Immune responses raise the levels of corticosteroids and activity of hypothalamic noradrenergic neurons. In addition, stress or lesions in various brain regions generate alterations in immune functions. It has been observed that stress leads to involution of the thymus, alters distribution patterns of T-cell subpopulations, decreases migration of bone marrow cells to the thymus. Stress induces increased levels of monoamines in the circulation, and decreases the content of noradrenalin in spleen and thymus. It has also been shown that noradrenalin reduces lymphocyte responses in vitro to mitogen stimulation and suppresses cellular immune functions, most likely via adrenergic receptors. Apparently, there is an extensive bidirectional communication between the central nervous system (CNS) and the immune system.

For a long time, it was assumed that bioactive substances produced and released by CNS and thymus are the exclusive means of communication between brain and thymus. However, elucidation of thymus innervation indicate that, beside the hormonal route, a neural route of communication exist between these organs as well. It is now clear that nerve fibers of the autonomic nervous system participate in the dialogue between brain and thymus.

Sympathetic nerve fibers in the thymus have been revealed by various techniques. It has been demonstrated that they are localized in the capsular and septal compartments, where the majority of nerve fibers are localized around the vasculature. Distribution patterns of these nerve fibers have been described as regional and specific in the cortex parenchyma of the thymus. However, nerve profiles in thymus do not make “classical” synaptic contacts with target cells such as thymocytes, mast cells, fibroblasts and epithelial cells.

On the basis of these considerations, we have investigated, at first whether chronic stress, induced by forced swimming, alters distribution patterns and density of nerve fibers containing monoamine and acetylcholinesterase (AChE) in the thymus of adult rats. Secondly, we examined whether changes in thymus innervation induce morphological alterations in thymus compartments.

Male rats of the imbred AO strain were used in the experiments. The animals were divided into two groups: one group of 65-days old rats was exposed to the stress procedure, and the other served as control. Each group of animals consisted of 10 rats. They were housed in polycarbonate cages in our vivarium, under standard laboratory conditions (22±1 °C with a 12-h light/dark cycle). Food and water were available ad libitum. Our Institutional Animal Care and Use Committee has approved this experimental protocol.

Swimming stress was conducted on the basis of a modification of the stress procedure described by Porsolt et al. (1977). Stress was exerted between 8:00 and 11:00 a.m. to avoid effects of circadian rhythms. The stress procedure was applied during 21 consecutive days. Rats were removed from their cages, transported to a separate treatment room before every session. The rats were plunged individually into vertical Plexiglas cylinders (35 cm high, 25 cm diameter) containing a water column with a height of 20 cm that was maintained at 25 °C. On the first day of the stress procedure, animals were held in the cylinders for 15 min and the following days for 5 min. Animals were observed continuonaly when they were in water. At the end of each stress session, the rats were dried briefly (15 min) at 32 °C before being returned to their cages. The water was changed after each session with an animal to avoid olfactory cues left by the previous animal. The rats were sacrificed when 86-days old by decapitation. The thymuses were carefully removed, snap frozen and stored at ?70 °C until sectioning using a cryostat (Reichert, Wien, Austria) at ?20 °C. Serial sections of the thymuses from various levels were mounted onto slides and used for morphometrical and enzyme/fluorescence histochemical analysis. Approximately, 50 sections (10 ?m thick) were not taken between each analyzed level. Consecutive thymus sections, at each level, were used for fluorescence histochemistry to determine distribution patterns of monoaminergic nerves profiles and cells, enzyme histochemistry to determine distribution patterns of AchE-positive nerve profiles and cells and histology after staining with hematoxylin and eosin to identify thymus compartments and to perform morphometry.

The SPG method described by De la Torre (1980) was used for the identification of monoaminergic nerve profiles and cells. Thymus sections (10-?m thick) were dipped into a solution containing 2% glyoxylic acid, 0.2 M sucrose and 0.236 M monobasic potassium phosphate (pH 7.4) for 10 min. Thymus sections were dried afterwards under a gentle warm airflow. We standardized the time between sectioning of thymuses and dipping sections in glyoxylic acid. Moreover, we used two hair dryers with standardized drying time and temperature to assure a constant treatment protocol. Sections were then covered with a drop of paraffin oil, heated at 95 °C for 2.5 min and immediately afterwards cover slipped. All sections were analyzed on the same day to prevent diffusion and/or photodecomposition of fluorescence. Sections were examined using a BH2 fluorescence photomicroscope (Olympus, Tokyo, Japan) equipped with exciting filter BP-405 and barrier filter Y-475.

For visualization of cholinergic innervation of the thymus, the direct thiocholine method was used. Thymus sections (20-?m thick) taken from different tissue levels were fixed for 1 min in 10% formaldehyde containing 1% calcium chloride. After fixation, sections were rinsed three times in distilled water. Sections were than dipped in a solution containing 5 mg acethylthiocholine iodide as substrate for AChE in acetate buffer (pH 5.5) and incubated for 1 hour at 37 °C. Tetraisopropylpyrophosphoramide (iso-OMPA, Sigma, St. Louis, MO, USA) was used as an inhibitor of non-specific esterase activity. For visualization of enzyme activity, sections were incubated with 0.5% diaminobenzidine (DAB) in 0.1 M sodium phosphate buffer at room temperature for 45 min in the dark. Control sections were incubated in the buffer in the absence of substrate and non-specific esterase inhibitor. The thymus sections were examined with an Olympus BH2 photomicroscope.

Hematoxylin and eosin-stained thymus sections (5-?m thick) were used for histological analysis using an Olympus BH2 microscope, and morphometrical analysis was performed using a multipurpose test system M 42 (42 test points; 21 test lines) attached to a Biovar microscope (Reichert, Wien, Austria). Schematic representation of the application of the multipurpose test system M 42 is shown in Fig. 1.