Mass Spectrometric Enzyme Histochemistry for Choline Acetyltransferase Reveals De Novo Acetylcholine Synthesis in Rodent Brain and Spinal Cord

Choline acetyltransferase (ChAT) is a necessary enzyme for the synthesis of acetylcholine, a neurotransmitter. It is a specific marker for cholinergic neurons. Various studies have been done on ChAT’s localization and activity in association with acetylcholinesterase. For example, in-tissue visualization of ChAT has been conducted using immunohistochemistry for the presence of the ChAT protein. These approaches, however, have some drawbacks. For example, immunohistochemistry may provide false-positive results because it recognizes molecules with antigens that are identical to those of the ChAT protein. Even though in situ hybridization visualizes an enzyme gene’s transcription location, mRNA and enzyme localization are not always constant. Moreover, transgenic mice approaches have drawbacks such as difficult production, phenotypes that aren’t necessarily identical to wild type. It also has a limited application range to other model animals.

Furthermore, none of these approaches directly demonstrate enzymatic activity, and their results do not indicate whether or not the observed cholinergic neuron is active. Enzyme histochemistry is used to visualize enzyme activity. It includes performing enzymatic reactions on tissue sections or under comparable conditions. After this procedure, the reaction products are seen via indirect methods. Conventional enzyme histochemical procedures need extra secondary reactions, and the product of the enzyme reaction must be opaque, coloured or fluorescent, before being observed under a microscope. One of the drawbacks of traditional enzyme histochemistry is the lack of secondary reactions for visualization.

Enzyme histochemistry has been used for hydrolases such as esterases and phosphatases, as well as a portion of oxidoreductases. In general, most transferases and ligases are difficult to study using traditional enzyme histochemistry. An enzyme histochemistry investigation was conducted to observe ChAT activity. This approach visualized coenzyme A generated by the breakdown of acetyl-CoA as mercaptide deposition. This reaction was indirect and unspecific for detecting ChAT activity.

In recent years, mass spectrometry imaging (MSI) has gained popularity as a new method for enzyme histochemistry. By using mass spectrometry on a tissue segment, MSI can obtain localised pictures of molecules like metabolites and proteins. MSI-based enzyme histochemistry should provide a view of diverse enzymatic processes without the need for additional reactions. Yet, MSI-based enzyme histochemistry is still in its early stages and has only been used to hydrolases like phospholipase, protease, and cholinesterase, as well as kinases and phosphatases. Osaka University scientists led by Associate professor Shuichi Shimma recently devised a semiquantitative MSI-based enzyme histochemical technique for cholinesterase (ChE) that use deuterium-labeled ACh as a substrate. The approach directly visualized ChE activity on mouse brain and fly tissues, revealing distinct and quantifiable ChE activity in great detail.

The research team were able to visualize ChAT activity in situ using MSI-based enzyme histochemistry, which is difficult with conventional enzyme histochemistry.  Excessive degradation of generated ACh-d9 was sufficiently inhibited by the addition of physostigmine. In this way, ChAT activity in the mouse brain and spinal cord could be observed. The resulting rough localization of ChAT activity is consistent with prior results, supporting this approach. Furthermore, for the first time, direct visualization in the tissues allowed us to elucidate the detailed distribution of ChAT activity in the striatum and cerebellum of the mouse brain. It also explained ChAT activity in the axon of the ventral white matter part connecting the anterior horn and ventral root of the spinal cord. Lastly, the devised approach was tested on SCI model mice to determine the site-specific influence on ChAT activity in SCI. The authors findings support the value of directly measuring ChAT activity in tissues.

The new quantitative ChAT enzyme histochemistry method is predicted to be a useful tool in the study of immunohistochemistry and cholinergic neurons. “Furthermore, the newly developed enzyme histochemistry using MSI is expected to apply difficult enzymatic reactions in the future because of its simple concept. In the field of enzyme histochemistry, the new enzyme histochemistry is expected to expand in the future in neuroscience, plant science, and insect science”, Assoc. Prof. Shimma said.