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Presynaptic muscarinic m2 receptors depress glutamatergic EPSCs in rat hypoglossal motoneurons (HMs)
1188
Abstracts
Vol. 60, Nos. 13/14,1997
52 REGULATION OF CA2+ INFLUX BY MUSCARINIC (mAChR) IN PC12D CELLS
ACETYLCHOLINE
RECEPTORS
D. Saffen and T. Ebihara, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan PC 12D is a subline of the rat pheochromocytoma-derived cell line PC 12 that is characterized by accelerated neuronal differentiation following exposure to nerve growth factor. We have previously shown that PC12D cells express mRNA’s encoding the ml and m4 subtypes of mAChR, and that the ml subtype alone mediates the rapid induction of the immediate-early gene zif268 following exposure to muscarinic agonists. This gene induction depends upon semiindependent intracellular signalling pathways that require protein kinase C and the influx of extracellular calcium, respectively; release of calcium from internal stores by itself is not a sufficient stimulus for induction of the zif268 gene. In the present study we have investigated the mechanisms by which mAChR regulate Ca2+ influx using PC12D cells loaded with the fluorescent dye Fura-2. Our data suggest the following conclusions: 1) mAChR-mediated Ca2+ We find no evidence for influx is a consequence of the emptying of internal Ca2+ stores. “receptor-dependent” Ca2+ influx distinct from influx stimulated by thapsigargin, which depletes intracellular Ca2+ stores by inhibiting the Ca2+ pump of the endoplasmic reticulum. 2) Influx of Ca2+ following exposure to mAChR agonists or thapsigargin is significantly reduced by depolarization of the cell membrane. This effect is at least in part due to a reduction in the electrochemical driving force for Ca2+ entry into the cells. 3) Activation of mAChR inhibits Ltype voltage-dependent calcium channels. This inhibition is a consequence of ml mAChRmediated activation of protein kinase C and is also observed following exposure to phorbol esters.
53 PRESYNAPTIC MOTONEURONS
MUSCARINIC (HMs).
M2
RECEPTORS
DEPRESS
MC. Bellingham’ & A.J. Berger., ‘Medical Biochemistry, Biophysics, University of Washington, Seattle, USA.
GLUTAMATERGIC
University of Newcastle,
EPSCs
IN
RAT
Callaghan, Australia 8
HYPOGLOSSAL
Physiology and
Cholinergic modulation of synaptic inputs is likely to be significant in statedependent regulation of motor responses. Whole-cell patch-damp recordings were made from young adult rat HMs (n = 17) visually identified using Nomarkski optics and infrared translumination of transverse brainstem slices. Glutamatergic excitatory postsynaptic currents (EPSCs), evoked by electrical stimulation lateral to the hypoglossal motor nudeus, were reversibly reduced in amplitude by bath application of carbachol (10 )JM, 37 f 3X, mean % of control f SD, n = 22) muscarine (10 IJM, 50 f 336, n q 12) or physostigmine (200 ~JM, 54 + 7%, n = 3) without significant change in holding current, input resistance or EPSC rise time and decay constant. The mean EC% for carbachol or muscarine was 152 nM and 704 nM respectively (n >= 3 for each dose). EPSC reduction was completely blocked by atropine (1 PM, n = 11) while antagonist inhibition curve analysis found a high mean apparent affinity constant (pKe) of 8.07 for methoctramine, a M2 antagonist, but a low mean pKe of < 7.0 for pirenzipine, a Ml antagonist, showing that depression of evoked EPSCs was mediated by muscarintc M2 receptors. With TTX present, the amplitude of postsynaptic currents evoked in HMs by focal pressure injection of L-glutamate (0.51 mM) was not reduced by carbachol or muscarine (n >= 5) while the frequency of spontaneous glutamatergic miniature EPSCs was significantly (P < 0.01) reduced without change in amplitude by carbachol or muscarlne, indicating that excitatory glutamatergic inputs to rat HMs are modulated by muscarinic receptors at a presynaptic site. Sequential blockade of N- and then P-type calcium channels did not significantly alter the reduction of the remaining EPSC by carbachd, suggesting that the mechanism underlying EPSC depression most probably involves steps in synaptic release occurring afler calcium entry into the presynaptic terminal. Forskotin and SQ22538, which respectively activate and inhibit adenylyl cyclase, or H7, a protein kinase AK inhibitor, did not occlude muscarinic reduction of the EPSC. Membrane-permeant cyclic monophosphate analogs (8bromo-cAMP or -cGMP), a guanytyl cyclase inhibitor (LY83583), or an inhibitor of calmodulin kinasedependent activation of cAMP-spec5c phosphodiesterase (calmidazolium) also did not ocdude muscan,.ic effects on the EPSC. These data suggest that modulation of CAMP levels does not mediate muscarinic effects on synaptic transmission. A marked loss of tongue tone is seen during rapid eye movement sleep, when central cholinergic neurons are active. We suggest that cholinergic depression of excitatory synapttc drive to HMs may partially underlit this loss. Supported by Parker B. Francis Foundation, NS 14857, HL 49657.
Abstracts
Vol. 60, Nos. 13/14,1997
52 REGULATION OF CA2+ INFLUX BY MUSCARINIC (mAChR) IN PC12D CELLS
ACETYLCHOLINE
RECEPTORS
D. Saffen and T. Ebihara, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan PC 12D is a subline of the rat pheochromocytoma-derived cell line PC 12 that is characterized by accelerated neuronal differentiation following exposure to nerve growth factor. We have previously shown that PC12D cells express mRNA’s encoding the ml and m4 subtypes of mAChR, and that the ml subtype alone mediates the rapid induction of the immediate-early gene zif268 following exposure to muscarinic agonists. This gene induction depends upon semiindependent intracellular signalling pathways that require protein kinase C and the influx of extracellular calcium, respectively; release of calcium from internal stores by itself is not a sufficient stimulus for induction of the zif268 gene. In the present study we have investigated the mechanisms by which mAChR regulate Ca2+ influx using PC12D cells loaded with the fluorescent dye Fura-2. Our data suggest the following conclusions: 1) mAChR-mediated Ca2+ We find no evidence for influx is a consequence of the emptying of internal Ca2+ stores. “receptor-dependent” Ca2+ influx distinct from influx stimulated by thapsigargin, which depletes intracellular Ca2+ stores by inhibiting the Ca2+ pump of the endoplasmic reticulum. 2) Influx of Ca2+ following exposure to mAChR agonists or thapsigargin is significantly reduced by depolarization of the cell membrane. This effect is at least in part due to a reduction in the electrochemical driving force for Ca2+ entry into the cells. 3) Activation of mAChR inhibits Ltype voltage-dependent calcium channels. This inhibition is a consequence of ml mAChRmediated activation of protein kinase C and is also observed following exposure to phorbol esters.
53 PRESYNAPTIC MOTONEURONS
MUSCARINIC (HMs).
M2
RECEPTORS
DEPRESS
MC. Bellingham’ & A.J. Berger., ‘Medical Biochemistry, Biophysics, University of Washington, Seattle, USA.
GLUTAMATERGIC
University of Newcastle,
EPSCs
IN
RAT
Callaghan, Australia 8
HYPOGLOSSAL
Physiology and
Cholinergic modulation of synaptic inputs is likely to be significant in statedependent regulation of motor responses. Whole-cell patch-damp recordings were made from young adult rat HMs (n = 17) visually identified using Nomarkski optics and infrared translumination of transverse brainstem slices. Glutamatergic excitatory postsynaptic currents (EPSCs), evoked by electrical stimulation lateral to the hypoglossal motor nudeus, were reversibly reduced in amplitude by bath application of carbachol (10 )JM, 37 f 3X, mean % of control f SD, n = 22) muscarine (10 IJM, 50 f 336, n q 12) or physostigmine (200 ~JM, 54 + 7%, n = 3) without significant change in holding current, input resistance or EPSC rise time and decay constant. The mean EC% for carbachol or muscarine was 152 nM and 704 nM respectively (n >= 3 for each dose). EPSC reduction was completely blocked by atropine (1 PM, n = 11) while antagonist inhibition curve analysis found a high mean apparent affinity constant (pKe) of 8.07 for methoctramine, a M2 antagonist, but a low mean pKe of < 7.0 for pirenzipine, a Ml antagonist, showing that depression of evoked EPSCs was mediated by muscarintc M2 receptors. With TTX present, the amplitude of postsynaptic currents evoked in HMs by focal pressure injection of L-glutamate (0.51 mM) was not reduced by carbachol or muscarine (n >= 5) while the frequency of spontaneous glutamatergic miniature EPSCs was significantly (P < 0.01) reduced without change in amplitude by carbachol or muscarlne, indicating that excitatory glutamatergic inputs to rat HMs are modulated by muscarinic receptors at a presynaptic site. Sequential blockade of N- and then P-type calcium channels did not significantly alter the reduction of the remaining EPSC by carbachd, suggesting that the mechanism underlying EPSC depression most probably involves steps in synaptic release occurring afler calcium entry into the presynaptic terminal. Forskotin and SQ22538, which respectively activate and inhibit adenylyl cyclase, or H7, a protein kinase AK inhibitor, did not occlude muscarinic reduction of the EPSC. Membrane-permeant cyclic monophosphate analogs (8bromo-cAMP or -cGMP), a guanytyl cyclase inhibitor (LY83583), or an inhibitor of calmodulin kinasedependent activation of cAMP-spec5c phosphodiesterase (calmidazolium) also did not ocdude muscan,.ic effects on the EPSC. These data suggest that modulation of CAMP levels does not mediate muscarinic effects on synaptic transmission. A marked loss of tongue tone is seen during rapid eye movement sleep, when central cholinergic neurons are active. We suggest that cholinergic depression of excitatory synapttc drive to HMs may partially underlit this loss. Supported by Parker B. Francis Foundation, NS 14857, HL 49657.