Intrinsic innervation anddopaminergic markers afterexperimental denervation in rat thymus F. Mignini,1 M. Sabbatini,2 V. D’Andrea,3

C. Cavallotti4

1Anatomia Umana, Dip. di MedicinaSperimentale e Sanità Pubblica,Università di Camerino, Camerino, Italy 2Anatomia Umana, Dip. Medicina Clinica eSperimentale, Università del PiemonteOrientale, Novara, Italy3Dip. di Chirurgia, Università La Sapienza,Roma, Italy4Dip. Scienze Cardiovascolari eRespiratorie, Università La Sapienza,Roma, Italy

Abstract

The aim of this study was to examine ratthymus innervation using denervation tech-niques and to explore the related micro-anatomical localization of dopamine, D1, D2receptors and dopamine membrane trans-porter (DAT).

In the thymus subcapsular region, theparenchymal cholinergic fibers belong exclu-sively to phrenic nerve branching. No somaticphrenic nerve branching was detected in anyother analysed thymus lobule regions.

In rats subjected to sympathetic or parasym-pathetic ablation, it was observed that cate-cholaminergic and cholinergic nerve fibersrespectively contributed to forming plexusesalong vessel walls.

In the subcapsular and septal region, noparenchymal nerve branching, belonging tosympathetic or parasympathetic nervous sys-tem was noted. Instead, in the deep corticalregion, cortico-medullary junction (CM-j) andmedulla, catecholaminergic and cholinergicnerve fibers were detected along the vesselsand parenchyma.

Dopamine and dopamine receptors werewidely diffused in the lobular cortico-medullary junction region and in the medulla,where the final steps of thymocyte maturationand their trafficking take place.

No variation in dopamine and DAT immunereaction was observed following total or partialparasympathectomy or phrenic nerve cutting.After chemical or surgical sympathectomyhowever, neither dopamine nor DAT immunereaction was noted again. Instead, D1 and D2dopamine receptor expression was not affect-ed by thymus denervation.

In rats subjected to specific denervation, itwas observed the direct intraparenchymalbranching of the phrenic nerve and sympathet-ic and parasympathetic fibers into thymusparenchyma along vessels. These findings onthe dopaminergic system highlight the impor-tance of neurotransmitter receptor expressionin the homeostasis of neuroimmune modula-tion.

Introduction

Experimental studies have shown that theneuronal pathway is involved in the control ofimmune responsiveness at several levels, fromcellular functions and interactions to overallimmunological responses.1-3

The anatomical innervation of the thymus israther complex, because it follows multiplenerve pathways. The sympathetic componentreaches the thymus through the perivascularplexus, which originates from the stellate gan-glion, which is in turn formed by the fusion ofthe third cervical ganglion together with thefirst thoracic ganglion. The parasympatheticcomponent reaches the thymus through thefibers running into the vagal branches of thesuperior and inferior (or recurrent) laryngealnerve. Another minor component of thymusnerve supply is the phrenic nerve, a somaticnerve originating in the cervical plexus.4-6

Sympathetic and parasympathetic nervefibers enter the thymus along with blood ves-sels and branch into the cortex, the capsularand septal system, the cortico-medullary junc-tion and the medulla.7 Some of the cateco-laminergic fibers leave vessels and thenbranch into thymus parenchyma.2

Up to date, the sympathetic system has beenconsidered the most important component ofthymus innervation in controlling thymusfunction, mainly via noradrenergic fibers.8 Theintraparenchymal branching nerve profile hasbeen well visualized2 and the related centralnervous system nuclei well mapped.9 However,recent evidence has pointed out an importantrole of the cholinergic pathways as well,10-15

although parasympathetic intraparenchymalinnervation is still questioned. In fact, anunquestionable neuroanatomical evidence of aparasympathetic or vagal nerve supply to thethymus has not yet been found. Whilst someauthors have reported anatomical mapping onvagal hindbrain nuclei,16 or the presence ofacethylcholinesterase-positive structures,17,18

others have not confirmed these findings9,19

and sentence about a substantial absence ofany vagal nerve supply to any immune organs.8

Similar disagreements concern data interpre-tation following the use of fluorescent tracerand horseradish peroxidase retrograde

labelling16 or retrograde transneuronal virustracing.9

Several classic neurotransmitters and neu-rotransmitter receptors have been found inthymocytes and other thymus cells, supportingthe bidirectional communication between thebrain and the immune systems.3 Pharma-cological experiments have provided evidenceof the muscarinic receptors role in differenti-ating thymocytes into cytolytic (CD8+) T lym-phocytes.15 It is thought that the mechanismresponsible for lymphocyte release from thethymus involves vagal fibers running in therecurrent laryngeal nerve and mediated bynicotinic receptors.11

Recent studies have found expression ofdopamine, dopamine receptors and trans-porters in thymus, suggesting a role ofdopamine in the maturation and selection oflymphocytes and the activation of immuneresponse.20,21 Indeed, other studies have point-ed out dopamine and noradrenaline co-releaseby sympathetic fibers innervating cortico-medullary and medullary regions.22,23

It has been observed that the effect ofdopamine on proliferation and cytotoxity ofCD4+ and CD8+ lymphocytes in vitro is mediat-ed by the D1 class of receptors,24 and expres-sion of D3 and D2 receptor subtypes has beenfound in lymphocyte outer membranes.25 D1and D2 receptors are the most widespreadreceptors types in the dopaminergic system,performing intracellular events, and acting bydifferent pathways.

The aims of this study were to examine therelationship between sympathetic and para-sympathetic intraparenchymal nerve distribu-tion in a rat model using experimental dener-vation techniques, as well as to performmicroanatomical localization of dopamine D1and D2 receptors and dopamine membranetransporter (DAT) in distinct regions of thy-

European Journal of Histochemistry 2010; volume 54:e17

Correspondence: Dr. Maurizio Sabbatini,Dip. Medicina Clinica e Sperimentale, Lab.Anatomia Umana, Università del PiemonteOrientale “A. Avogadro”, via Solaroli, 1728100 Novara, Italy.Fax: +39.0321.660632 - Tel: +39.0321.3733532E-mail: maurizio.sabbatini@med.unipmn.it

Key words: thymus, sympathetic nerve, parasympa-tethic nerve, phrenic nerve, dopamine receptors.

Received for publication: 11 November 2009.Accepted for publication: 17 February 2010.

This work is licensed under a Creative CommonsAttribution 3.0 License (by-nc 3.0).

©Copyright F. Mignini et al., 2010Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2010; 54:e17doi:10.4081/ejh.2010.e17

[European Journal of Histochemistry 2010; 54:e17] [page 81]

[page 82] [European Journal of Histochemistry 2010; 54:e17]

mus lobules. This work sought to address thediscrepant observations about thymus innerva-tion,8,19 using histochemical techniques andmonolateral surgical or chemical removal ofthymus innervation, and to resolve the dis-agreement about some methodological analy-ses, too.19,26

Materials and Methods

AnimalsExperiments were performed on 28

Sprague-Dawley male rats (2 months old;weighing 238±21 g; Charles River, Calco,Como, Italy). Animals were housed for fourweeks before the experiments, and kept undera constant light-dark cycle (7:00 a.m. to 7:00p.m. light period), at an ambient temperatureof 22±1°C, with food and water available adlibitum.

Animals were handled according to interna-tionally accepted principles for care of labora-tory animals (E.E.C. Council Directive 86/609,OJL358,1; Dec. 12,1987).

Rats were anaesthetized with pentobarbitalsodium (50 mg/kg i.p.); the monitoring eyelidand paw pinch reflexes assessed the adequacyof anaesthesia. When all reflexes were absent,rats were subjected to monolateral surgical orchemical removal of thymus innervation.

Denervation experiments were performedwith 6 experimental groups, 4 rats per group.One group was sham operated and used ascontrol group, while the other five groups weretreated as follows: surgical sympathectomy,chemical sympathectomy, surgical partialparasympathectomy, surgical parasympathec-tomy and surgical somatectomy, respectively.

Surgical proceduresSurgical sympathectomy was performed by

removal of the star ganglion, the third ganglionof the sympathetic lateral-vertebral chain, inthe neck region, on the right side of the verte-bral column.

Chemical sympathectomy was obtained bytreatment with 6-OH-dopamine (6-OH-DA).The destruction of thymus sympathetic nervefibers was achieved by partially modifying theguidelines proposed by Angeletti and Levi-Montalcini.27 Briefly, each rat received anintra-peritoneum injection of 6-OH-DA inphysiological solution with 0.5% ascorbic acidas solvent, at doses of 100 mg/Kg/day for 3days. After 3-5 days, this procedure destroyedalmost all the adrenergic nerve fibers of thebody including those of the thymus.

The surgical monolateral partial parasympa-thectomy was performed by right laryngealsuperior nerve cutting. The nerve was

approached in its location in the right lateralregion of the neck, near the vascular envelop-ment and the thyroid gland.

The surgical monolateral parasympathecto-my was performed by cutting the vagus nervein its upper course, before it supplies the supe-rior laryngeal nerve.

The surgical monolateral denervation of thesomatic nerve was performed by phrenic nervecutting, approaching the nerve along its coursebetween the mediastinic pleura and the peri-cardium.

All nerves were cut by removing about 2-3mm of their length, thus avoiding early regen-eration of the fibers. After denervation, ani-mals were allowed to survive for 2 weeks. Theefficiency of ganglionectomy was assessed bythe onset of ptosis of the eyelid.

Tissue treatmentAnimals were anaesthetized and killed by

decapitation. The thymus right lobe wasremoved from each animal, washed in coldphosphate-buffered saline (PBS), then embed-ded in a cryoprotectant medium and frozen inisopentane cooled with liquid nitrogen. Fromthis specimen, serial sections (10 μm thick,one section per slide) were cut on a cryostat at-20°C. Sections were subdivided into fourgroups of eight slides each.

The first section of each slide group wasprocessed for hematoxilyn and eosin stainingto obtain histopathologic visualization of thetissue.

The second section of each slide group wasstained using the Bodian method for frozensections,28 being this method quite useful instaining all nerve fibers and neuro-fibrils inthe thymus parenchyma.17 Nerve fibers andneuro-fibrils were stained in black.

The third section of each slide group wasprocessed for histofluorescence staining ofcatecholaminergic nerve fibers by the methoddescribed by Quayyum and Fatani,29 using theglyoxylic acid-induced fluorescence technique.Further details are reported in previous papersof our group.30,31

The fourth section of each slide group wasprocessed for histoenzymatic staining of AChEpositive nerve fibers, in accordance to the thy-ocoline method protocol.32 Further details arereported in a previous paper of our group.33

All sections were analyzed on the same dayto prevent diffusion and/or fluorescence pho-todecomposition.

ImmunohistochemistryThe fifth, sixth, seventh and eighth sections

of each slide group were used for the immuno-histochemical detection of dopamine (mousemonoclonal antibody, dil. 1:50; Abcam pic, codeab8892; Cambridge, UK), DAT (goat polyclonal

antibody, dil. 1:1000; Santa Cruz Biotechno-logy, Santa Cruz, Ca, USA), D1 (rabbit poly-clonal antibody, dil. 1:500; Chemicon, No. AB1765P), and D2 (goat polyclonal antibodySanta Cruz Biothecnology, No. sc-7522).Sections were incubated for 1 h at 4°C in a 3%normal donkey serum dissolved in 0.1 M phos-phate buffered saline (PBS) and 0.3% TritonX-100 to prevent non-specific binding of IgGs.Sections were then incubated overnight at 4°Cwith primary antibodies. Goat anti-mouse(Chemicon, No. AP124B) or donkey anti-goat(Vectastain ABC kit, Vector, No. PK6105) ordonkey anti-rabbit (Vectastain ABC kit, Vector,No. PK6101) biotynilated secondary antibodywas used with a biotin-streptavidin immunos-taining kit employing diamino benzidine(DAB) as a chromogen (Vectastaine Elite kit,Vector Laboratories). Further details arereported in a previous paper of our group.20

Control sections were processed as above,but using a non-immune IgG instead of the pri-mary antibody. No positive reaction wasobserved under these conditions.

Counting and morphometryFor each slide, analysis was performed on 5

randomly chosen thymus lobules. Thymus lobules were subdivided into the

subcapsular, septa, cortex, cortico-medullaryjunction and medulla regions. For each region,three sampling fields of analysis (eachfield=150.0¥103 μm2) were randomly chosenand for each of them the nerve fiber branchingarea was detected by microscope connected todigital camera, using image analysis computersoftware (Qwin, Leica). The nerve branchingis characterised by heterogeneous thickness-es, therefore in image analysis the digitalisedmeasurements of the nerve fiber branchingareas reliably avoid variance among samples.

Data were collected and reported as num-bers expressing the area of nerve branching inthe field of analysis (=150.0¥103 μm2).

Neural plexuses enveloped along intra-parenchymal arteries were specifically investi-gated along their course into the subcapsularzone, septa, cortex and medulla.

Fiber positive nerves, in accordance withBodian’s protocol, were analysed under micro-scope in a light field, while a fluorescencemicroscope was employed for other fluores-cence techniques.

Data analysis Data are presented as mean ± S.E.M. calcu-

lated from values detected in the individualanimals of each experimental group. The nor-mal distribution of data was assessed bymeans of the Kolgomorov-Smirnov test.Statistical differences among experimentalgroups for each lobular region investigated

Original paper

[European Journal of Histochemistry 2010; 54:e17] [page 83]

were then assessed by two-way ANOVA, withthe different experimental groups as the firstsource of variance, and the different tech-niques evidencing the nerve fibre componentas the second one; the Newman-Keuls post hoctest was performed. Statistical procedureswere performed with the Prism 2.01 statisticalsoftware (GraphPad Software Inc., CA, U.S.A.).

Results

Nerve fibers distribution Quantitative results of nerve fiber distribu-

tion in thymus vessels and parenchyma of thelobular subcapsular region are summarised inFigure 1. In the subcapsular region of the thy-mus, the nerve fibers of sham-operated ratsdisplayed wide intense branching alongperivascular sites, with a smaller number offibers branching in the parenchyma (Figure 2).

The rats subjected to chemical or surgicalsympathectomy had a strong decrease of nervefibers localized in perivascular sites, in com-parison with the sham-operated ones, whileintraparenchymal fibers were not affected byspecific denervation (Figures 1 and 2).Instead, in rats subjected to total or partialparasympathectomy, we observed a moderateor very moderate decrease respectively inperivascular fibers only, and again, intra-parenchymal fibers were not affected byparasympathectomy. In rats subjected tosomatic nerve axotomy, we detected the disap-pearance of intraparenchymal fibers only,while perivascular fibers were not affected(Figures 1 and 2). Collecting all the morpho-metric data obtained by specific nerve cuttingand the Bodian technique, we observed thatsympathetic, parasympathetic and somaticnerves had a distribution of 60%, 30% and 10%of total nerve fibers, respectively.

Histochemical techniques in the subcapsu-lar region of sham-operated rats identifiedcathecolaminergic nerve fibers in perivascularsites only, while histochemical cholinergicnerve fiber visualization revealed vascular andparenchymal presence of these fibers (Figure3). The application of histochemical tech-niques following specific denervation con-firmed the findings above detailed: cholinergicparenchymal branches disappeared afterphrenic nerve cutting (Figures 1 and 3E),while vagus and laryngeal nerve cutting indi-cated that the vascular branches belong to theparasympathetic nerve system (Figure 1).After phrenic nerve cutting, we observed novariation in perivascular, cholinergic, or cate-cholaminergic histofluorescence (Figure 1).These findings indicate that in the subcapsu-lar region, the parenchymal cholinergic fibers

Original paper

Figure 1. Analysis of thymus lobularsubcapsular region. Bar graph show-ing morphometric analysis of nervefibers running along vessels andbranching into the parenchyma.Data are reported as Area of nervefibers ¥103 μm2, evaluated on stan-dard field of analysis (see Materialsand Methods section). White bar:Bodian’s method positive nerve fibers(p.f.); Black bar: AChE p.f.; Grey bar:cathecolaminergic p.f.. Sym:Sympathectomised experimentalgroup; T-Psy: Total Parasympa-thectomised experimental group; P-Psy: Partial Parasympathectomisedexperimental group; Phr: phrenec-tom-ised experimental group.Statistical significance between datarelated to experimental groups:a=P<0.05 vs sham group; b=P<0.05vs sham and sympathectomy groups;c=P<0.05 vs sham, sympathectomyand total parasympathectomygroups; d=P<0.05 vs sham, sympa-thectomy, total and partial parasym-pathectomy groups; e=P<0.05 vssympathectomy, total and partialparasympathectomy groups;f=P<0.05 vs total and partialparasympathectomy groups.Statistical significance between dataobtained by different techniquesused, in the same experimentalgroup: °=P<0.05 vs Bodian p.f.;°°=P<0.05 vs AChE and Cathecol p.f.

Figure 2. Microphotographpanel of nerve fibers in thesubcapsular region of thy-mus. Bodian’s method. Notethe different distribution ofnerve fibers according to thedifferent nerve cuttings per-formed. (A) Sham-operatedrat; (B) Surgical sympathec-tomized rats; (C) Phren-ectomised rat; D: Partialparasympathectomised rat(laryngeal nerve cutting); E:Total parasympathecto-mized rat (nerve vagus cut-ting). Calibration bar:(A,B,E)= 40 μm; (C,D)= 60μm.

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belong exclusively to phrenic nerve branching. Quantitative results of nerve fiber distribu-

tion in thymus vessels and parenchyma of sever-al lobular regions are summarised in Figure 4.

In the septal region no nerve branching wasobserved in the parenchyma; instead, it seemsthat all fibers detected contribute to formingplexuses along vessel walls. In rats subjectedto sympathectomy or total parasympatectomywe detected a reduction of 60% and 40% oftotal fibers respectively, compared with sham-operated rats (Figure 4). The histochemicalvisualization revealed that all sympatheticfibers were cathecolaminergic, while theparasympathetic fibers were cholinergic. Inrats subjected to partial parasympatehctomy,we detected only a moderate reduction incholinergic fibers. No evidence was observedin animals subjected to phrenic nerve axoto-my (data not shown).

In the deep cortical region, cortico-medullary junction (CM-j) and medulla, thenerve fibers were observed branching alongperivascular sites, except for a few fibers leav-ing perivascular sites, branching into theparenchyma.

In rats subjected to specific denervation,the results were similar to the ones observedin the septal region. The sympathectomy and

total parasympathectomy decreased the nervefibers by 60% and 40% respectively, comparedwith sham-operated rats, in both perivascularfibers and parenchymal fibers (Figure 4). Thehistochemical visualization confirmed that allthe sympathetic fibers were cathecolaminer-gic, while the parasympathetic fibers werecholinergic (data not shown).

In particular, in these regions, after dener-vation and histochemical techniques, theabsolute number of nerve fibers branchinginto parenchyma was similar to the sympa-thetic catecholaminergic and parasympatheticcholinergic fibers detected. In terms of rela-tive distribution between vessels andparenchyma, the presence of parasympatheticcholinergic fibers was instead slightly greaterin the parenchyma, when compared with therelative distribution of sympathetic cate-cholaminergic nerve fibers (Figure 4).Furthermore, the parasympathetic cholinergicparenchymal branching was more widespreadin the deep cortex region than in the otherregions.

Microanatomical localization ofdopamine markers

The cortico-medullary junction and medulla

were the thymus areas with the greatestdopaminergic expression.

Dopamine immunoreactivity was observedin the CM-j and in the medulla, whereas noimmune reaction was observed in subcapsu-lar, septal or deep cortex region. In theimmunoreactive areas, dopamine displayedreticular localization (Figure 5A). No DATimmunoreactivity was noticeable in thethymic cortex, whereas immune reaction wasobserved in medulla and in CM-j. At highermagnification, immune reaction showedreticular localization (Figure 5C). No varia-tion was observed following total or partialparasympathectomy or phrenic nerve cutting,while after chemical or surgical sympathecto-my neither dopamine nor DAT immune reac-tion was observed again (Figure 5B, D).

Dopamine D1 and D2 receptor immunore-activity was observed in the CM-j region to aslighter extent in the medulla, whereas noimmune reaction was observed in subcapsu-lar, septal or deep cortex regions (Figure 6).

Several thymus denervation protocols didnot change the D1 and D2 receptor immunore-activity pattern observed in non-denervatedrats (data not shown).

Original paper

Figure 3. Microphotographpanel showing cathecolaminer-gic fluorescent nerve fibers(A,B,C) and histoenzymaticAChE positive nerve fibers(D,E,F). A: Sham-operated rat,subcapsular ragion. Note theexclusive distribution of fibersforming a rich perivascularplexus. B: Surgical sympathec-tomised rat, subcapsular region.Note the absence of catheco-laminergic fluorescent nervefibers. C: Sham-operated rat,medulla. Note the distributionof parallel nerve fibers runningalong vascular profile and sever-al branching fibers running intoparenchyma. D: Sham-operatedrat, subcapsular ragion. Note thedistribution of positive nervefibers running along vessels andbranching into parenchyma. E:Phrenectomised rat, subcapsularregion. Note the absence ofparenchymal fibers, while theAChE perivascular plexusremain well observable. F:Phrenic denerved rat, medulla.Note the occurrence of positivenerve fibers (parasympatheticfibers) running along vessels andbranching into parenchyma.Calibration bar: 45 μm.

[European Journal of Histochemistry 2010; 54:e17] [page 85]

Discussion

Several findings indicate the occurrence ofbranching of the somatic phrenic nerve, vagusand sympathetic nerves into the thymus.2,34

Although a more specific immunohistochem-istry technique currently provides fine visuali-zation of nerve profiles, the use of the Bodiantechnique together with other techniques isuseful in visualizing intricate intraparenchy-mal nerve branching development and itsquantitative depletion after denervation exper-iments. There are no fibrillary components ofthymus parenchyma that cause aspecific reac-tions and limit Bodian technique specificity inmarking nerve fibers.17,31,33

Thymus innervation by the phrenic nerve isan old acquisition from embriogenic4 andanatomical studies.5 This finding about thy-mus anatomy is reported in several texts andtreatises.35-37 Nevertheless, we have not beenable to find further studies about intra-parenchymal phrenic nerve thymus innerva-tions in the literature.

In the present work, the intraparenchymalbranching of the phrenic nerve has been visu-alized, following specific denervation, as anAChE-positive nerve component exclusivelydistributed in the intraparenchymal subcapsu-lar region of thymus lobules. Unlike otherintraparenchymal nerve components, thephrenic nerve does not participate to form theperivascular plexus as the access way of nervefibers. The phrenic nerve seems to access theintraparenchymal subcapsular region directlythrough the connectival capsula of thymus lob-ules. These findings agree well with the

Original paper

Figure 4. Analysis of regions inwhich thymus lobule has beensubdivided (see Materials andMethods section for detail).Bar graph showing morpho-metric analysis of nerve fibersrunning along vessels andbranching into the parenchy-ma. Data are reported asFigure 1. White bar: totalnerve fibers; Black bar:perivascular fibers; Grey bar:intraparenchymal fibers.Legend of statistical signifi-cance as in Figure 1.

Figure 5. Microphotograph panel of immu -nohistochemistry evidence of dopamine(A,B) and DAT (C,D) presence into thymus.A: Sham-operated rat. Note the occurring ofpositive dopamine immunohistochemistryinto medullary parenchyma. B: Chemicalsympathectomised rat. Note the absence ofdopamine immunohistochemistry reaction.C: Sham-operated rat. Note the occurrenceof positive DAT immunohistochemistryfibers into medullary parenchyma. D:Chemical sympathectomised rat. Note theabsence of DAT positive reaction. c: deepcortical region; m= medulla. Calibration bar:50 μm.

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embryonal origin of subcapsular thymicepithelial cells.4,38 Indeed, Mićić and col-leagues10 have found AChE positive nerve pro-files in the subcapsulary region mainly in closeproximity to the thymic epithelial cells. Thesefindings have stimulated further evaluation ofthe functional involvement of the phrenicnerve in the cortico-lympho-epitelial complex,in which thymic epithelial cells interact withimmature lymphocytes (CD4– and CD8–, doublenegative, thymocytes).39

Vagus nerve thymus innervation is anotherwidely questioned and important point.8 Ineffect, the mass of evidence about parasympa-thic innervation of the thymus is based on thefindings of an AChE positive nerve fiber profilein the thymus7,10,23 and on the influence ofcholinergic pathways in thymus function,14,15

but their belonging to effective vagal branch-ing is questioned.8,19

Some findings about direct thymus innerva-tion and influences by vagus nerve have beenreported.5,11,40 Nevertheless, mapping of brainvagal nuclei is still uncertain. In fact, someauthors reported the presence of brainstemnuclei that give rise to efferent vagal fibersthat reach the thymus gland,16,41,42 but otherstudies did not detect them.9 Indeed, Trotterand his collaborator9 excluded from their studyanimals that showed labelling neurons inNucleus Tractus Solitarii and NucleusAmbiguus because the authors consideredthese findings an indication of tracer(pseudorabies virus) leakage from the thymusto the esophagus.

To avoid these difficulties, we used AChEand cathecolamine histochemistry associatedwith specific denervation experiments, so as toinvestigate the organization of sympatheticand parasympathetic thymus nerve fibers intheir specific running and branching.

We have observed in rats subjected to specif-ic denervation the disappearance of the sym-pathetic and parasympathetic fibers that pene-trate into thymus parenchyma along vesselsaccompanying tissue connective septa (septalregion). Few fibers of either system leave ves-

sel profiles to branch into parenchyma at deep-er cortical region and lower region of thymuslobule. Many fibers of either systems remain toform plexuses along vessels branching into themedullary region. This organization may sup-port the supposed involvement of the sympa-thetic and parasympathetic systems in theblood-thymic barrier and thymocyte traffickingas reported by other authors.43,44

Comparing the quantity of sympathetic andparasympathetic fibers, we have observed thatthere are proportionally more intraparenchy-mal fibers of the parasympathetic system thanof the sympathetic one. The majority of the lat-ter remain perivascular. These findings maysupport the hypothesis of Mićić and col-leagues10 that parasympathetic system isinvolved in the thymocyte maturation process.

Previous findings of other authors haveindicated that the thymus dopaminergic sys-tem is related to the organization of the sym-pathetic nervous visceral system.21,45,46

DAT is an important marker indicatingdopamine reuptake; it is normally expressed incells involved in the use of the dopaminergicsystem.47 In the present work, we observed thatdopamine and DAT have similar immune reac-tive appearance and localization in thymusparenchyma, confirming the presence of thedopaminergic system in the thymus.Furthermore, our findings after specific nerveaxotomy show that it cannot be excluded thatthe dopaminergic system is present in sympa-thetic nerve fibers.

The same authors have also posited non-synaptic release of neurotransmitter by sympa-thetic fibers.46 Indeed, neurotransmitterrelease diffusion in a paracrine fashion mayexplain the reticular appearance of dopamineimmune reaction.

The occurrence of D1 and D2 receptorimmune reaction in thymus medulla, notaffected by denervation protocols, indicatesthe location of these dopamine receptors insites different from nerve fibers. Resolutionlimitations imposed by the microanatomicaltechniques employed in this study made it

impossible to identify the cellular populationswith dopamine concentrations significantenough to be measured. From a morphologicalpoint of view, these structures can correspondto processes of epithelial thymic cells as wellas to thymocytes strictly close to these cellularprocesses. We have begun to clarify this crucialpoint through the use of cytofluorimetry.

To summarize, in the present work, the dis-tribution of the dopaminergic system has beenobserved mainly in the thymus medulla wherethymocytes have their last maturation steps.These findings may be related to observationsof dopamine receptors on outer membrane ofcirculating lymphocytes,24,25 suggesting that thedopaminergic system plays a functional role onthymocytes resident in the thymus medulla.Further experiments are underway to definethe role of the dopaminergic system in thefunctional biology of thymocytes and maturelymphocytes.

References

1. Elenkov IJ, Wilder RL, Chrousos GP, ViziES. The sympathetic nerve. An integrativeinterface between two supersystems: thebrain and the immune system. PharmacolRev 2000;52:595-638.

2. Mignini F, Streccioni V, Amenta F.Autonomic innervation of immune organsand neuroimmune modulation. AutonAutacoid Pharmacol 2003;23:1-25.

3. Wrona D. Neural-immune relations: Anintegrative view of the bidirectional rela-tionship between the brain and theimmune systems. J Neuroimmunol 2006;172:38-58.

4. Hammar JA. Konstitutionsanatomischestudien über die neurotisierung des men-schenebryos; über die innervationsver-hältnisse der inkretorgane und der thymusbis in den 4. Fötalmonat Z Mikrosp-AnatForsch 1935;38:253-93.

5. Bulloch K, Pomerantz W. Autonomic nerv-ous system innervation of thymic-relatedlymphoid tissue in wild type and nudemice. J Comp Neurol 1984;228:57-68.

6. Kendall MD. Functional anatomy of thethymic microenvironment. J Anat 1991;177:1-29.

7. al-Shawaf AA, Kendall MD, Cowen T.Identification of neural profiles containingvasoactive intestinal polypeptide, acetyl-cholinesterase and catecholamines in therat thymus. J Anat 1991;174:131-43.

8. Nance DM, Sanders VM. Autonomic inner-vation and regulation of the immune-sys-tem (1987-2007). Brain Behav Immun2007;21:736-45.

9. Trotter RN, Stornetta RL, Guyenet PG,

Original paper

Figure 6. Microphotograph panel of immunohistochemistry evidence of D1 dopamine recep-tor (A) and D2 dopamine receptor (B) presence in the thymus. Legends as in Figure 5. c: deepcortical region; CM-j= cortico-medullary junction; m= medulla. Calibration bar: 50 μm.

[European Journal of Histochemistry 2010; 54:e17] [page 87]

Original paper

Roberts MR. Transneuronal mapping ofthe CNS network controlling sympatheticoutflow to the rat thymus. Auton Neurosci2007;131:9-20.

10. Mićić M, Leposavić G, Ugresić N, BogojevićM, Isaković K. Parasympathetic innerva-tion of the rat thymus during first life peri-od: histochemical and biochemical study.Thymus 1992;19:173-82.

11. Antonica A, Ayroldi E, Magni F, Paolocci N.Lymphocyte traffic changes induced bymonolateral vagal denervation in mousethymus and peripheral organs. J Neuro -immunol 1996;64:115-22.

12. Tracey KJ. The inflammatory reflex.Nature 2002; 420:853-859.

13. Pavlov VA, Wang H, Czura CJ, Bogojević M,Isaković K. The cholinergic antinflamma-tory pathway: a missing link in neuro-immunomodulation. Mol Med 2003;9:125-34.

14. Saeed RW, Varma S, Peng-Nemeroff T,Sherry B, Balakhaneh D, Huston J et al.Cholinergic stimulation blocks endo-the-lial cell activation and leukocyte recruit-ment during inflammation. J Exp Med2005;201:1113-23.

15. Zimring JC, Kapp LM, Yamada M et al.Regulation of CD8+ cytolytic T lymphocytedifferentiation by a cholinergic pathway. JNeuroimmunol 2005;164:66-75.

16. Dovas A, Lucchi ML, Bortolami R et al.Collaterals of recurrent laringeal nervefibres innervate the thymus: a fluorescenttracer and HRP investigation of efferentvagal neurons in the rat brainstem. BrainRes 1998;809:141-8.

17. Cavallotti D, Artico M, Iannetti G,Cavallotti C. Quantification of acetyl-cholinesterase-positive structures inhuman thymus during development andaging. Neurochem Int 2000;36:75-82.

18. Cavallotti D, Artico M, Cavallotti C et al.Acetylcholinesterase activity in rat thymusafter immunostimulation with interleukinβ. Ann Anat 2000;182:243-8.

19. Nance DM, Hopkins DA, Bieger D. Re-investigation of the innervation of the thy-mus gland in mice and rats. Brain BehavImmun 1987;1:134-47.

20. Mignini F, Traini E, Tomassoni D, AmentaF. Dopamine plasma membrane trans-porter (DAT) in rat thymus and spleen: animmunochemical and immunohistochem-ical study. Auton Autacoid Pharmacol

2006;26:183-9.21. Mignini F, Tomassoni D, Traini E, Amenta

F. Dopamine, vesicular transporters anddopamine receptor expression and local-ization in rat thymus and spleen. JNeuroimmunol 2009; 206:5-15.

22. Torres KC, Antonelli LR, Souza AL et al.Norepinephrine, dopamine and dexam-ethasone modulate discrete leukocyte sub-populations and cytokine profiles fromhuman PBMC. J Neuroimmunol 2005;166:144-57.

23. Živković I, Rakin A, Petrović-Djergović DMet al. The effect of chronic stress on thy-mus innervation in the adult rat. ActaHistochem 2005;106:449-58.

24. Saha B, Mondal AC, Majumder J et al.Physiological concentrations of dopamineinhibit the proliferation and cytotoxicity ofhuman CD4+ and CD8+ T cells in vitro: areceptor-mediated mechanism. Neuro-immunomodulation 2001;9:23-33.

25. Levite M, Chowers Y, Ganor Y et al.Dopamine interacts directly with its D3and D2 receptors on normal human T cells,and activates beta 1 integrin function. EurJ Immunol 2001;31:3504-12.

26. Bellinger DL, Lorton D, Hamill RW et al.Acetylcholinesterase staining and cholineacetyltransferase activity in the youngadult rat spleen: lack of evidence forcholinergic innervation. Brain BehavImmun 1993;7:191-204.

27. Angeletti PU, Levi-Montalcini R.Sympathetic nerve cell destruction in new-born mammals by 6-hydroxydopamine.PNAS 1970;65:114-21.

28. Herr BE, Coleman PD, Griggs RC. A Bodianmethod for mounted frozen sections. StainTechnol 1976;51:261-5.

29. Qayyum MA, Fatani JA. Use of glyoxylicacid in the demonstration of autonomicnerve profiles. Experientia 1985;41:1389-90.

30. Artico M, Cavallotti C, Iannetti G, Caval -lotti D. Effect of interleukin 1b on rat thy-mus microenvironment. Eur J Histochem2001;45:357-66.

31. Artico M, Cavallotti C, Cavallotti D.Adrenergic fibres and mast cells: correla-tion in rat thymus. Immunol Lett 2002;84:69-76.

32. Karnowsky MJ, Roots L. A “direct-coloring”thiocholine method for cholinesterases. JHistochem Cytochem 1964;12:219-21.

33. Artico M, Cavallotti C, Tranquilli Leali FMet al. Effect of interferon on thymusmicroenvironment. Immunol Lett 2003;85:19-27.

34. Meredith EJ, Chamba A, Holder MJ et al.Close encounters of the monoamine kind:immune cell betray their nervous disposi-tion. Immunology 2005;115:289-95.

35. Grey’s Anatomy, by P.L. Williams XXXVIIIedition, Pearson Professional Limited,London, 1995.

36. Ritter MA, Crispe JN. The thymus. JRLpress, Oxford, 1992.

37. Anastasiadis K, Ratnatunga C. The thymusgland – Diagnosis and Surgical manage-ment. Springer Verlag, Berlin, 2007

38. Sultana DA, Tomita S, Hamada M et al.Gene expression profile of third pharyn-geal pouch reveals role of mesenchymalMafB in embryonic thymus development.Blood 2009;113:2976-87.

39. Pezzano M, Samms M, Martinez M,Guyden J. Questionable thymic nurse cell.Microbiol Mol Biol Rev 2001;65:390-403.

40. Niijima A, Hori T, Katafuchi T, Ichijo T. Theeffect of interleukin-β on the efferentactivity of the vagus nerve to the thymus. JAuton Nerv Syst 1995;54:137-44.

41. Bulloch K, Moore RY. Innervation of thethymus gland by brain stem and spinalcord in mouse and rat. Am J Anat1981;162:157-66.

42. Fatani JA, Qayyum MA, Metha L, Singh U.Parasympathetic innervation of the thy-mus: a histochemical and immunocyto-chemical study. J Anat 1986;147:115-9.

43. Kendall MD, al-Shawaf A. Innervation ofthe rat thymus gland. Brain Behav Immun1991;5:9-28.

44. Goldschneider I. Cyclical mobilization andgated importation of thymocyte progeni-tors in the adult mouse: evidence for a thy-mus-bone marrow feedback loop. ImmunolRev 2006;209:58-75.

45. Katz DM, Adler JE, Black IB. Catecho -laminergic primary sensory neurons: auto-nomic targets and mechanisms of trans-mitter regulation. Fed Proc 1987;46:24-9.

46. Vizi ES, Elenkov IJ. Nonsynaptic noradren-aline release in neuro-immune responses.Acta Biol Hung 2002;53:229-44.

47. Giros B, Caron MG. Molecular characteriza-tion of the dopamine transporter. TrendsPharmacol Sci 1993;14:43-9.