. A similar reaction occurs with melanin or its precursors. These substances can all be sources of artifacts. Therefore, the results of this method should be evaluated with caution.

• Q4: What are the issues that need to be paid attention to when using fluorescein neuron tracing?

  A：

  (1) Path selection: Fluorescein can also be used for anterograde tracing, but the labeling is weak, so it is not recommended.

  (2) Labeling method: Different fluorophores have different excitation wavelengths and emission wavelengths, so double labeling or multiple labeling methods can be used for labeling. However, the retrograde transport speeds of different fluoresceins vary greatly, so the fluorescein can be injected in two surgeries during the experiment.

  (3) Diffusion problem: After reaching the cell body, some fluorophores tend to diffuse out of the neuron cell body and stain the surrounding glial cells. Therefore, the selection of appropriate survival time is very important.

  (4) Effective injection site: Due to the smaller molecular weight of fluorescein, retrograde tracing is easier to diffuse, so it is more difficult to determine the effective injection site than the HRP method. There is also the issue of passing fiber intake, which requires extra attention when analyzing the results.

  (5) Fading problem: Fluorescein fades quickly under excitation light, so the observation time is short. Even under low temperature and dark conditions, the storage time of slices is still limited and cannot be stored for a long time.

  (6) Optimization method: Adding 2.5% triethylene diamine to the mounting medium prepared with a mixture of 50% glycerol and 50% PBS can effectively extend the observation and storage time. Fluorescein can also be wrapped in latex microspheres as a tracking agent to reduce the problems of diffusion, passing fibers and rapid fading.

• Q5: How is the time specificity of conditional gene knockout achieved?

  A: The inducible Cre-loxP system mediates time-specific gene knockout. By controlling the time of administration of inducer, the occurrence of gene knockout is artificially controlled.

  (1) Tetracycline-inducible tetO-Cre combines the tetracycline regulatory system with the Cre-loxP system and generally requires mating of two transgenic mice: one is a Cre tool mouse controlled by a tetracycline responsive promoter element (TRE or tetO) (TRE-Cre, also called tetO-Cre); the other is a mouse that expresses the tetracycline transcriptional activator rtTA or tTA driven by a tissue-specific promoter. In this process, rtTA or tTA with transcriptional activation function binds to tetO (tetO cannot independently drive downstream gene expression) and activates the expression of Cre. At the same time, this binding is regulated by tetracycline or the tetracycline derivative doxycycline (Dox), so in tetO-Cre and tissue-specific rtTA (or tTA) double transgenic positive mice, Cre can be controlled by giving or withdrawing Dox The time when recombinase is produced in a specific tissue.

  (2) Interferon-inducible Mx1-Cre Interferon induction is achieved through the response of the Mx1 gene promoter to interferon. Mx1-Cre mice do not express Cre recombinase under normal conditions, but can be induced to express Cre by treatment with interferon-α, interferon-β or the synthetic double-stranded RNA analog poly I:C. This method is limited to cells that respond to interferon, and its gene knockout efficiency depends on the level of tissue or cell response to interferon or the number of interferon-responsive cells.

  (3) Estrogen-inducible Cre-ER fuses the ligand-binding region of the estrogen receptor (ER) with Cre recombinase to form a fusion protein (Cre-ER) located in the cytoplasm. In this way, by controlling the injection time of estrogen, time-specific regulation of gene recombination can be achieved. In order to avoid the interference of endogenous estrogen, a point mutation (G521R) was made in the ligand-binding region of human ER, so that Cre-ER only responds to the induction of exogenous synthetic estrogens (such as Tamoxifen, 4-OHT). The Cre-ER system, especially the Cre-ERT system, is currently the most widely used inducible Cre system. By simply designing Cre-ERT2 behind a tissue-specific promoter and mating it with flox mice, you can ultimately achieve spatiotemporal-specific knockout of the target gene by administering Tamoxifen at specific time points.