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20 Collaborations with: Prof. E.J. Nouwen Laboratory of Neurobiology and Neuropharmacology Department of Medicine Universitaire Instelling Antwerpen (UIA - UA) B-2610 Wilrijk Antwerpen.
Part 1: Characterisation of noradrenergic large dense-cored vesicles It is generally accepted that neurons can secrete a cocktail of peptide and non-peptide or classical neurotransmitters from at least two types of organelles, i.e. the small synaptic vesicles (SSV) and the large dense-cored vesicles (LDV). According to the current views, the classical transmitters, such as acetylcholine (ACh), noradrenaline (NA), glutamate and GABA, are stored in and released from the SSV. On the other hand, the neuropeptides are stored in and released from the LDV. Exocytosis from SSV and LDV is considered to be differentially regulated and to take place at different release sites. However, there are indications to believe that this general concept of neurotransmitter release is oversimplified and that different neural systems with different physiological functions in the organism have developed variations on this commonly accepted model. This is certainly the case for the peripheral noradrenergic neurons, the subject of our research. Noradrenergic neurons store their classical transmitter NA not only in the small dense-cored vesicles (SDV), but, together with the neuropeptides, also in LDV. In addition, in the previous years we were able to demonstrate that in noradrenergic neurons NA is exclusively released from the LDV. Despite their numerical majority, the SDV are not directly involved in noradrenergic neurotransmission. Now we want to come to an identification of all proteins present on the LDV-membrane in order to obtain a detailed view of the intraneuronal dynamics of this vesicle-population, which seems to be of crucial importance for the noradrenergic neurotransmission. In a first approach LDV were purified using sucrose/D 2O gradient centrifugation. This method is actually considered as being the most efficient procedure to purify LDV. This purified LDV fraction was analysed with 2D-gelelectrophosesis. Via Western-Blot with antibodies to well known membrane-bound vesicle-proteins we were able to assign the identity of several spots. In order to obtain the identity of the unknown spots, we subjected them to amino-acid analysis. Unfortunately, it turned out that most of these spots were due to mitochondrial contamination. We had to conclude that the current purification procedure for LDV was insufficient to obtain our goals and that a more specific procedure had to be developed. A new purification procedure for LDV based on immuno-affinity chromatography was set up. For this purpose polyclonal antibodies to synaptobrevin, a protein known to be present on LDV-membranes, were prepared and coupled to CNBr-activated Sepharose beads. These beads were then packed in a column on which a post-mitochondrial supernatant obtained from bovine splenic nerves was loaded. However, the amount of purified LDV obtained by this approach was too limited to perform an elaborate analysis. An alternative procedure in which we labeled the LDV with magnetic beads, was more succesful. In this procedure a post-mitochondrial supernatant of bovine splenic nerves was incubated with polyclonal antibodies to synaptobrevin and the monoamine transporter VMAT2 (also prepared in house), followed by an incubation with magnetic beads on which anti-rabbit IgG’s were coupled. The magnetically labeled LDV were then separated from the contaminating organelles in an magnetic field. In order to estimate the degree of purity of the LDV-fraction obtained by this method, the enrichment of different LDV-membrane markers has to be calculated. Therefore, ELISAs to dopamine-beta-hydro- 21
Page 1 and 2: Geneeskundige Stichting Koningin El
Page 3 and 4: Fondation Médicale Reine Elisabeth
Page 5 and 6: L’abondance et la qualité des pu
Page 7 and 8: Prof. Dr. A. Bossuyt Secrétaire du
Page 9 and 10: Prof. Dr. G. Orban Lab. voor Neuro-
Page 11: Reports of the University Research
Page 14 and 15: 14 Members of the team: Christophe
Page 16 and 17: 16 domains, most notably that of N-
Page 19: Report of the Research Group of Pro
Page 23 and 24: Part 2 : Chromogranins as neuro-imm
Page 25: Publications 2000 : • J. Depreite
Page 28 and 29: 28 Collaborations with: A. Volny-Lu
Page 30 and 31: 30 MATERIALS AND METHODS Surgery an
Page 32 and 33: 32 RESULTS Recordings were obtained
Page 34 and 35: 34 Responses to brush stimuli The s
Page 36 and 37: 36 DISCUSSION This experimental stu
Page 39 and 40: Report of the Research Group of Pro
Page 41 and 42: PART 1: Molecular Pharmacology of A
Page 43 and 44: c) Most insurmountable AT 1 recepto
Page 45 and 46: dy tested biphenyl-tetrazole derive
Page 47 and 48: ii) More distant functional respons
Page 49 and 50: References •Aiyar N, Baker E, Vic
Page 51 and 52: •Olins GM, Chen ST, McMahon EG, P
Page 53 and 54: PART 2: The use of Endothelin 1 in
Page 55 and 56: vasoconstriction, peaked 20 minutes
Page 57: ACCEPTED • Vanderheyden P.M.L., F
Page 60 and 61: 60 Prof. Dr. Christophe Erneux I.R.
Page 62 and 63: 62 tion forming Ins(1,3,4,5)P 4 and
Page 64: 64 Bibliography •Berridge, M.J. a
Page 68 and 69: 68 University of Mons-Hainaut Labor
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70 Works carried out in 2000 1. We
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Report of the Research Group of Pro
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Genetic definition of brain tumours
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Analysing this way these 48 tumours
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Genetic determinants of mouse brain
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Genomic organization of the Dab1 ge
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In vitro studies of the hypothalami
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vitro GnRH release by intact male g
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Study on the functional regulation
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Elucidation of the DAT primary sequ
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The role of presenilin-1 in the gam
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INTRODUCTION As usual, this activit
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The acquisitions for this study sho
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not lead to the integrative process
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REFERENCES Thesis •Steven Laureys
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• Positron emission tomography in
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Before scanning, subjects practised
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RESEARCH PROGRAM ON MEMORY FUNCTION
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Corticobasal degeneration, motor sl
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Figure 2. Brain Areas Disclosed Dur
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• Cognitive Neuropsychological an
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Neuregulin signaling regulates neur
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tein present in neural precursor ce
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Cell biology for prevention and tre
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Functional significance of neurorec
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Spinal Cord Traumatic Lesion Martin
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A.Schulman, and T.R.Van De Water, B
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•Identification of PSF, the PTB-a
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143
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Brain activations in a dot pattern
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References •R Vogels, G Sary, P D
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A Scientific activities 1. Overview
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4. Purinergic receptors. Purinergic
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mals also scored higher in anxiety
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cell clones have been used in these
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•Ledent C, Valverde O, Cossu G, P
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• Snell BJ, Short JL, Drago J, Le
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I. Physiology and physiopathology o
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nuclei, which are involved in respo
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Natl. Acad. Sci. USA, 96: 5257-5262
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in those who did not. In rats sacri
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(Poster). Abstract of the 4th EURON
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Positional cloning of the neuronal
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fragments, 18 expressed-sequence-ta
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Philadelphia, USA, 3-7 October 2000
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I. FMRI in Awake Behaving Macaques
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gressively higher levels of the cor
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MION was injected into the femoral
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Future Possibilities Future contras
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FIGURES Figure 1: FMRI responses in
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Figure 5: Average percent signal ch
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FUNCTIONAL MAGNETIC RESONANCE IMAGI
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explain these search processes. Par
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205
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Investigation of the molecular mech
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tral) cortex compared to non-depriv
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By comparing the percentual increas
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altered amino acid residue is equal
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BIBLIOGRAPHY - 2000 Publications in
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Soc. Neurosci. Abstr., 2000, vol. 2
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1. Coding of 3D-shape in macaque in
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3. Position sensitivity of macaque
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Publications: •JANSSEN P., VOGELS
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