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Inhibition of acetylcholine outflow from guinea-pig cerebral cortex following locus coeruleus stimulation
Neuroscience Letters, 14 (1979) 97--100
97
© Elsevier/North-Holland Scientific Publishers Ltd.
INHIBITION OF ACETYLCHOLINE O U T F L O W F R O M GUINEA-PIG C E R E B R A L C O R T E X FOLLOWING LOCUS C O E R U L E U S STIMULATION
C. BIANCHI, G. SPIDALIERI, P. GUANDALINI, S. TANGANELLI and L. BEANI Department of Pharmacology, University of Ferrara (G.S. & P.G.) Institute of Human Physiology of Ferrara (Italy)
(Received December llth, 1979) (Revised version received May 21st, 1979) (Accepted May 21st, 1979)
SUMMARY
Experiments were performed in unanaesthetized guinea-pigs in order to obtain direct evidence that the noradrenergic projections from locus coeruleus (L.C.) to cerebral cortex inhibit the acetylcholine (ACh) release. The animals were provided with left and right epidural cups, to collect outflowing ACh and with a stimulating electrode, positioned in the left L.C. The electrical stimulation of L.C. caused a significant reduction of ACh outflow from b o t h parietal areas. This effect was abolished, reduced or prevented by phentolamine, phenoxybenzamine or reserpine plus a-mt pretreatment, hence showing to be a-mediated.
Recent reports show that Noradrenaline (NA) inhibits, by means of a-receptors, acetylcholine (ACh) release from the guinea-pig cerebral cortex [1,8]. The present experiments were performed to ascertain whether this inhibition could be reproduced by electrically stimulating the locus coeruleus (L.C.) in unrestrained, unanaesthetized animals. As previously described [ 1], t w o small epidural cups were implanted on the left and right parietal areas of guinea-pigs, weighing between 380 g and 500 g, anaesthetized with pentobarbital. A monopolar or concentric bipolar stimulating electrode (exposed tip, 150/~m; outer diameter 100--200 pm; semi-micro series of Rhodes Med. Instr., CA, U.S.A.) was placed in the left L.C., according to the stereotaxical coordinates: AP = 00 (ear-bar); L = 0 . 7 5 ; H = + 0 . 5 5 , t h e t o o t h bar of the K o p f frame being in the same horizontal plane as the ear rods. The cups and the electrode were firmly fastened to the skull by means of dental cement. Two days after surgery, the experiment was carried o u t as follows: the eserinized saline solution (0.2 ml), placed in both cups, was renewed every 30 min and immediately bioassayed for its ACh content [ 1]. After ascertaining a constant outflow of the transmitter for 2 h, L.C. was stimulated every 3 sec, for 30 min with trains of 30 negative pulses (0.25 msec
98 duration, 80 pA, 30/sec) delivered from a Grass stimulator. Three hours later, a monoamine antagonist was administered i.p. and L.C. stimulation was repeated. When using a monopolar electrode, the metal grid of the cage was earthed. Spread of current was assumed to be negligible as no signs of activation of motor structures near the L.C. (e.g., mesencephalic trigeminal tract) w~re observed. In any case, similar results were obtained with monopolar and concentric electrodes (see below). At the end of each experiment the brains were processed for histological examination {fixation in 10% formalin, paraffin-embedded blocks, 30 ~m slices, cresyl-violet staining) and the site of the electrode tip was verified. On two of the guinea-pigs, Falk-Hillarp's histochemical technique [3] was employed. Biochemical evidence for correct localization of L.C. was also checked in animals submitted to discrete electrolytic lesion of L.C. and processed for forebraln monoamine content [2] 8--10 days later. The lesion, (1.5 mA anodal current for 15 sec) was confined to the desired area and caused a selective fall of NA content to a b o u t 50%, which was restricted to the ipsilateral cortex. Figure 1A, shows the effect of L.C. stimulation (L.C.S.) in seven animals. Since similar results were obtained with monopolar (5 expts.) and concentric electrodes (2 expts.), the data were pooled together. During and 30 min after L.C.S. ACh outflow diminished above all in the ipsilateral cortex. In addition L.C.8. caused the animals to become quiet or even drowsy. A rebound increase of the overflow was evident 90 min later, associated with enhanced l o c o m o t o r and exploratory activity. As already reported and discussed, phentolamine 5 mg/kg enhanced per se the ACh outflow [ 1 ] and cancelled the effects of L.C.S. both on ACh outflow and animal behaviour. With the aim to add evidence that L.C.S. elicited a noradrenergic, ~-mediated inhibition on the cholinergic corticopetal structures, other treatments were performed (two experiments for each drug, i.p.). Phentolamine 2 mg/ kg and phenoxybenzamine 2 mg]kg nearly halved the effect of L.C.S., while reserpine plus ~-methyl-p-tyrosine (5 mg/kg 18 h, plus 200 mg/kg 2 h before L.C.S.) prevented it. Conversely, propranolol 10 mg/kg and spiroperidol 2 mg/kg were ineffective. When the electrode tip was positioned a few hundreds of g m away from the correct site, the ACh outflow, instead of being reduced as normally happens with L.C.S., was unaffected or increased (Fig. 1B). These results show (i) that L.C.S. reproduces the picture previously obtained with NA, injected into the lateral ventricles of the guinea-pig (1) and {ii) that the control on the cholinergic neurones is exclusively ~-mediated. This finding is in conflict with recent reports [ 5,6] showing that L.C.S. inhibition in the rat cerebral cortex is solely ~-mediated. At present we cannot establish, whether the effect depends on the diminished firing rate of corticopetal cholinergic fibers and/or on the reduction of ACh release/pulse at the nerve endings, being such evidence supported by the direct effect of NA on electrically-stimulated cortical slices [ 1 ].
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Fig. 1. (A) A b s o l u t e values o f ACh release from the left (e i ) and right (A. . . . A) parietal cortex caused by 30 min stimulation o f left locus coeruleus in unanaesthetized guinea-pigs, before and after phentolamine, 5 m g / k g i . p . (7 expts). Vertical bars, S.E.M., * significantly different (P < 0 . 0 5 or lower) from pre-treatment values. Student's t-test for paired data. (B) Absolute changes of ACh release from the left (o o) and right (A . . . . A) parietal cortex, caused by 30 rain stimulation o f a region 3 5 0 ~ m more lateral and 4 5 0 u m more cranial than locus coeruleus.
In any case, the noradrenergic control is long-lasting, suggesting some modulatory (metabolic?) influences of the monoamine on the cholinergic cells [4,8]. Furthermore, the spreading of the inhibition to the site controlateral to the electrode raises the point of crossed linkages, which could ensure positive cooperation between the two L.C. ACKNOWLEDGEMENTS
This work was partly supported by a grant from the Consiglio Nazionale delle Ricerche, Roma (No. 77.01634.04). REFERENCES 1 Beani, L., Bianchi, C., Giacomelli, A. and Tamberi, F., Noradrenaline inhibition of acetylcholine release from guinea-pig brain, Europ. J. Pharmacol., 48 (1978) 179--193. 2 Cox, R.H. and Perhach, J.L., A sensitive, rapid and simple method for the simultaneous spectrophotofluorimetric determinations of noradrenaline, dopamine, 5-hydroxy-
I00 triptamine and 5-hydroxyindolacetic acid in discrete areas of brain, J. Neurochem., 20 (1973) 1777--1780. 3 Falck, B., Hillarp, N.A., Thieme, G. and Thorp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348--354. 4 Korf, J. and Sebens, J.B., Cyclic AMP in the rat cerebral cortex after activation of noradranaline neurons of the locus coeruleus, J. Neurochem., 32 (1979) 463--468. 5 0 l p e , H.R. and Koella, W.P., The effect of ~- and ~-receptor blocking drugs on locus coeruleus elicited inhibition of cingulate cortical cells, Neurosci. Lett., Suppl., 1 (1978) $301. 6 Phillis, J.W. and Kostopoulus, G.K., Activation of a noradrenergic pathway from the brain stem to rat cerebral cortex, Gen. Pharmac., 8 (1977) 207--211. 7 Reader, T.A., Ferron, A., Descarries, L. and Jasper, H.H., Modulatory role for biogenic amines in the cerebral cortex microiontophoretic studies, Brain Res., 160 (1979) 217--229. 8 Vizi, E.S., Interaction between adrenergic and cholinergic system: presynaptic inhibitory effect of noractrenaline on acetylclioline release, J. Neural. Trans., Suppl. XI {1974) 61--78.
97
© Elsevier/North-Holland Scientific Publishers Ltd.
INHIBITION OF ACETYLCHOLINE O U T F L O W F R O M GUINEA-PIG C E R E B R A L C O R T E X FOLLOWING LOCUS C O E R U L E U S STIMULATION
C. BIANCHI, G. SPIDALIERI, P. GUANDALINI, S. TANGANELLI and L. BEANI Department of Pharmacology, University of Ferrara (G.S. & P.G.) Institute of Human Physiology of Ferrara (Italy)
(Received December llth, 1979) (Revised version received May 21st, 1979) (Accepted May 21st, 1979)
SUMMARY
Experiments were performed in unanaesthetized guinea-pigs in order to obtain direct evidence that the noradrenergic projections from locus coeruleus (L.C.) to cerebral cortex inhibit the acetylcholine (ACh) release. The animals were provided with left and right epidural cups, to collect outflowing ACh and with a stimulating electrode, positioned in the left L.C. The electrical stimulation of L.C. caused a significant reduction of ACh outflow from b o t h parietal areas. This effect was abolished, reduced or prevented by phentolamine, phenoxybenzamine or reserpine plus a-mt pretreatment, hence showing to be a-mediated.
Recent reports show that Noradrenaline (NA) inhibits, by means of a-receptors, acetylcholine (ACh) release from the guinea-pig cerebral cortex [1,8]. The present experiments were performed to ascertain whether this inhibition could be reproduced by electrically stimulating the locus coeruleus (L.C.) in unrestrained, unanaesthetized animals. As previously described [ 1], t w o small epidural cups were implanted on the left and right parietal areas of guinea-pigs, weighing between 380 g and 500 g, anaesthetized with pentobarbital. A monopolar or concentric bipolar stimulating electrode (exposed tip, 150/~m; outer diameter 100--200 pm; semi-micro series of Rhodes Med. Instr., CA, U.S.A.) was placed in the left L.C., according to the stereotaxical coordinates: AP = 00 (ear-bar); L = 0 . 7 5 ; H = + 0 . 5 5 , t h e t o o t h bar of the K o p f frame being in the same horizontal plane as the ear rods. The cups and the electrode were firmly fastened to the skull by means of dental cement. Two days after surgery, the experiment was carried o u t as follows: the eserinized saline solution (0.2 ml), placed in both cups, was renewed every 30 min and immediately bioassayed for its ACh content [ 1]. After ascertaining a constant outflow of the transmitter for 2 h, L.C. was stimulated every 3 sec, for 30 min with trains of 30 negative pulses (0.25 msec
98 duration, 80 pA, 30/sec) delivered from a Grass stimulator. Three hours later, a monoamine antagonist was administered i.p. and L.C. stimulation was repeated. When using a monopolar electrode, the metal grid of the cage was earthed. Spread of current was assumed to be negligible as no signs of activation of motor structures near the L.C. (e.g., mesencephalic trigeminal tract) w~re observed. In any case, similar results were obtained with monopolar and concentric electrodes (see below). At the end of each experiment the brains were processed for histological examination {fixation in 10% formalin, paraffin-embedded blocks, 30 ~m slices, cresyl-violet staining) and the site of the electrode tip was verified. On two of the guinea-pigs, Falk-Hillarp's histochemical technique [3] was employed. Biochemical evidence for correct localization of L.C. was also checked in animals submitted to discrete electrolytic lesion of L.C. and processed for forebraln monoamine content [2] 8--10 days later. The lesion, (1.5 mA anodal current for 15 sec) was confined to the desired area and caused a selective fall of NA content to a b o u t 50%, which was restricted to the ipsilateral cortex. Figure 1A, shows the effect of L.C. stimulation (L.C.S.) in seven animals. Since similar results were obtained with monopolar (5 expts.) and concentric electrodes (2 expts.), the data were pooled together. During and 30 min after L.C.S. ACh outflow diminished above all in the ipsilateral cortex. In addition L.C.8. caused the animals to become quiet or even drowsy. A rebound increase of the overflow was evident 90 min later, associated with enhanced l o c o m o t o r and exploratory activity. As already reported and discussed, phentolamine 5 mg/kg enhanced per se the ACh outflow [ 1 ] and cancelled the effects of L.C.S. both on ACh outflow and animal behaviour. With the aim to add evidence that L.C.S. elicited a noradrenergic, ~-mediated inhibition on the cholinergic corticopetal structures, other treatments were performed (two experiments for each drug, i.p.). Phentolamine 2 mg/ kg and phenoxybenzamine 2 mg]kg nearly halved the effect of L.C.S., while reserpine plus ~-methyl-p-tyrosine (5 mg/kg 18 h, plus 200 mg/kg 2 h before L.C.S.) prevented it. Conversely, propranolol 10 mg/kg and spiroperidol 2 mg/kg were ineffective. When the electrode tip was positioned a few hundreds of g m away from the correct site, the ACh outflow, instead of being reduced as normally happens with L.C.S., was unaffected or increased (Fig. 1B). These results show (i) that L.C.S. reproduces the picture previously obtained with NA, injected into the lateral ventricles of the guinea-pig (1) and {ii) that the control on the cholinergic neurones is exclusively ~-mediated. This finding is in conflict with recent reports [ 5,6] showing that L.C.S. inhibition in the rat cerebral cortex is solely ~-mediated. At present we cannot establish, whether the effect depends on the diminished firing rate of corticopetal cholinergic fibers and/or on the reduction of ACh release/pulse at the nerve endings, being such evidence supported by the direct effect of NA on electrically-stimulated cortical slices [ 1 ].
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Fig. 1. (A) A b s o l u t e values o f ACh release from the left (e i ) and right (A. . . . A) parietal cortex caused by 30 min stimulation o f left locus coeruleus in unanaesthetized guinea-pigs, before and after phentolamine, 5 m g / k g i . p . (7 expts). Vertical bars, S.E.M., * significantly different (P < 0 . 0 5 or lower) from pre-treatment values. Student's t-test for paired data. (B) Absolute changes of ACh release from the left (o o) and right (A . . . . A) parietal cortex, caused by 30 rain stimulation o f a region 3 5 0 ~ m more lateral and 4 5 0 u m more cranial than locus coeruleus.
In any case, the noradrenergic control is long-lasting, suggesting some modulatory (metabolic?) influences of the monoamine on the cholinergic cells [4,8]. Furthermore, the spreading of the inhibition to the site controlateral to the electrode raises the point of crossed linkages, which could ensure positive cooperation between the two L.C. ACKNOWLEDGEMENTS
This work was partly supported by a grant from the Consiglio Nazionale delle Ricerche, Roma (No. 77.01634.04). REFERENCES 1 Beani, L., Bianchi, C., Giacomelli, A. and Tamberi, F., Noradrenaline inhibition of acetylcholine release from guinea-pig brain, Europ. J. Pharmacol., 48 (1978) 179--193. 2 Cox, R.H. and Perhach, J.L., A sensitive, rapid and simple method for the simultaneous spectrophotofluorimetric determinations of noradrenaline, dopamine, 5-hydroxy-
I00 triptamine and 5-hydroxyindolacetic acid in discrete areas of brain, J. Neurochem., 20 (1973) 1777--1780. 3 Falck, B., Hillarp, N.A., Thieme, G. and Thorp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348--354. 4 Korf, J. and Sebens, J.B., Cyclic AMP in the rat cerebral cortex after activation of noradranaline neurons of the locus coeruleus, J. Neurochem., 32 (1979) 463--468. 5 0 l p e , H.R. and Koella, W.P., The effect of ~- and ~-receptor blocking drugs on locus coeruleus elicited inhibition of cingulate cortical cells, Neurosci. Lett., Suppl., 1 (1978) $301. 6 Phillis, J.W. and Kostopoulus, G.K., Activation of a noradrenergic pathway from the brain stem to rat cerebral cortex, Gen. Pharmac., 8 (1977) 207--211. 7 Reader, T.A., Ferron, A., Descarries, L. and Jasper, H.H., Modulatory role for biogenic amines in the cerebral cortex microiontophoretic studies, Brain Res., 160 (1979) 217--229. 8 Vizi, E.S., Interaction between adrenergic and cholinergic system: presynaptic inhibitory effect of noractrenaline on acetylclioline release, J. Neural. Trans., Suppl. XI {1974) 61--78.