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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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Agrin-mediated decrease in gap junction–dependent intercellular communication between chromaffin cells in neonates. (A) Reduced number of electrically and LY-coupled chromaffin cells in neonatal agrin-treated slices. *, P < 0.01 when compared with control slices. (B) Monitoring of electrical coupling between chromaffin cell pairs in control and agrin-containing saline showing examples of robust and weak coupling. Cells were current-clamped at −65 mV. A robust coupling was less frequently observed in agrin-treated slices. (C) Histograms illustrating the wide distribution range of the coupling ratio calculated in 26 control and 18 agrin-treated cells from voltage-clamp measurements of Ij (holding potential −60 mV, transjunctional potential from −120 to +60 mV, 150 ms duration). Insets: pooled data of Gj calculated in 11 control cell pairs and 4 agrin-treated cell pairs.
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fig5: Agrin-mediated decrease in gap junction–dependent intercellular communication between chromaffin cells in neonates. (A) Reduced number of electrically and LY-coupled chromaffin cells in neonatal agrin-treated slices. *, P < 0.01 when compared with control slices. (B) Monitoring of electrical coupling between chromaffin cell pairs in control and agrin-containing saline showing examples of robust and weak coupling. Cells were current-clamped at −65 mV. A robust coupling was less frequently observed in agrin-treated slices. (C) Histograms illustrating the wide distribution range of the coupling ratio calculated in 26 control and 18 agrin-treated cells from voltage-clamp measurements of Ij (holding potential −60 mV, transjunctional potential from −120 to +60 mV, 150 ms duration). Insets: pooled data of Gj calculated in 11 control cell pairs and 4 agrin-treated cell pairs.

Mentions: Studies were next undertaken to examine the effect of agrin on both gap junction–mediated electrical and metabolic coupling between neonate chromaffin cells. Metabolic coupling was assessed using Lucifer yellow (LY) to label coupled cells, whereas electrical coupling was evidenced by dual whole-cell patch-clamp recording of intercellular macroscopic junctional currents (Ij). The probability of LY spreading between chromaffin cells was significantly reduced in agrin-treated slices compared with untreated slices (Fig. 5 A; 31 vs. 57%, P < 0.01). In the presence of the anti–rat agrin-neutralizing pAb, agrin failed to decrease LY diffusion between neonate chromaffin cells (58.3%, n = 24; unpublished data). Similarly, the dye coupling was not modified by inactive agrin0,0 (61.5%, n = 13; unpublished data). In adults, agrin did not change the incidence of LY diffusion (37.9% in treated-slices, n = 29 vs. 44% under control conditions, P > 0.01; unpublished data). Agrin decreased the percentage of electrically coupled cell pairs (Fig. 5 A; 53.8%, n = 26 pairs in control slices vs. 22.2%, n = 18 pairs in agrin-treated slices, P < 0.01). In addition, agrin reduced the incidence of robustly coupled cells (Fig. 5 B). The voltage changes in response to depolarizing current injected into the stimulated cell were robustly reflected in the unstepped cell in 64.3% (n = 9/14 cell pairs) and 50% (n = 4 cell pairs) in control and agrin-treated slices, respectively (P < 0.01). The resulting coupling ratios exhibited a wide distribution range from 0.01 to 0.97 (Fig. 5 C). This strongly suggests the presence of two chromaffin cell populations—a weakly coupled cell population and a highly coupled cell population. Recordings of Ij in chromaffin cell pairs voltage clamped at −60 mV confirmed these findings (unpublished data). Delivering voltage steps with command pulses of both polarities triggered Ij in the unstepped cell that exhibited variable degrees of attenuation. In control slices, 81.8% recorded pairs (n = 11), Ij amplitude could reach 1–2 nA, whereas in the remaining pairs Ij did not exceed several picoamperes. In agrin-treated slices, the percentage of coupled cell pairs exhibiting a high Ij amplitude dropped to 50%. When plotted as a function of the transjunctional potential, the I/V curve of Ij displayed a linear relationship within the membrane potential range of −60 to +120 mV. The curve used to fit the data was derived from a linear regression, given a macroscopic junctional conductance (Gj) of 43 and 60 pS for the weak coupling and 12 and 13 nS for the robust coupling (Fig. 5 C, insets).


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

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

Agrin-mediated decrease in gap junction–dependent intercellular communication between chromaffin cells in neonates. (A) Reduced number of electrically and LY-coupled chromaffin cells in neonatal agrin-treated slices. *, P < 0.01 when compared with control slices. (B) Monitoring of electrical coupling between chromaffin cell pairs in control and agrin-containing saline showing examples of robust and weak coupling. Cells were current-clamped at −65 mV. A robust coupling was less frequently observed in agrin-treated slices. (C) Histograms illustrating the wide distribution range of the coupling ratio calculated in 26 control and 18 agrin-treated cells from voltage-clamp measurements of Ij (holding potential −60 mV, transjunctional potential from −120 to +60 mV, 150 ms duration). Insets: pooled data of Gj calculated in 11 control cell pairs and 4 agrin-treated cell pairs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171940&req=5

fig5: Agrin-mediated decrease in gap junction–dependent intercellular communication between chromaffin cells in neonates. (A) Reduced number of electrically and LY-coupled chromaffin cells in neonatal agrin-treated slices. *, P < 0.01 when compared with control slices. (B) Monitoring of electrical coupling between chromaffin cell pairs in control and agrin-containing saline showing examples of robust and weak coupling. Cells were current-clamped at −65 mV. A robust coupling was less frequently observed in agrin-treated slices. (C) Histograms illustrating the wide distribution range of the coupling ratio calculated in 26 control and 18 agrin-treated cells from voltage-clamp measurements of Ij (holding potential −60 mV, transjunctional potential from −120 to +60 mV, 150 ms duration). Insets: pooled data of Gj calculated in 11 control cell pairs and 4 agrin-treated cell pairs.
Mentions: Studies were next undertaken to examine the effect of agrin on both gap junction–mediated electrical and metabolic coupling between neonate chromaffin cells. Metabolic coupling was assessed using Lucifer yellow (LY) to label coupled cells, whereas electrical coupling was evidenced by dual whole-cell patch-clamp recording of intercellular macroscopic junctional currents (Ij). The probability of LY spreading between chromaffin cells was significantly reduced in agrin-treated slices compared with untreated slices (Fig. 5 A; 31 vs. 57%, P < 0.01). In the presence of the anti–rat agrin-neutralizing pAb, agrin failed to decrease LY diffusion between neonate chromaffin cells (58.3%, n = 24; unpublished data). Similarly, the dye coupling was not modified by inactive agrin0,0 (61.5%, n = 13; unpublished data). In adults, agrin did not change the incidence of LY diffusion (37.9% in treated-slices, n = 29 vs. 44% under control conditions, P > 0.01; unpublished data). Agrin decreased the percentage of electrically coupled cell pairs (Fig. 5 A; 53.8%, n = 26 pairs in control slices vs. 22.2%, n = 18 pairs in agrin-treated slices, P < 0.01). In addition, agrin reduced the incidence of robustly coupled cells (Fig. 5 B). The voltage changes in response to depolarizing current injected into the stimulated cell were robustly reflected in the unstepped cell in 64.3% (n = 9/14 cell pairs) and 50% (n = 4 cell pairs) in control and agrin-treated slices, respectively (P < 0.01). The resulting coupling ratios exhibited a wide distribution range from 0.01 to 0.97 (Fig. 5 C). This strongly suggests the presence of two chromaffin cell populations—a weakly coupled cell population and a highly coupled cell population. Recordings of Ij in chromaffin cell pairs voltage clamped at −60 mV confirmed these findings (unpublished data). Delivering voltage steps with command pulses of both polarities triggered Ij in the unstepped cell that exhibited variable degrees of attenuation. In control slices, 81.8% recorded pairs (n = 11), Ij amplitude could reach 1–2 nA, whereas in the remaining pairs Ij did not exceed several picoamperes. In agrin-treated slices, the percentage of coupled cell pairs exhibiting a high Ij amplitude dropped to 50%. When plotted as a function of the transjunctional potential, the I/V curve of Ij displayed a linear relationship within the membrane potential range of −60 to +120 mV. The curve used to fit the data was derived from a linear regression, given a macroscopic junctional conductance (Gj) of 43 and 60 pS for the weak coupling and 12 and 13 nS for the robust coupling (Fig. 5 C, insets).

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