functional role of these brush cells, and of ACh in particular, inchemosensation in health and disease.

doi:10.1016/j.autneu.2013.05.140

Poster 20.15

Acetylcholine levels and cholinergic mechanisms in PRiMAdeficient mice

F. Mohr (Department of Pharmacology, Goethe University, Frankfurt,Germany), E. Krejci (Université Paris Descartes CNRS UMR 8194,Paris, France), M. Zimmermann, J. Klein (Department of Pharmacol-ogy, Goethe University, Frankfurt, Germany)

The proline-rich membrane anchor (PRiMA), a transmembraneprotein, directs acetylcholinesterase (AChE) to the cell surface andorganizes AChE into its tetrameric and functional form. In PRiMA KOmice striatal AChE activity is decreased to less than 10% of wild type(WT) activity. As cholinergic dysfunction plays a major role in avariety of diseases (e.g. Alzheimer’s disease, Myasthenia gravis), it isof general interest how AChE activity is regulated and if PRiMA maybe a new target for the treatment of cholinergic dysfunction. In thepresent study, we analyzed extracellular striatal and hippocampalacetylcholine (ACh) levels in PRiMA KO mice. ACh samples werecollected by in vivo microdialysis in awake mice. Samples wereanalyzed for ACh by HPLC. Basal levels in the striatum of ACh in WTmice were 3.5 ± 2.5 fmol/5 μl. In PRiMA KO mice, ACh levels weredramatically increased by 350fold (1446 ± 704 fmol/5 μl; N ≥ 22each). Intrastriatal infusion of the specific AChE inhibitor BW284c51caused a rise of ACh levels in both WT and KO mice. No modificationsof ACh levels were detected after treatment with the specific BChEinhibitor bambuterol suggesting that BChE does not play a major rolein adaptive mechanisms. Although ACh levels are extremely high andcan be further raised by AChE inhibitors, PRiMA KO mice do not showany noticeable phenotype. In further experiments, a dysfunction ofmuscarinic M2 receptors was revealed by intrastriatal infusion of themuscarinic agonist, oxotremorine (100 μM). Behavior tests indicatethat PRiMA KO mice do not have altered cognitive function. Ourfindings of dramatically increased ACh levels in PRiMA KO mice, inthe absence of an overt phenotype, suggest that inhibition of thePRiMA-AChE interaction by drugs may compensate for centralcholinergic dysfunction in the absence of serious side effects.

doi:10.1016/j.autneu.2013.05.141

Poster 20.17

Chronic skin inflammation is associated with functionallyrelevant NNCS changes in the skin

Eva M.J. Peters (Psychoneuroimmunology Laboratory, Department ofPsychosomatic Medicine and Psychotherapy, Justus-Liebig-University,Giessen, Germany), Anna V. Michenko (Department of Dermatology,University Hospital Giessen, Giessen, Germany), Jörg Kupfer (Institute ofMedical Psychology, Justus-Liebig University, Giessen, Germany),Wolfgang Kummer (Institute of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany), Susanne Tumala, BadrinarayananRaghavan (Psychoneuroimmunology Laboratory, Department of Psycho-somatic Medicine and Psychotherapy, Justus-Liebig-University, Giessen,Germany), Uwe Gieler (Department of Dermatology, University HospitalGiessen, Giessen, Germany)

The stress response to inflammatory as well as psychoemotionalchallenges may involve cholinergic signaling not only on a systemiclevel but also locally in peripheral tissues. Here we investigate theeffect of a short and rather acute psychoemotional stress challenge(Trier Social Stress Test [TSST]) on neuro-immune plasticity andmembers of the non-neuronal cholinergic system (NNCS) of the skinin patients with atopic dermatitis (AD) compared to skin-healthycontrols. Immunohistomorphometry (IHM) of nerve fiber-densityand -immune contacts revealed that lesional skin of AD patientscompared to healthy control skin showed significantly highernumbers of PGP 9-5+ nerve fibers and lower numbers of tryptase+mast cells in the upper dermis. After stress reduced nerve fibernumbers were accompanied by decreased neuronal-plasticity (Gap43and NGF IHM) and decreased neuro-immune contacts between PGP9.5+ nerve fibers and mast cells solely in lesional AD skin while innon-lesional AD skin, nerve-mast cell contacts showed a tendency toincreased numbers. By quantitative RT-PCR we found that α7nAChRlevel was lower in AD skin than in controls and that stress showed atendency to increase it solely in lesional AD skin. At the same time,α3nAChR level was lower in non-lesional and higher in lesional ADskin compared to controls and stress decreased it in healthy skinwhile it was increased it in AD skin. Secreted mammal Ly-6/urokinase-type plasminogen activator receptor-related protein(SLURP) 1 (endogenous α7nAChR ligand) mRNA level was lower innon-lesional AD than in healthy controls and SLURP-2 (endogenousα3nAChR ligand) showed a like tendency while lesional AD skin didnot show much difference to control. After stress, the expression ofSLURP-1 and -2 decreased in healthy skin but increased in lesionalAD skin. By contrast, in non-lesional AD skin after stress SLURP levelsremained low. Clinical/pathological outcome parameters revealedhighest epidermal thickness in lesional AD skin which was reducedafter stress experience along with improved disease activity asassessed by the SCORAD. By contrast, in non-lesional AD skin, alongwith the described tendency towards higher numbers of nerve-mastcell contacts, epidermal thickness was increased after stress alongwith a tendency towards higher itch perception. We conclude thatpsychoemotional stress has differential NNCS effects in healthy andAD skin with pro-inflammatory effects in not yet affected skin andanti-inflammatory effects in inflamed skin.

doi:10.1016/j.autneu.2013.05.142

Poster 20.18

Stress alters cytokine profile of the skin: prospective links tostress-induced NNCS regulation in skin

Frank Risto Rommel, Badrinarayanan Raghavan, Susanne Tumala(Psychoneuroimmunology Laboratory, Department of PsychosomaticMedicine and Psychotherapy, Justus-Liebig-University, Giessen, Germany),Uwe Gieler (Department of Dermatology, University Hospital Giessen,Giessen, Germany), Eva M.J. Peters (Psychoneuroimmunology Labora-tory, Department of Psychosomatic Medicine and Psychotherapy,Justus-Liebig-University, Giessen, Germany)

Organs at the self-environment interface are confronted with aplethora of exogenous and endogenous challenges. Adaptation tothese challenges appears to be a distinct feature of the cholinergicsystem. The skin is known to possess an intrinsically acting stress-cascade, in which acetylcholine may play a vital role in the regulationof peripheral inflammation and disease. Thus it is essential to addressthe role of stress perception and subsequent non-neuronal choliner-gic system (NNCS) induced modulation of the skin in response todifferent kinds of stressors. By microarray analysis we compared skin

Abstracts / Autonomic Neuroscience: Basic and Clinical 177 (2013) 1–6564

of mice exposed to 24 hrs of noise stress with skin from unstressedmice. We found a specific pattern in cytokine release potentiallyinfluenced by muscarinic and nicotinic acetylcholine receptor (m-and n-AChR) activation. In stressed mouse skin, we observed asignificant downregulation of TNFα receptor 1 (TNFR1, 2.576 fold)and upregulation of TNFR5 (1.194 fold) as well as regulation of IL1.Mast cells, being the predominant source of dermal TNFα, mayexpress various cholinergic receptors. They are a prime effector cellpopulation of inflammatory as well as perceived stress. So weinvestigated the expression of cytokines like TNFα IL-1β, TGF-β, IL-10 and NNCS markers such as choline acetyl transferase (ChAT) andnicotinic receptor α7 (α7nAChR) in mast cells of mice withexperimentally induced atopic dermatitis-like allergic dermatitis(AlD). Pro-inflammatory cytokines TNFα and IL-1β in the mast cellsof AlD mouse skin were upregulated while additional noise stressreversed this upregulation. In concordance, the anti-inflammatorycytokines TGF-β and IL-10 were downregulated in AlD skin andupregulated by additional noise stress. Moreover we found TNFα andIL-1β expression in single nerve fibers and nerval plexi in mouse skin.The number and staining intensity of positive nerve fibers for boththe cytokines was increased in the skin of noise stressed mice andreduced in AlD and noise stress + AlD. Finally, a downregulation ofChAT and upregulation of α7nAChR was observed in the mast cells ofAlD mouse skin which was reversed by additional noise stress. Allthese data infer that in mast cells and in cutaneous neuro-immunenetwork, NNCS mediators play a key role in the regulation of stress-sensitive inflammatory responses. Further studies are needed toelucidate the underlying molecular mechanisms and the potential ofpharmacological intervention.

doi:10.1016/j.autneu.2013.05.143

Poster 20.19

Testicular somatic cells express functional acetylcholine receptors

Sylvia Schirmer (Institute for Anatomy & Cell Biology, Justus-Liebig-University Giessen, Germany; Anatomy & Histology, Centre for

Neuroscience, Flinders University, Australia), Petra Hartmann,Wolfgang Kummer (Institute for Anatomy & Cell Biology, Justus-Liebig-University Giessen, Germany), Rainer Haberberger (Anatomy& Histology, Centre for Neuroscience, Flinders University, Australia),Andreas Meinhardt (Institute for Anatomy & Cell Biology, Justus-Liebig-University Giessen, Germany)

The cholinergic system consists of the transmitter moleculeacetylcholine (ACh), its synthesizing enzyme, transporters andreceptors. Within recent years it has been demonstrated that thissystem is not restricted to neurons but plays an important role instructure and function of non-neuronal tissues. Previously we couldshow the existence of the ACh-system within rodent testicular cells.

We determined the expression pattern of the cholinergic systemin peritubular cells (PTC), Sertoli cells (SC) and testicular macro-phages (TM) of rats. Total RNA was isolated from these cell types andused for standard RT-PCR (n = 3). Here we could detect mRNAs forthe choline transporter 1 (CHT1) and choline acetyltransferase(ChAT) whereas VAChT and OCT2 were absent in these cellpopulations. Several known α- and β-subunits of nicotinic receptorsand most muscarinic receptor subtypes were expressed.

Subsequently the function of ACh-receptors in primary isolatedtesticular cell populations was analyzed using calcium-imaging andthe fluorescent dye Fura-2 AM. Stimulation of ACh-receptors resultedin calcium-influx in PTC but not SC and TM. PTC showed a responseto ACh (10 μM) and muscarine (10 μM), but not to nicotine (100 μM).The ATP-induced response in PTC was similar before and after Ach-receptor stimulation. SC and TM did not respond to ACh and/or (ant)agonist but showed increased intracellular calcium-concentration inresponse to treatment with ATP (100 μM).

However, SC and TM showed an ATP-induced calcium increasewhich changed in response to pre-treatment with ACh, muscarine ornicotine. The experiments show the presence of cell-specific patternof synthesis and response to ACh in peritubular cells, macrophagesand Sertoli cells.

doi:10.1016/j.autneu.2013.05.144

Abstracts / Autonomic Neuroscience: Basic and Clinical 177 (2013) 1–65 65