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Presynaptic membrane movement in the cat cervical sympathetic ganglion in situ , electrically stimulated at a low frequency
S176
25. Neurotransmitters
IV. Presynaptic mechanisms
PRESYNAPTIC MEMBRANE MOVEMENT IN THE CAT CERVICAL STIMULATED AT A LOW FREQUENCY.
SYMPATHETIC
GANGLION
IN SITU,
ELECTRICALLY
MASASHI HORI .I, TOMOKO KADOTA 2, MITSUAKI FUJITA .2 and KEN KADOTA I, IDepartment of Neurochemistry, Psychiatric Research Institute of Tokyo, Setagaya-ku, T-~kyo i--5~, and 2Department of Anatomy, School of Medicine, Chiba University, Chiba 280, Japan. Presynaptic morphological changes following electrical stimulation at 0.2 Hz were studied in the cat superior cervical sympathetic ganglion (SCG) in situ, under a normal blood supply. Cats were anesthetized by intraperitoneal administration of sodium pentobarbital, 35-40 mg/kg, and both sides of SCG were prepared. Bilateral preganglionic trunks were electrically stimulated at 0.2 Hz for periods of 5-240 min. The ganglion was fixed by perfusion via the lingual artery and processed for electron microscopy. Ultrastructural changes in presynaptic terminals were examined and morphometrically analyzed. In some cases, horseradish peroxidase (HRP) was administered to examine endocytotic processes accompanying transmitter release. The number of the synaptic vesicles (SVs) did not change even after 4 hours of stimulation. There was an enlargement of the terminal area, resulting from the exocytosis of SVs. The number of macropinocytstic vacuoles and coated vesicles (CVs) increased as the stimulation period was prolonged. HRP was incorporated into these endocytotic structures. The marker substance was also taken up into a few SVs, morphologically indistinguishable from the core structure of the CVs, filled with HRP. No profile suggesting the budding-off of SVs was observed in the macropinocytotic vacuoles. The HRP-laden SVs amounted to I-3 % of the total number of SVs. The SVs containing HRP did not always exist near the active zone. These experiments suggest that the recycling route of SVs is limited to the conversion of CVs to SVs in the terminal of the preganglionic nerve of the cat SCG in vivo. The supply of SVs along this route is at a very low rate. The recycling of SVs via CVs seems to replace only a minor part of the SVs in the presynaptic terminal. A greater part of SVs, 97-99 % of the total, may not be supplied from the membranous structures of endocytotic origin.
ULTRASTRUCTURAL IN SITU
CHANGES
AT FACILITATED
SYNAPSES
OF CAT SYMPATHETIC
GANGLION
TETANICALLY
STIMULATED
1 * TOMOKO KADOTA , MA~ASHI HORI 2 and KEN KADOTA 2, iDepartment of Anatomy, Chiba University Medical School, Chiba 280,-Division of Neurochemistry, Psychiatric Research Institute of Tokyo, Setagaya, Tokyo 166. Morphological changes at a facilitated synapse (in vivo) were ultrastructurally and morphometrically studied in the cat superior cervical ganglion under a normal blood supply. Bilateral preganglionic trunks were electrically stimulated at i0 Hz for periods of 1-30 min. The postganglionic action potential (PGP) was recorded. The ganglion was rapidly fixed by local perfusion via the lingual artery and processed for electron microscopy. The PGP transiently increased in amplitude up to about 35% above the single response (control level) within 10-30 sec after the onset of stimulation. Then, it gradually reduced, and reached a steady state range, about 10% higher than the control level, after 5 min of stimulation. The synaptic vesicles (SVs), distributing in an area near the active zone, increased about 40% in density above the control level 10-30 sec after starting stimulation, and then decreased to a plateau level, approximately 10% higher than the control, after B min of stimulation. The SVs became smaller in size as the stimulation did. The perimeter of the terminal profile showed a transient decrease within 1 min of stimulation and then a gradual and continuous increase. Electrophysiologists have shown that tetanic stimulation at the cholinergic synapses enhances the excitatory postsynaptic potential (EPSP). This "facilitation" was observed in the cat sympathetic ganglion. Corresponding to the increase of PGP, the increase of SV density near the active zone was observed in the present experiment, suggesting a transmitter release from SV by exocytosis. Following the vesicle hypothesis, this change of the SV density may relate to basic mechanisms for synaptic facilitation. The increase of the smaller SV in size matched our previous findings (Kadota & Kadota, 1985) that the tubular structure of the smooth endoplasmic reticulum, serving as a sourse for SV supply, augumented in number in nerve terminals in response to activation at the same frequency.
25. Neurotransmitters
IV. Presynaptic mechanisms
PRESYNAPTIC MEMBRANE MOVEMENT IN THE CAT CERVICAL STIMULATED AT A LOW FREQUENCY.
SYMPATHETIC
GANGLION
IN SITU,
ELECTRICALLY
MASASHI HORI .I, TOMOKO KADOTA 2, MITSUAKI FUJITA .2 and KEN KADOTA I, IDepartment of Neurochemistry, Psychiatric Research Institute of Tokyo, Setagaya-ku, T-~kyo i--5~, and 2Department of Anatomy, School of Medicine, Chiba University, Chiba 280, Japan. Presynaptic morphological changes following electrical stimulation at 0.2 Hz were studied in the cat superior cervical sympathetic ganglion (SCG) in situ, under a normal blood supply. Cats were anesthetized by intraperitoneal administration of sodium pentobarbital, 35-40 mg/kg, and both sides of SCG were prepared. Bilateral preganglionic trunks were electrically stimulated at 0.2 Hz for periods of 5-240 min. The ganglion was fixed by perfusion via the lingual artery and processed for electron microscopy. Ultrastructural changes in presynaptic terminals were examined and morphometrically analyzed. In some cases, horseradish peroxidase (HRP) was administered to examine endocytotic processes accompanying transmitter release. The number of the synaptic vesicles (SVs) did not change even after 4 hours of stimulation. There was an enlargement of the terminal area, resulting from the exocytosis of SVs. The number of macropinocytstic vacuoles and coated vesicles (CVs) increased as the stimulation period was prolonged. HRP was incorporated into these endocytotic structures. The marker substance was also taken up into a few SVs, morphologically indistinguishable from the core structure of the CVs, filled with HRP. No profile suggesting the budding-off of SVs was observed in the macropinocytotic vacuoles. The HRP-laden SVs amounted to I-3 % of the total number of SVs. The SVs containing HRP did not always exist near the active zone. These experiments suggest that the recycling route of SVs is limited to the conversion of CVs to SVs in the terminal of the preganglionic nerve of the cat SCG in vivo. The supply of SVs along this route is at a very low rate. The recycling of SVs via CVs seems to replace only a minor part of the SVs in the presynaptic terminal. A greater part of SVs, 97-99 % of the total, may not be supplied from the membranous structures of endocytotic origin.
ULTRASTRUCTURAL IN SITU
CHANGES
AT FACILITATED
SYNAPSES
OF CAT SYMPATHETIC
GANGLION
TETANICALLY
STIMULATED
1 * TOMOKO KADOTA , MA~ASHI HORI 2 and KEN KADOTA 2, iDepartment of Anatomy, Chiba University Medical School, Chiba 280,-Division of Neurochemistry, Psychiatric Research Institute of Tokyo, Setagaya, Tokyo 166. Morphological changes at a facilitated synapse (in vivo) were ultrastructurally and morphometrically studied in the cat superior cervical ganglion under a normal blood supply. Bilateral preganglionic trunks were electrically stimulated at i0 Hz for periods of 1-30 min. The postganglionic action potential (PGP) was recorded. The ganglion was rapidly fixed by local perfusion via the lingual artery and processed for electron microscopy. The PGP transiently increased in amplitude up to about 35% above the single response (control level) within 10-30 sec after the onset of stimulation. Then, it gradually reduced, and reached a steady state range, about 10% higher than the control level, after 5 min of stimulation. The synaptic vesicles (SVs), distributing in an area near the active zone, increased about 40% in density above the control level 10-30 sec after starting stimulation, and then decreased to a plateau level, approximately 10% higher than the control, after B min of stimulation. The SVs became smaller in size as the stimulation did. The perimeter of the terminal profile showed a transient decrease within 1 min of stimulation and then a gradual and continuous increase. Electrophysiologists have shown that tetanic stimulation at the cholinergic synapses enhances the excitatory postsynaptic potential (EPSP). This "facilitation" was observed in the cat sympathetic ganglion. Corresponding to the increase of PGP, the increase of SV density near the active zone was observed in the present experiment, suggesting a transmitter release from SV by exocytosis. Following the vesicle hypothesis, this change of the SV density may relate to basic mechanisms for synaptic facilitation. The increase of the smaller SV in size matched our previous findings (Kadota & Kadota, 1985) that the tubular structure of the smooth endoplasmic reticulum, serving as a sourse for SV supply, augumented in number in nerve terminals in response to activation at the same frequency.