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Aging of cholinergic synapses in the avian iris
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149
CHRONIC ADMINISTRATION OF PYRIDOSTIGMINE (P) AND/OR EXHAUSTIVE EXERCISE CHANGE MORPHOLOGY AND PHYSIOLOGY OF RAT MUSCLE AND NEUROMUSCULAR JUNCTION. Fishman, R.H.B., Chipman, M.4 Silman 2, I. ~ Argov, Z. Dept. Neurol., Hadassah Med. 0rg. FOB 12OOO, Jerusalem; ~Dept. Neurobiol., Weizman Insti., Rehovot; Israel. Female Fisher rats (110-170g) were given P in drinking water to obtain 12.5mg/kg/d. for 3,7,14,21, or 28d. P and control (non-dosed) rats ran on a threadmill, I hr/d. 31 m/min., a full gallop. A shocking plate, delivering O.5-4ma's, at the rear of the track reinforced running; the shocks measured the ability to maintain required effort. Whole blood acetylcholinesterase (ACHE) was taken at the start and end of each experiment for internal reference (radiometric assay; ACh-H 3 substrate; Johnson & Russell, 1975, Analyt. Biochem. 64:229). The gastrocnemiue (G) was processed routinely for TEM after en bloc AChE stain. Force output and EMG of G were measured after stimulating the sciatic N. (trains of 8,16,32 stimuli at IOOHz every IOsec. for 10 min.). P didn't change drinking (ca. 20ml/rat/d.) but tended to increase weight gain; running tended to decrease it. Running indicates a bimodal drug response with time: P rats appear more vigorous, resistant to fatigue than controls after 3d; thereafter, the trend reverses as controls improve while P rats don't. In contrast, P rats seem to show greater power output over time than controls, under these stimulus conditions. TEM shows swollen, ruptured muscle mitochondria, damage typical of exhaustive running. P is found with Z-line swelling, eubjunctional muscle disintegration, and evidence of axon terminal retraction and/or degeneration. The extent, time course and longevity of these changes is being investigated.
150AGING OF CHOLINERGIC SYNAPSES IN THE AVIAN IRIS Giacobini, E., *Mussini, I. and Mattio, T. Dept. Pharmacology, Southern l i t . Univ. Sch. Med., Springfield, I l l . 62708 and *C.S. Biol. Fisiopat. Musc., Ist. Patologia Generale, Universita di Padova, 35100 Padova, I t a l y We have made use of the c i l i a r y ganglion-iris preparation of the aging (1.5-9 yrs) chicken as a model of senescent peripheral cholinergic synapses. Neuromuscular junctions in the i r i s of aging chickens show early (1.5 yrs) morphological signs of damage such as, reduction and polymorphism of synaptic vesicles and increase of neurofilaments and mitochondria. Accumulations of cytoplasmic organelles and lysosomes are seen in the axoplasm of the nerve fiber. At later stages (5-9 yrs), the nerve ending is enveloped by Schwann cells i n f i l t r a t i n g and f i l l i n g the synaptic c l e f t . Quantit a t i v e morphometric changes in the ratio describing the relationship between volumes of terminals and volumes of synaptic vesicles show a progressive decrease in the volu~ne occupied by synaptic vesicles. Th@ a b i l i t y of the cholinergic synapses to take u~ JH-choline and release the formed JH-acetylcholine (ACh) in response to high ~T-depolarization is impaired at 5 yrs resulting in a significant depletion of the °H-ACh releasable pool. These experiments seem to point out for the f i r s t time a selective functional defect in the cholinergic synapse during aging. (Supported by AFOSR Grant NL-144 and by Nowatski Eye Research Fund to E.G.)
1 5 1 PHARMACOLOGICAL AND MORPHOLOGICAL CHARACTERIZATION OF NORMALAND DIFFERENTIATED HUMAN NEUROBLASTOMA CELL LINE. C. Gotti, E. Sher, D. Cabrini, D. Fornasari
and F. Clementi
We used the IMR.32 human neuroblastoma cell line as a tool for studying the cholinergic neuronal receptors, in normal conditions and after pharmacological induced djfferentiation. In these cells we have detected a specific and saturable binding of l~bI-wBungaro toxin ~Bgtx); with a Kd and a B max of 9 nM and 40 fmol/mg of protein,respectively.Th~ ~Bgtx binding was specificallX inhibited by nicotinic agonists and antagonists. A speci f i c and saturable binding of OH-QNB, a muscarinic antagonist, was also present; the Kd of the binding was 250 pM and the B max of 27 fmol/mg of protein. The binding was inhibited with high a f f i n i t y by classical muscarinic agonists and antagonists and withlower a f f i n i t y by nicotinic drugs. We also studied the regulation of these two recepto~during cellular differentiation induced by dibutyryl-cAMP and by PgEl. We detected in differen tiated cells a more than six fold increase in 1251-wBgtx binding sites (287 fmol/mg oT protein), while the Kd was not significantly affected. In contrast with this result, no increase in 3H-QNB binding sites was detected in differentiated cells. Morphological ob servations at the optical and scanning electron microscope revealed that only part o7 the cells underwent a clear morphological differentiation with axons of more than 50 u length. Differentiated cells showed in their axons numerous dense-core and clear vesi ~le$. Further t ies fe in pro res in order to Glarifv the ce]]ular and.molecular asls or tnls ~l~eren(latlon In@uce~ regumatlon or neurOtransmltter receptors.
149
CHRONIC ADMINISTRATION OF PYRIDOSTIGMINE (P) AND/OR EXHAUSTIVE EXERCISE CHANGE MORPHOLOGY AND PHYSIOLOGY OF RAT MUSCLE AND NEUROMUSCULAR JUNCTION. Fishman, R.H.B., Chipman, M.4 Silman 2, I. ~ Argov, Z. Dept. Neurol., Hadassah Med. 0rg. FOB 12OOO, Jerusalem; ~Dept. Neurobiol., Weizman Insti., Rehovot; Israel. Female Fisher rats (110-170g) were given P in drinking water to obtain 12.5mg/kg/d. for 3,7,14,21, or 28d. P and control (non-dosed) rats ran on a threadmill, I hr/d. 31 m/min., a full gallop. A shocking plate, delivering O.5-4ma's, at the rear of the track reinforced running; the shocks measured the ability to maintain required effort. Whole blood acetylcholinesterase (ACHE) was taken at the start and end of each experiment for internal reference (radiometric assay; ACh-H 3 substrate; Johnson & Russell, 1975, Analyt. Biochem. 64:229). The gastrocnemiue (G) was processed routinely for TEM after en bloc AChE stain. Force output and EMG of G were measured after stimulating the sciatic N. (trains of 8,16,32 stimuli at IOOHz every IOsec. for 10 min.). P didn't change drinking (ca. 20ml/rat/d.) but tended to increase weight gain; running tended to decrease it. Running indicates a bimodal drug response with time: P rats appear more vigorous, resistant to fatigue than controls after 3d; thereafter, the trend reverses as controls improve while P rats don't. In contrast, P rats seem to show greater power output over time than controls, under these stimulus conditions. TEM shows swollen, ruptured muscle mitochondria, damage typical of exhaustive running. P is found with Z-line swelling, eubjunctional muscle disintegration, and evidence of axon terminal retraction and/or degeneration. The extent, time course and longevity of these changes is being investigated.
150AGING OF CHOLINERGIC SYNAPSES IN THE AVIAN IRIS Giacobini, E., *Mussini, I. and Mattio, T. Dept. Pharmacology, Southern l i t . Univ. Sch. Med., Springfield, I l l . 62708 and *C.S. Biol. Fisiopat. Musc., Ist. Patologia Generale, Universita di Padova, 35100 Padova, I t a l y We have made use of the c i l i a r y ganglion-iris preparation of the aging (1.5-9 yrs) chicken as a model of senescent peripheral cholinergic synapses. Neuromuscular junctions in the i r i s of aging chickens show early (1.5 yrs) morphological signs of damage such as, reduction and polymorphism of synaptic vesicles and increase of neurofilaments and mitochondria. Accumulations of cytoplasmic organelles and lysosomes are seen in the axoplasm of the nerve fiber. At later stages (5-9 yrs), the nerve ending is enveloped by Schwann cells i n f i l t r a t i n g and f i l l i n g the synaptic c l e f t . Quantit a t i v e morphometric changes in the ratio describing the relationship between volumes of terminals and volumes of synaptic vesicles show a progressive decrease in the volu~ne occupied by synaptic vesicles. Th@ a b i l i t y of the cholinergic synapses to take u~ JH-choline and release the formed JH-acetylcholine (ACh) in response to high ~T-depolarization is impaired at 5 yrs resulting in a significant depletion of the °H-ACh releasable pool. These experiments seem to point out for the f i r s t time a selective functional defect in the cholinergic synapse during aging. (Supported by AFOSR Grant NL-144 and by Nowatski Eye Research Fund to E.G.)
1 5 1 PHARMACOLOGICAL AND MORPHOLOGICAL CHARACTERIZATION OF NORMALAND DIFFERENTIATED HUMAN NEUROBLASTOMA CELL LINE. C. Gotti, E. Sher, D. Cabrini, D. Fornasari
and F. Clementi
We used the IMR.32 human neuroblastoma cell line as a tool for studying the cholinergic neuronal receptors, in normal conditions and after pharmacological induced djfferentiation. In these cells we have detected a specific and saturable binding of l~bI-wBungaro toxin ~Bgtx); with a Kd and a B max of 9 nM and 40 fmol/mg of protein,respectively.Th~ ~Bgtx binding was specificallX inhibited by nicotinic agonists and antagonists. A speci f i c and saturable binding of OH-QNB, a muscarinic antagonist, was also present; the Kd of the binding was 250 pM and the B max of 27 fmol/mg of protein. The binding was inhibited with high a f f i n i t y by classical muscarinic agonists and antagonists and withlower a f f i n i t y by nicotinic drugs. We also studied the regulation of these two recepto~during cellular differentiation induced by dibutyryl-cAMP and by PgEl. We detected in differen tiated cells a more than six fold increase in 1251-wBgtx binding sites (287 fmol/mg oT protein), while the Kd was not significantly affected. In contrast with this result, no increase in 3H-QNB binding sites was detected in differentiated cells. Morphological ob servations at the optical and scanning electron microscope revealed that only part o7 the cells underwent a clear morphological differentiation with axons of more than 50 u length. Differentiated cells showed in their axons numerous dense-core and clear vesi ~le$. Further t ies fe in pro res in order to Glarifv the ce]]ular and.molecular asls or tnls ~l~eren(latlon In@uce~ regumatlon or neurOtransmltter receptors.