A Master in the Histochemistry of the Cholinergic Synapse – Insight for Future Researches

Citation: Philippe Anglade , Yamina Larabi-Godinot (2019) Shigeru Tsuji (1936-2008): A Master in the Histochemistry of the Cholinergic Synapse – Insight for Future Researches. J Neurophysiol Neurol Disord 5: 1-11. *Corresponding author: Philippe Anglade, 28 bis Allée Piketty, Saint-Fargeau-Ponthierry, 77310 Saint-Fargeau-Ponthierry, France; Tel: 33-1-01 60 65 75 15; E-mail: philippe-anglade@orange.fr

. He gave his contribution to pioneering works on the molecular species of AChE in the laboratory of Jean Massoulié (Physical and Chemical Biology Institute, Paris) [6]. He worked on the putative role of intra-cellular AChE in the laboratory of Ladislas Tauc (Laboratory of Cellular and Molecular Biology, Gif-sur-Yvette) [7]. These years passed in a very stimulating atmosphere provided Tsuji with a strong formation for advanced research. Thus, his expertise in electron microscopy largely contributed to the basic study performed by Denise Cade (Pierre et Marie Curie University) on the in vitro de-differentiation of the proximal tubules of the bovine nephrons [8]. Tsuji was hence armed to begin his original contribution to the understanding of the cholinergic synapse. This started when he attempted to localize AChE on 35 nm thick ultra-thin frozen sections by a negatively stained immune complex. By this technical tour-de-force, he obtained an AChE immuno-staining on the basal lamina of the synaptic cleft in the electric organ of electric eel [9].
The cellular localization of the cholinergic agents: a rather hard challenge for histochem-

ists.
Degradation of acetylcholine released from nerve terminals was considered as an essential step for the fine regulation of the action of the neurotransmitter on the target cells.
Thus, thorough researches were undertaken to localize and identify AChE, the agent of acetylcholine degradation [for review see 10]. In situ localization of AChE activity became possible thanks to the Koelle and Friedenwald's method [11] that used acetylthiocholine as an artificial substrate of AChE.
Thiocholine was formed as the reaction product and precipitated at the enzymatic site with copper ions. Thus the final precipitates of cupro-thiocholine (converted in cupric sulphide, a black precipitate) revealed in situ the sites of AChE activity, providing controls of the specificity for AChE. After its subsequent improvement to decrease the artifactual diffusion of the precipitates [3], AChE histochemistry became an important tool for the in situ study of the cholinergic nervous system. A method, based on the formation of the metallic precipitate of cupric ferrocyanide at the site of AChE activity, was proposed by Karnovsky and Roots [12]. The reaction of Karnovsky and Roots allowed reliable observation of enzymatic activity at the ultra-structural level, and, thus, gave a new impulse in the histochemistry of the cholinergic nervous system. It was shown that AChE activity was located in the synaptic cleft associated with cholinergic neurons [13,14]. This was confirmed when AChE itself was localized by immuno-cytologic procedures using specific antibodies directed against the purified enzyme [15]. However, AChE activity was currently detected not only in the cholinergic but also in the non-cholinergic neuronal cell bodies. Since histochemical detection of AChE was not suitable to identify unequivocally the cell bodies of the cholinergic neurons, it was considered as a last resource, only useful for general staining of nerve circuits. Moreover, in situ detection of choline acetyltransferase (ChAT), the synthesis enzyme of acetylcholine, and of its enzymatic activity, remained a puzzling question, especially in the peripheral nervous system. At last, despite the breakthrough made in the research on acetylcholine receptors [16], the study of cholinergic synapse in situ was hampered by the impossibility to precipitate acetylcholine itself for a suitable localization [see 17]. All these obstacles were certainly much more than sufficient to engage an eager histochemist, as was Tsuji, to gather his strengths for the scientific challenge of the cholinergic synapse! ( Figure.

Histochemical detection of AChE activity: when old methods shed light on new problems
Thanks to their modification of the original Koelle and Friedenwald's method, Couteaux and Taxi obtained a satisfactory localization of AChE activity in the frog neuromuscular junction [3]. When these authors observed, at light microscope level, AChE activity underlying the folds of the sarcolemma outside of the nerve endings, they proved the relevance of histochemistry for the investigation of the nervous system. This all the more as Karnovsky and Roots devised, a decade later, a method enabling suitable localization of AChE activity under electron microscope [12]. Tsuji began his active research in cholinergic nerve system in this hinge period.
He rapidly understood that the sharpness of the localization of AChE activity could still be improved by obtaining a minimal diffusion of the histochemical precipitates. He, thus, began thorough methodological research that will last on more than 30 years, with ups and downs before obtaining a fine result at the end.
Following a trend of his personality, Tsuji looked at first for elucidating the components formed in the "soups" of the histochemical reactions. He liked to compare maliciously histochemistry with good cooking and a beautiful painting. This humor masked a profound exigence towards experimental results. Furthermore, Tsuji always thought that science without joy and beauty is not more than a poor and sad thing ( Figure.  Fe ++ (CN) 6 ), the well-known precipitate of Karnovsky and Roots. It was demonstrated that, in the medium of Karnovsky and Roots, cuprous thiocholine iodide reacted with Fe +++ (CN) 6 to form Cu + 3 Fe +++ (CN) 6 . Thus, the reaction of Karnovsky and Roots actually resulted in the formation of two precipitates, Cu ++ 2 Fe ++ (CN) 6 and Cu + 3 Fe +++ (CN) 6 (26). Moreover, these two products may be transformed into each other by alternating intra-molecular oxide-reduction [19]. However, these significant in vitro  (From Tsuji, 1998) The aptitude of Tsuji for re-using and renewing the neglected methods was particularly well illustrated in the field of AChE histochemistry. Indeed, he used the precipitation of organic non-metallic compounds, such as red α-naphtyl azo dye [23], to reveal AChE activity after silver nitrate impregnation [24]. Moreover, employing these non-metallic compounds for the histochemical reaction, he devised a method combining the localization of AChE activity and neuropeptide immunoreactivity [25].  [27]or induction of AChE during apoptosis [28].Tsuji participated with enthusiasm for this renewal. Thus, in collaboration with morphologists working on the central nervous system, he provided ultra-structural data suggesting AChE release from the dendrites of the dopaminergic neurons in the rat substantia nigra [29]. Moreover, using immunocytochemistry, Tsuji detected AChE in structures J Neurophysiol Neurol Disord 2019 | Vol 5: 102 JScholar Publishers 4 identified as dendritic spinules. These data suggested a putative role of AChE in synaptic plasticity, in particular in the remodeling of the cholinergic synapses [30].
The discovery of the role of AChE in the pathogenesis of Alzheimer's disease could be of therapeutic importance. Until now, Alzheimer's disease is most commonly treated by AChE inhibitors in order to reduce the symptoms due to the loss of cholinergic neurons. However, AChE might intervene in the pathological process by favoring the aggregation of beta-amyloid peptide and modulating tau-phosphorylation [31][32][33]. Therefore, the proposition of new AChE inhibitors specifically targeting beta-amyloid deposition and abnormal tau-phosphorylation may lead to an improvement in the cure of Alzheimer's disease [31][32][33]. In this context, Tsuji showed, thanks to the fruitful sup-

Ionic fixation of acetylcholine-like cations: sun and rain on original research
This is now a key point of Tsuji's work. It is the best and the most disputable part of his research. The first wish of this report is that the best be highly considered for future investigations, whereas most disputable be simply seen as the reverse of the medal.
Tsuji wrote in a first paper: "A chromatography technique which characterizes choline by phosphomolybdic acid (…) an analogous molybdenum compound of PTA (phosphotungstic acid), gave us the idea of using an heteropoly tungstic acid for cytochemistry of diffusible substances in nerve terminals. " [35]. Thus, he understood that tungstic heteropolyanions might precipitate acetylcholine in situ, providing the use of suitable experimental conditions. And, indeed, after using phosphomolybdic or silicotungstic acid (STA) at low pH, he regularly    Further works allowed to precise both the limits and the relevance of ionic fixation for specific detection of cholinergic nerve terminals. By using this method, the plasticity of the nerve afferents to nigrostriatal dopaminergic neurons was evidenced in Parkinson's disease, although the cholinergic nature of all the stained terminals could not be assessed [42] (Figure. 7).
Moreover, ionic fixation seemed more specific for acetylcholine in the peripheral nerve system [37,41] [43]. This is all the more interesting as ionic fixation is compatible with immuno-cytochemical reaction [43].In the same way, an important capture of [ 3 H] choline was evidenced in fibrocytes surrounding the frog motor end-plates [44] (Figure.  In one word, ionic fixation is a major contribution for a reliable visualization of quaternary ammonium compounds used as histochemical markers, such as [ 3 H] choline and d-tubocurarine [45]. Its relative specificity for acetylcholine in situ remains a question to be precisely estimated. This method must be a sound and useful tool for future studies in the domain of the cholinergic synapse. This is certainly the best wish that Tsuji would transmit to the next generation of researchers.

When research and friendship gathered…
The ways of scientific research are often going by human friendship. Thus, Tsuji met Shomatsu Yokoyama in the laboratory of Greven, at the time when he was a young physiologist who was studying the nerve conductor system of the frog heart.
In these years, Yokoyama was already an experimented scien- Tsuji was endowed with a particular sense for elucidating the chemical interactions between ions or molecules in the cellular tissues. It is, thus, not surprising that he felt a great interest for the book written by Pascal Mentré on the practically unknown (but probably basic) role of cellular water in the intermolecular reactions [51]. He, himself, devoted huge energy to promote and achieve the Japanese translation of this original work thanks to the unconditional support and contribution of his Japanese scientific friends [51]. in the living brain [54][55][56].
Once, Maurice Israël (Laboratory of cellular and molecular biology, CNRS, Gif-sur-Yvette) proposed a name for the laboratory that his friend had settled in his house of Bagneux: "Room René Couteaux". This is a summary of the scientific inspiration of Tsuji who was proud to be considered as a true pupil of Couteaux, the founder of the modern school of French neurocytology. The pupil paid homage to his master in the last publication [57], and in a symposium organized in 2007 in Paris by Jean-Gaël Barbara (REHSEIS Laboratory, CNRS UMR 7596) on the history of the French school of neuroanatomy [58]. In turn, Couteaux had, long before, confirmed that the work of Tsuji was a major contribution to neuroscience. Indeed, one day, using poetic humor towards his pupil, Couteaux told him: "As you know, the art of the Japanese painter Leonard Foujita a is a foreign flower which bloomed on a foreign land"… a Tsuguharu Fujita (also called Leonard Foujita) was born in 1886 in Tokyo. He was an expressionist Japanese painter who came and settled in France before the First World War. He