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Agrin mediates a rapid switch from electrical coupling to chemical neurotransmission during synaptogenesis.

Martin AO, Alonso G, Guérineau NC - J. Cell Biol. (2005)

Bottom Line: When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms.This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases.Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR5203, INSERM U661, Université Montpellier I, Département d'Endocrinologie, Institut de Génomique Fonctionnelle, 34094 Montpellier Cedex 5, France.

ABSTRACT
In contrast to its well-established actions as an organizer of synaptic differentiation at the neuromuscular junction, the proteoglycan agrin is still in search of a function in the nervous system. Here, we report an entirely unanticipated role for agrin in the dual modulation of electrical and chemical intercellular communication that occurs during the critical period of synapse formation. When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms. Specifically, it led to decreased gap junction-mediated electrical coupling that preceded an increase in nicotinic synaptic transmission. This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases. Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

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Activation of Src family–related tyrosine kinases is required for agrin effects on both gap junctional coupling and synaptic transmission. (A) Double-immunofluorescent staining showing that MuSK is constitutively expressed in both adult and neonatal chromaffin cells, and colocalized with the Z+ agrin variant in adults. Arrows indicate the sites of colocalized MuSK with the agrin Z+ variant. (B) PP2, a specific Src family tyrosine kinase inhibitor (5 μM, 20 min before agrin) fully blocked the effect of agrin on electrical coupling. By contrast, PP3 pretreatment (5 μM, 20 min before agrin), the inactive structural analogue of PP2, did not antagonize the agrin-mediated decrease in Ij. (C) Blockade of the agrin-mediated increase in sEPSC amplitude in PP2-containing saline. *, P < 0.01 when compared with untreated slices.
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fig8: Activation of Src family–related tyrosine kinases is required for agrin effects on both gap junctional coupling and synaptic transmission. (A) Double-immunofluorescent staining showing that MuSK is constitutively expressed in both adult and neonatal chromaffin cells, and colocalized with the Z+ agrin variant in adults. Arrows indicate the sites of colocalized MuSK with the agrin Z+ variant. (B) PP2, a specific Src family tyrosine kinase inhibitor (5 μM, 20 min before agrin) fully blocked the effect of agrin on electrical coupling. By contrast, PP3 pretreatment (5 μM, 20 min before agrin), the inactive structural analogue of PP2, did not antagonize the agrin-mediated decrease in Ij. (C) Blockade of the agrin-mediated increase in sEPSC amplitude in PP2-containing saline. *, P < 0.01 when compared with untreated slices.

Mentions: As shown in Figs. 6 and 7, agrin acted sequentially on both electrical coupling and synaptic transmission. Do these effects require activation of a unique or different intracellular signaling pathways? To address this issue we first looked for the expression of the muscle-specific kinase (MuSK) in both adult and neonate adrenal medulla. This question was prompted by the finding that at the NMJ agrin activates a multicomponent receptor complex including MuSK (Glass et al., 1996). As illustrated in Fig. 8 A, MuSK is highly expressed in neonate and adult chromaffin cells. The subsequent colocalization with the Z+ agrin variant in adults strongly supports the hypothesis that agrin might activate MuSK in the adrenal gland. Additional experiments were then performed to determine whether agrin effects depended on the activation of Src family–related kinases, as reported at the NMJ (Mittaud et al., 2001). This was achieved by incubating adrenal neonatal slices with the specific Src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (5 μM, 20 min; Hanke et al., 1996) before agrin addition. As illustrated in Fig. 8 B, agrin failed to reduce Ij in the presence of PP2 (n = 8 cell pairs). By contrast, the use of the inactive structural analogue 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3) (5 μM, 20 min) did not prevent agrin-mediated Ij decrease (n = 3 cell pairs). Similarly, PP2 pretreatment impaired the agrin-mediated increase in sEPSC amplitude (Fig. 8 C). Even after 150 min of agrin exposure, sEPSC amplitude did not significantly differ from that found in control slices (78.6 ± 18.3 pA, n = 5 cells in PP2-treated slices vs. 51.8 ± 2.6 pA, n = 17 cells in control, P > 0.01). Together, these results indicate that both agrin-induced changes in cell–cell communication depend on activation of Src family tyrosine kinase–mediated intracellular pathway.


Agrin mediates a rapid switch from electrical coupling to chemical neurotransmission during synaptogenesis.

Martin AO, Alonso G, Guérineau NC - J. Cell Biol. (2005)

Activation of Src family–related tyrosine kinases is required for agrin effects on both gap junctional coupling and synaptic transmission. (A) Double-immunofluorescent staining showing that MuSK is constitutively expressed in both adult and neonatal chromaffin cells, and colocalized with the Z+ agrin variant in adults. Arrows indicate the sites of colocalized MuSK with the agrin Z+ variant. (B) PP2, a specific Src family tyrosine kinase inhibitor (5 μM, 20 min before agrin) fully blocked the effect of agrin on electrical coupling. By contrast, PP3 pretreatment (5 μM, 20 min before agrin), the inactive structural analogue of PP2, did not antagonize the agrin-mediated decrease in Ij. (C) Blockade of the agrin-mediated increase in sEPSC amplitude in PP2-containing saline. *, P < 0.01 when compared with untreated slices.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171940&req=5

fig8: Activation of Src family–related tyrosine kinases is required for agrin effects on both gap junctional coupling and synaptic transmission. (A) Double-immunofluorescent staining showing that MuSK is constitutively expressed in both adult and neonatal chromaffin cells, and colocalized with the Z+ agrin variant in adults. Arrows indicate the sites of colocalized MuSK with the agrin Z+ variant. (B) PP2, a specific Src family tyrosine kinase inhibitor (5 μM, 20 min before agrin) fully blocked the effect of agrin on electrical coupling. By contrast, PP3 pretreatment (5 μM, 20 min before agrin), the inactive structural analogue of PP2, did not antagonize the agrin-mediated decrease in Ij. (C) Blockade of the agrin-mediated increase in sEPSC amplitude in PP2-containing saline. *, P < 0.01 when compared with untreated slices.
Mentions: As shown in Figs. 6 and 7, agrin acted sequentially on both electrical coupling and synaptic transmission. Do these effects require activation of a unique or different intracellular signaling pathways? To address this issue we first looked for the expression of the muscle-specific kinase (MuSK) in both adult and neonate adrenal medulla. This question was prompted by the finding that at the NMJ agrin activates a multicomponent receptor complex including MuSK (Glass et al., 1996). As illustrated in Fig. 8 A, MuSK is highly expressed in neonate and adult chromaffin cells. The subsequent colocalization with the Z+ agrin variant in adults strongly supports the hypothesis that agrin might activate MuSK in the adrenal gland. Additional experiments were then performed to determine whether agrin effects depended on the activation of Src family–related kinases, as reported at the NMJ (Mittaud et al., 2001). This was achieved by incubating adrenal neonatal slices with the specific Src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (5 μM, 20 min; Hanke et al., 1996) before agrin addition. As illustrated in Fig. 8 B, agrin failed to reduce Ij in the presence of PP2 (n = 8 cell pairs). By contrast, the use of the inactive structural analogue 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3) (5 μM, 20 min) did not prevent agrin-mediated Ij decrease (n = 3 cell pairs). Similarly, PP2 pretreatment impaired the agrin-mediated increase in sEPSC amplitude (Fig. 8 C). Even after 150 min of agrin exposure, sEPSC amplitude did not significantly differ from that found in control slices (78.6 ± 18.3 pA, n = 5 cells in PP2-treated slices vs. 51.8 ± 2.6 pA, n = 17 cells in control, P > 0.01). Together, these results indicate that both agrin-induced changes in cell–cell communication depend on activation of Src family tyrosine kinase–mediated intracellular pathway.

Bottom Line: When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms.This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases.Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR5203, INSERM U661, Université Montpellier I, Département d'Endocrinologie, Institut de Génomique Fonctionnelle, 34094 Montpellier Cedex 5, France.

ABSTRACT
In contrast to its well-established actions as an organizer of synaptic differentiation at the neuromuscular junction, the proteoglycan agrin is still in search of a function in the nervous system. Here, we report an entirely unanticipated role for agrin in the dual modulation of electrical and chemical intercellular communication that occurs during the critical period of synapse formation. When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms. Specifically, it led to decreased gap junction-mediated electrical coupling that preceded an increase in nicotinic synaptic transmission. This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases. Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

Show MeSH
Related in: MedlinePlus