In humans and non-human primates, the locus ceruleus (LC) corresponds to the region in the dorsolateral portion of the rostral mesencephalic and caudal mesencephalic tegmentum [1]. In the mouse, this nucleus is localized to the lateral part of the periventricular gray matter and is composed of closely packed, medium-sized nerve cells [2]. The LC contains noradrenaline neurons that project widely throughout the central nervous system [3]. When we stained paraffin sections from mouse LC with the Holmes silver impregnation method, we noticed small, silver-positive structures [4]. Furthermore, using hematoxylin-eosin staining, we also observed faintly eosinophilic small bodies in the cytoplasm [4]. The existence of nucleolus-like inclusion bodies has been demonstrated in nerve cells of the central nervous system in normal mice and rats using electron microscopy (EM) [5,6]. These inclusion bodies are devoid of a limiting membrane and comprise aggregates of granular and/or filamentous materials. It has been reported that intracytoplasmic inclusion bodies contain protein and some RNA [4,7-9]. Holmes’s silver-positive bodies were identified in neuronal cytoplasm with silver staining of paraffin sections. The nucleolus-like inclusion bodies were demonstrated using EM [4,10]. We proved that the silver-positive bodies identified using the Holmes method and the inclusion bodies demonstrated with EM were the same. We subsequently modified the Holmes method to shorten the reaction time for Holmes silver staining [11,12].

It is well known that primates have melanin granules in the perikarya and dendrites of neurons in the LC in the brain [13]. Furthermore, neurons in the LC contain spherical protein bodies (acidophilic bodies) containing proteins and/or peptides, which are distributed in the soma and dendrites of human monoamine neurons [14-16].

The present study aimed to compare mouse intracytoplasmic inclusion bodies with human spherical protein bodies.

Seven normal ddY male mice (body weight, about 20 g) were used for this experiment. All animals were deeply anesthetized with ether then perfused with fixative solution. The protocol for the use of these animals was approved by the Animal Care and Use Committee of Fujita Health University.

In the present study, the PTAH method and the Mallory method were used. The PTAH stain has a high affinity for basic amino acids (lysine, arginine, histidine) [17,18]. The PTAH staining of the nucleolus and protein bodies reflects the high concentrations of arginine in those structures [19-21]. The inclusion bodies of mouse (observed in this study) and protein bodies of human [16] have identical histochemical reactions in brain tissue, where they are easily detected by acid fuchsine in the Mallory method as well as by the PTAH method. Although the presence of acidophilic components in the inclusion and protein bodies has been suggested by previous experiments [4,14-16], the nature of these molecules remains unclear.

From our studies, the inclusion bodies appeared to be globes of 1.0 - 3.0 μm in diameter. The fine structure lacked a limiting membrane, and indicated a small accumulation of electron-dense balls. Inclusion bodies were reportedly formed from rER and free ribosomes in cultured mouse cells that include an insertion of the Huntington’s disease gene, using in situ hybridization-electron microscopy [22]. We also thought the inclusion body to be composed of the product of the rER and free ribosomes from our EM observations of hamster LC [23]. The protein bodies from the human were composed of the dense homogeneous substances and a dense rim and a limiting double membrane [26]. They stand out and are differentiated from all other neuronal inclusions, especially melanin. In earlier EM observations, spherical dense bodies that originated inside mitochondria were found in the LC of Japanese macaque monkeys [24] and humans [25,26]. The findings of all previous studies have consistently reported that the dense homogeneous substance of protein bodies is stored gradually in the matrix of the mitochondria and finally a round homogeneous mass remains and is surrounded by the two mitochondrial membranes [27]. Therefore, the fine structure of the inclusion bodies and protein bodies was quite different.

We reported that the preferential occurrence of inclusion bodies in mouse seems to be in the LC, some hypothalamic nuclei and other parts of the autonomic and limbic systems as well as in the circumventricular structures [28]. Consistent with our observations, it was reported that the protein bodies exist in the aminergic cell groups [15,16].

Our previous studies used experimental conditions such as dehydration/fasting or reserpine administration for mice, and the numbers of inclusion bodies decreased [29]. It was reported that the protein bodies were found to be severely depleted or missing from the neurons of substantia nigra (SN) and LC in Parkinsonian brains [15] and disrupted in the LC in depression [30]. These findings point to the importance of the metabolic economy of catecholamine neurons since protein bodies in the SN and LC were lost in parallel with the dopamine loss in the SN, with the reduced concentrations of norepinephrine in the LC reported in Parkinsonian brains [31,32] and with disturbed metabolism of norepinephrine in cerebrospinal fluid in depression [33].

Based on the present findings, we can conclude that although both inclusion bodies and protein bodies are formed with the same composition (arginine), their place of origin and fine structure were different. In addition, the numbers of both the inclusion bodies and protein bodies decrease in various disease states. The argininerich basic protein of the spherical bodies, it is argued that, may be involved in the modulation of excitability of the catecholamine neurons in the rodent and the human. To better understand the functional significance of the protein bodies and inclusion bodies, further study will be necessary.