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209 Development of synaptic plasticity in the cerebral ganglion of Aplysia
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EFFECTS OF THE F3 CELL ADHESION MOLECULE ON NEURITE OUTGROWTH AND FASCICULATION ARE CELL-TYPE SPECIFIC.
Characterization of Cells Expressing NGF and TrkA in the Developing Chicken Retina. Miriam Karlsson (I). Francis Lcfcort (2). LOUIS Reichardt (2). Harvey Kartcn (3) and Finn HallbBo;k (I). (I) Dept. Dcvl. Neurosci., Uppsala University, Sweden. (2) Dept. Physiology, UCSF, USA. (3) Dept. Neurosci. UCSD. USA.
M. Buttiglione, J. M Revest, V. Matranga*, G. Rougon, C. Faivre-Sarrailh. Institut de Biologie du DCveloppement. CNRS 9943; 13288 Marseille FR and *CNR, Palermo IT.
Many neurons dcpcnd on trophic support during periods of naturally occuring celldeath. In the retina, ganglion cells undergo celldeath between embryomc day 13-17 (El3-El7), hut less IS known about other celltypes. Members of the neurotrophin family and their receptors, are involved I” support of survival and neuritogenesis during neuronal selection. To clarify the role of nerve growth factor (NGF) and its tyrosine kinase receptor TrkA during development of the retina, we have studied frkA mRNA expression and immunoreactivity. Using in situ hybridization, we found rrkA and NGF labeled cells in the outer part of the inner nuclear layer (INL) as early as E7. By E8-9 labeling for trkA, but not for NGF. could be seen over the inner part of the INL. Using RNase protection assay we show that rrkA mRNA levels peak around E15. TrkA immunohistochemistry confirmed the in situ results and we could Identify labeled horizontal cells and amacrine cells. Labeled cells start to occur al E7. and by El3 most TrkA lrnmunoreactive amacrine cells disappear, whereas the horizontals persist. Smce NGF is only expressed where horizontals arc located, we suggest that NGF support horizontals, hut not amacrine cells during a period of cell death. In summary, these data suggest that localized expression of neurotrophins, i.e. NGF, support certam populations of retinal neurons defined by their ability to express the cognate neurotrophin receptor during specific timeperiods in the developing chicken retma.
In previous studies we demonstrated that F3, a GPIanchored molecule of the immunoglobulin (Ig) super family made of 6 Ig-like domains and 4 FNIII repeats, can play a role in cell adhesion and control of neurite outgrowth. We already showed that F3 either presented as a component of the surface of transfected cells or in soluble form stimulated neurite outgrowth of sensory neurons. We will show that F3 or F3-Ig domains expressed on a monolayer of transfected cells induce a strong reduction of neurite outgrowth of isolated cerebellar neurons together with a high fasciculation of neurites when the same cells are grown as reaggregates. A recombinant F3Fc chimeric molecule compraising its Ig-like domains used as a coated substrate has the same effect on neurite fasciculation but not its soluble form. Thus F3 can exert stimulating or inhibitory effects on neurite outgrowth depending on the neurons tested. Such functional diversity can be mediated by neuronal cell-type specific receptors; alternatively, it might result from the triggering of different transducing pathways.
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210
DEVELOPMENT OF SYNAPTIC PLASTICITY IN THE CEREBRAL GANGLION OF APLYSIA.
A NEURAL SPECIFIC PROMOTER DRIVES THE EXPRESSION OF THE MOUSE AXONAL ADHESIVE GLYCOPROTEIN F3 *.
S M Fredman. Departmcnl of Physiology, Mrh3ny College. Nnshvdle. TN 37298, USA
MedIcal
G. Cangiano’, P.D’Errico”‘, M. Ambrosini’, Harold Creme?, M. Buttiglione’.‘, F. A&r&* and G. Gennarmi’. Istituto di Fisiologia Umana, Universiti di Ban’, Dipartimento ch Genetica, Blologia e Chimica Medica.Universitadi Torino’, Italy and Institut de Biologic du D&eloppement de Marseille3, France.
The monosynnplic connecl~on between the A and B neurons m the cerebral ganghon of Ap!vsra exhibits two prommmt forms of activitydependent synaptic plashcity Stimulnuon of individual preslmaptic A neurons at a.01 Hz results in low frequency synaptic depression (LFSD). a progressive reduction in the amplitude of EPSPs evoked in postsqnaptic B neurons. Brief high frequency stimulation (2 set @ 20 Hz) produces slow developing potentiatlon (SDP), an mcrease m EPSP amplitude which reaches a peak 5 mm tier the tetanizmg train and lasts -30 min. Both forms of plasticity appear to he due to changes in tmnsmitter release by the A neurons and are mediated hy distinct second messenger/ slgnnl lransduct~on pathways These forms ofplnstlcity appear at ddYercnt times during development. In juvenile .4p!vs10, IS5 days alter hatching and weighmg -7 5 g, A-B neuron synapses exhlhlt LFSD but not SDP Over the neu 14-2 I days, two changes take place. First, a weak form of SDI’ appears Following the trtanizmg train, EPSP nmphtudrs increase, hut decay hack to control levels in cl0 mm Second, as development progresses. more A neurons display SDP In slightly older and/or Ial-ger Ap/,vsra (I 65 days, I2- 14g) the potvntiation IS both greater m amphtude and longer lastmg. By the time animals reach an age of -175 days and a weight of -18g. SDP 1s mdistmb~~shable Tom that seen m the adult. We are presently trymg to detcnmne the cellular mechanisms underlying this dcvelopmenlal plasc~c~ty Supported hy NINDS grant NS28 I99 ,o SMF and the Mehnny NSF/MRCIZ in Cell and Molecular Biology
F3 is a mouseaxonal glycoproteininvolved in phenomenaof key developmental significance as neurite outgrowth and
fasciculation. Accordingly, F3 expression is developmentally regulated on neuronal subpopulations. To investigate the mechamsminvolvedin the regulationof F3 geneexpresslon,we have isolated the most 5’ 50 kb of the F3 gene and identified therein a functional promoter. F3 promoter was exclusively active in cellsof neuronatderivation like the F3 expressingnd26 cells while significant lower activities were found tn the non
expressingNzA neuroblastoma.By contrastthe F3 promoterwas silent in 3T3 fibroblasts. F3 promoter was able to drive neural specific reporter geneexpressionin transgenicmice. Transgene expressionwas essentially detectedon neuronalsubpopulation thus indicating that elementsresponsiblefor cell type-specific expressionwere locatedwithin the isolated F3 gene upstream region. The functional dissectionof the F3 promoter revealed both positive
and negative
elements scattered within
1.3 kb of
upstreamregion and within the first exon. CAMPand retinoic acid responsivesequenceswere also mapped. mdicatmg that pathwaysinvolving those signalling moleculesare potentially Involved in the regulation of F3 gene expressIon during developmentanddifferentiation. * Supportedby EEC and Telethon Italy grants.
101
208
EFFECTS OF THE F3 CELL ADHESION MOLECULE ON NEURITE OUTGROWTH AND FASCICULATION ARE CELL-TYPE SPECIFIC.
Characterization of Cells Expressing NGF and TrkA in the Developing Chicken Retina. Miriam Karlsson (I). Francis Lcfcort (2). LOUIS Reichardt (2). Harvey Kartcn (3) and Finn HallbBo;k (I). (I) Dept. Dcvl. Neurosci., Uppsala University, Sweden. (2) Dept. Physiology, UCSF, USA. (3) Dept. Neurosci. UCSD. USA.
M. Buttiglione, J. M Revest, V. Matranga*, G. Rougon, C. Faivre-Sarrailh. Institut de Biologie du DCveloppement. CNRS 9943; 13288 Marseille FR and *CNR, Palermo IT.
Many neurons dcpcnd on trophic support during periods of naturally occuring celldeath. In the retina, ganglion cells undergo celldeath between embryomc day 13-17 (El3-El7), hut less IS known about other celltypes. Members of the neurotrophin family and their receptors, are involved I” support of survival and neuritogenesis during neuronal selection. To clarify the role of nerve growth factor (NGF) and its tyrosine kinase receptor TrkA during development of the retina, we have studied frkA mRNA expression and immunoreactivity. Using in situ hybridization, we found rrkA and NGF labeled cells in the outer part of the inner nuclear layer (INL) as early as E7. By E8-9 labeling for trkA, but not for NGF. could be seen over the inner part of the INL. Using RNase protection assay we show that rrkA mRNA levels peak around E15. TrkA immunohistochemistry confirmed the in situ results and we could Identify labeled horizontal cells and amacrine cells. Labeled cells start to occur al E7. and by El3 most TrkA lrnmunoreactive amacrine cells disappear, whereas the horizontals persist. Smce NGF is only expressed where horizontals arc located, we suggest that NGF support horizontals, hut not amacrine cells during a period of cell death. In summary, these data suggest that localized expression of neurotrophins, i.e. NGF, support certam populations of retinal neurons defined by their ability to express the cognate neurotrophin receptor during specific timeperiods in the developing chicken retma.
In previous studies we demonstrated that F3, a GPIanchored molecule of the immunoglobulin (Ig) super family made of 6 Ig-like domains and 4 FNIII repeats, can play a role in cell adhesion and control of neurite outgrowth. We already showed that F3 either presented as a component of the surface of transfected cells or in soluble form stimulated neurite outgrowth of sensory neurons. We will show that F3 or F3-Ig domains expressed on a monolayer of transfected cells induce a strong reduction of neurite outgrowth of isolated cerebellar neurons together with a high fasciculation of neurites when the same cells are grown as reaggregates. A recombinant F3Fc chimeric molecule compraising its Ig-like domains used as a coated substrate has the same effect on neurite fasciculation but not its soluble form. Thus F3 can exert stimulating or inhibitory effects on neurite outgrowth depending on the neurons tested. Such functional diversity can be mediated by neuronal cell-type specific receptors; alternatively, it might result from the triggering of different transducing pathways.
209
210
DEVELOPMENT OF SYNAPTIC PLASTICITY IN THE CEREBRAL GANGLION OF APLYSIA.
A NEURAL SPECIFIC PROMOTER DRIVES THE EXPRESSION OF THE MOUSE AXONAL ADHESIVE GLYCOPROTEIN F3 *.
S M Fredman. Departmcnl of Physiology, Mrh3ny College. Nnshvdle. TN 37298, USA
MedIcal
G. Cangiano’, P.D’Errico”‘, M. Ambrosini’, Harold Creme?, M. Buttiglione’.‘, F. A&r&* and G. Gennarmi’. Istituto di Fisiologia Umana, Universiti di Ban’, Dipartimento ch Genetica, Blologia e Chimica Medica.Universitadi Torino’, Italy and Institut de Biologic du D&eloppement de Marseille3, France.
The monosynnplic connecl~on between the A and B neurons m the cerebral ganghon of Ap!vsra exhibits two prommmt forms of activitydependent synaptic plashcity Stimulnuon of individual preslmaptic A neurons at a.01 Hz results in low frequency synaptic depression (LFSD). a progressive reduction in the amplitude of EPSPs evoked in postsqnaptic B neurons. Brief high frequency stimulation (2 set @ 20 Hz) produces slow developing potentiatlon (SDP), an mcrease m EPSP amplitude which reaches a peak 5 mm tier the tetanizmg train and lasts -30 min. Both forms of plasticity appear to he due to changes in tmnsmitter release by the A neurons and are mediated hy distinct second messenger/ slgnnl lransduct~on pathways These forms ofplnstlcity appear at ddYercnt times during development. In juvenile .4p!vs10, IS5 days alter hatching and weighmg -7 5 g, A-B neuron synapses exhlhlt LFSD but not SDP Over the neu 14-2 I days, two changes take place. First, a weak form of SDI’ appears Following the trtanizmg train, EPSP nmphtudrs increase, hut decay hack to control levels in cl0 mm Second, as development progresses. more A neurons display SDP In slightly older and/or Ial-ger Ap/,vsra (I 65 days, I2- 14g) the potvntiation IS both greater m amphtude and longer lastmg. By the time animals reach an age of -175 days and a weight of -18g. SDP 1s mdistmb~~shable Tom that seen m the adult. We are presently trymg to detcnmne the cellular mechanisms underlying this dcvelopmenlal plasc~c~ty Supported hy NINDS grant NS28 I99 ,o SMF and the Mehnny NSF/MRCIZ in Cell and Molecular Biology
F3 is a mouseaxonal glycoproteininvolved in phenomenaof key developmental significance as neurite outgrowth and
fasciculation. Accordingly, F3 expression is developmentally regulated on neuronal subpopulations. To investigate the mechamsminvolvedin the regulationof F3 geneexpresslon,we have isolated the most 5’ 50 kb of the F3 gene and identified therein a functional promoter. F3 promoter was exclusively active in cellsof neuronatderivation like the F3 expressingnd26 cells while significant lower activities were found tn the non
expressingNzA neuroblastoma.By contrastthe F3 promoterwas silent in 3T3 fibroblasts. F3 promoter was able to drive neural specific reporter geneexpressionin transgenicmice. Transgene expressionwas essentially detectedon neuronalsubpopulation thus indicating that elementsresponsiblefor cell type-specific expressionwere locatedwithin the isolated F3 gene upstream region. The functional dissectionof the F3 promoter revealed both positive
and negative
elements scattered within
1.3 kb of
upstreamregion and within the first exon. CAMPand retinoic acid responsivesequenceswere also mapped. mdicatmg that pathwaysinvolving those signalling moleculesare potentially Involved in the regulation of F3 gene expressIon during developmentanddifferentiation. * Supportedby EEC and Telethon Italy grants.
101