Effects of electrical stimulation on acetylcholine levels in central cholinergic nerve terminals

Brain Research, 81 (1974) 243-251

243

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

E F F E C T S OF E L E C T R I C A L S T I M U L A T I O N ON A C E T Y L C H O L I N E LEVELS IN C E N T R A L C H O L I N E R G I C N E R V E T E R M I N A L S

HANS ROMMELSPACHER* AND MICHAEL J. KUHAR Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)

(Accepted July 18th, 1974)

SUMMARY

These studies explored some of the characteristics of the cholinergic septalhippocampal neurons. Acetylcholine (Ach) levels were measured after various conditions of electrical stimulation o f the medial septal area. Stimulation (40 Hz) for varying periods of duration up to 60 min did not alter hippocampal Ach levels. However, when 10 #g of hemicholinium-3 bromide (HC-3) were injected into the lateral ventricles immediately prior to stimulation, there was a maximal 50 ~o depletion of Ach levels after 7.5 min of stimulation. There was no significant depletion of Ach when 1 or 5 #g of HC-3 was utilized, and there was no further depletion (beyond that observed with 10/~g) when 15 or 20 #g were injected. In experiments where the stimulation frequencies were varied, it was observed that 0.4 and 1 Hz did not lower Ach levels, whereas frequencies between 10 and 100 Hz resulted in a similar 50 ~ depletion at 7.5 min. Three days after cessation of stimulation (40 Hz, 10/~g HC-3), Ach levels had returned to that of untreated controls. Stimulation of the corpus striatum did not have any effect on hippocampal Ach levels.

INTRODUCTION While the location of all cholinergic tracts in the brain are unknown, a variety of biochemical and histochemical experiments indicate the presence of a septalhippocampal acetylcholine-containing tract. Briefly, these experiments involve placement of lesions in the septum and fimbria which result in a large, long lasting depletion of acetylcholine (Ach), choline acetyltransferase activity and the high affinity choline uptake in the hippocampus 16-18. Electrophysiological methods have revealed that * Permanent address: The Free University of Berlin, West Berlin, G.F.R.

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H. ROMMELSPACHER AND M. J. K U I | A R

hippocampal pyramidal cells are sensitive to iontophoretically applied Ach ",ev. These observations in conjunction with the finding that release of Ach from the hippocampus increases after electrical stimulation of the surface of the septum 25 provides strong evidence that Ach is a neurotransmitter in some septal-hippocampal neurons. In an attempt to explore some of the characteristics of these central cholinergic neurons, we have examined the effect of electrical stimulation of the septum on the Ach levels in the hippocampus. METHODS

Male, Sprague-Dawley rats (180-200 g) were anesthetized with 8% chloral hydrate (0.5 ml/100 g) and mounted in a David K o p f stereotaxic apparatus. Body temperature was monitored using a rectal thermoprobe and maintained at approximately 37 °C with a heat lamp. After opening the skull in the appropriate position, a concentric bipolar electrode was inserted to the medial septum, A8380, VO, LO coordinates according to K6nig and KlippeP 5. Stimulation was performed utilizing a Grass $44 stimulator, connected to a Grass constant current unit (200 #A). The duration of the monophasic rectangular stimuli were 2 msec; the polarity was reversed every 15 sec except when stimulation frequencies greater than 40 Hz were used, the reversal time being 10 sec. In all cases, the forebrains were sectioned (50#m) and stained (cresyl violet) to precisely locate the tip of the stimulating electrode. A typical location is shown in Fig. 1. In many experiments, hemicholinium-3 bromide (HC-3), a compound known to cause a reduction in Ach levels in braina,'~,7,13 and peripheral nerves 1,2°, was injected into the anterior horn of the lateral ventricle in physiological saline. The volume of this injection was 10/~1 in all cases. After treatment, the animals were rapidly decapitated and their brains removed to pentane bath at - - 5 °C. The hippocampus was removed and homogenized in 10 volumes of 1 N formic acid-acetone mixture (15:85) and Ach levels were measured with the enzymatic-isotopic method of Goldberg and McCaman 9. HC-3 and other drugs did not appear to interfere with the assay since addition of external standards to tissue extracts resulted in quantitative detection of Ach. RESULTS

Hippocampal acetylcholine levels after medial septal stimulation: time course and effects of HC-3 When animals were anesthetized with chloral hydrate for stereotaxic manipulation, we found that their Ach levels were increased significantly (45 %) over untreated animals 10 min post injection. Anesthetic-induced increases in Ach levels have been previously reported 8. When we stimulated the septum (40 Hz) in chloral hydrate anesthetized animals for 3 min, hippocampal Ach levels were lowered but only to that of untreated control values. Stimulation for longer times, 7.5, 15, 30 and 60 min, did not result in a further significant reduction in Ach levels (Fig. 2).

CENTRAL CHOLINERGIC NEURONS

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Fig. 1. Coronal section (stained with cresyl violet) of rat brain showing position of tip of stimulating electrode (arrow). Bar -- 1 mm.

Since we have previously found that intraventricular injection of HC-3 caused a dose-dependent, gradual depletion of hippocampal Ach levels2z, we injected varying amounts of HC-3 into the lateral ventricle to see if electrical stimulation would accelerate the rate o f depletion of Ach. After 1 #g of HC-3, there was still no significant depletion of hippocampal Ach levels below that of untreated controls after septal stimulation for varying times. However, when 10/,g of HC-3 were injected there was a large depletion of hippocampal Ach levels after 7.5 min of stimulation. There was no further depletion when the periods of stimulation were extended up to 30 min (Fig.

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Fig. 2. Effects of septal stimulation and administration of varying doses of HC-3 on hippocampal Ach levels. Points and error bars represent the mean -t- S.E.M. (n) respectively. Asterisk (*) implies a statistically significant difference from animals with no treatment, to at least the level of P < 0.05. 2). In agreement with our earlier studies, we observed that there was no significant depletion of Ach after 7.5 min in nonstimulated animals that were administered 10 #g o f HC-3 (see ref. 22).

Dose-response effects of HC-3 after stimulation Since it seemed necessary to inject HC-3 into the ventricles to cause a stimulusinduced depletion of hippocampal Ach levels, we examined various doses of HC-3 and the effects of stimulation. All animals in these groups received a 40-Hz stimulation for 7.5 min. We found no significant depletion o f Ach after administration of 1 or 5 #g o f HC-3. As mentioned above, 10 Fg caused a large (approximately 50 ~,,,) depletion o f Ach. There was no further depletion after 15 or 20/~g HC-3 (Fig. 3).

Effects of various stimulation frequencies on the depletion of hippocampal Ach after HC-3 administration In the above studies, all animals were stimulated at 40 Hz. In the following experiments, we examined effects of various stimulation frequencies in animals given 20 Fg of HC-3. All animals were sacrificed after 7.5 rain of stimulation. In animals that were anesthetized, administered 20/~g HC-3, but not stimulated, the hippocampal Ach concentrations were approximately 28 nmoles/g. As mentioned above, this increase after anesthesia is typical. After stimulation at 0.4 or 1 Hz, there was a lowering of Ach to levels near untreated control values. However, at stimulation frequencies

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Fig. 3. Effects of various doses of HC-3 on hippocampal Ach levels after stimulation (40 Hz) for 7.5 min. Points and error bars show mean ~: S.E.M. (n) respectively.Asterisks (*) indicate a statistically significant difference from 0/~g HC-3 to at least P < 0.05 level. of 10--100 Hz, there was a large (approximately 50 %), maximum depletion of hippocampal Ach levels (Fig. 4).

Recovery of Ach levels after adm&istration of HC-3 and electrical stimulation We wished to see if these conditions of stimulation caused permanent, electrolytic damage to these cholinergic neurons. In this case, the reduction in Ach levels would be long lasting due to degenerative changes 16. Accordingly, we examined the recovery of acetylcholine levels in the hippocampus at various times after stimulation (40 Hz) o f the medial septal area and administration o f 10/zg HC-3. In this group of animals, stimulation of the septum for 30 min resulted in a large (approximately 50 %) depletion of hippocampal Ach levels. This depletion of hippocampal Ach was maintained when animals were sacrificed 30 and 60 min after the cessation of the stimulation. This result was expected as HC-3 alone would cause a reduction at these times z2. Three days later, the Ach levels in these animals had returned to that of untreated controls, suggesting that these stimulation conditions do not cause permanent damage to the cholinergic septal-hippocampal tract (Table I).

Experiments demonstrating the anatomical specificity of the stimulation-induced effects Throughout the above experiments, we have assumed that the changes in the hippocampus are due to neuronal impulses induced in the axons connecting the septum and hippocampus. To verify this we stimulated the septal area and examined Ach levels in the striatum, a region which does not receive any cholinergic input from the septum. We have also stimulated the striatum at coordinates directly lateral to the septal area and assayed Ach levels in the hippocampus. After stimulation of the septal area, there was no reduction of the striatal Ach levels. After stimulation of the striatum, there was no reduction o f the hippocampal Ach levels in these anesthetized animals as observed in above experiments when the septum was stimulated (Table II). These data are consistent with the notion that a

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Fig. 4. Effects of varying frequencies of septal stimulation on hippocampal Ach levels. Points and error bars show mean ± S.E.M. (n) respectively. Asterisks (*) denote a statistically significant difference from nonstimulated animals to at least the P < 0.05 level.

direct a n a t o m i c a l c o n n e c t i o n m u s t exist to cause a stimulus-induced depletion o f Ach levels. DISCUSSION The objective o f this investigation was to examine Ach levels in cholinergic nerve terminals in the h i p p o c a m p u s after increasing the rate o f n e u r o n a l impulse flow. W h e n the septum in anesthetized a n i m a l s was stimulated at 40 Hz, a frequency causing a large, rapid loss o f h i p p o c a m p a l Ach after HC-3 a d m i n i s t r a t i o n , Ach levels fall only to the levels observed in u n t r e a t e d a n i m a l s a n d m a i n t a i n this level even after

TABLE I RECOVERY

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ACETYLCHOLINE

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MEDIAL SEPTAL AREA

Conditions

Ach levels (nmoles/g)

Untreated controls 30 min stimulation 30 min after cessation of stimulation 60 min after cessation of stimulation 3 days after cessation of stimulation

23.4 ± 12.5 ± 12.8 ± 11.5 ± 21.8 ±

0.3 (14) 1.9 (6)* 1.5 (4)* 1.8 (4)* 1.5 (4)

Values are mean ± S.E.M. (n). * P < 0.001 significant deviation from untreated controls.

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CENTRAL CHOLINERGIC NEURONS T A B L E II

EFFECT OF STIMULATIONOF THE SEPTAL AREA AND THE STRIATUM ON THE ACETYLCHOLINECONCENTRATION IN THE H1PPOCAMPUS AND THE STRIATUM RESPECTIVELY The striatum (STR) and the septal area (SA) respectively were stimulated for 7.5 min at 40 Hz. CH, chloral hydrate, S T I M , stimulation; HIP, hippocampus.

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23.4 32.7 34.5 41.3 76.2 73.8

-+- 0.3 dz 1.3 ± 2.0 ± 4.2 2:1.5 2z 5.5

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Values are mean ± S.E.M. (n). 60 min of stimulation. These findings suggest that cholinergic neurons in the brain can synthesize Ach quite rapidly to keep up with high rates of neuronal stimulation. Compatible with this is the finding that Ach turnover in brain is very rapidla,23, ~6. Similar experiments utilizing the superior cervical sympathetic ganglia have revealed that stimulation of preganglionic trunk results in a small depletion of Ach after which there is no further depletion of Ach in the ganglia, even after prolonged periods of stimulation 1. Thus it appears that these central neurons are quite similar to those peripheral cholinergic neurons in that it is difficult to lower Ach levels by direct stimulation. However, since there is no knowledge of the normal, physiological rate of impulse flow in these neurons, it is possible that conditions (perhaps involving more than simply alteration of impulse flow) could occur resulting in a decrease of Ach levelsg, 21. Just as in experiments with ganglia, where the presence of HC-3 was required to cause depletions of Ach 1, we found it necessary to inject HC-3 into the lateral ventricles to cause a stimulus-induced depletion of Ach in cholinergic terminals. Under our conditions, we did not see a significant depletion until 10 #g of HC-3 were injected. Intraventricular injection of HC-3 in non-stimulated animals caused a gradual, reversible depletion of hippocampal Ach levels5,7,13. In these present studies, we combine electrical stimulation with HC-3 administration and we find a large depletion at 7.5 min, a time at which without stimulation there would be no significant decrease. Since we find a greater rate of Ach depletion after injection o f HC-3, we conclude that stimulation under these conditions causes an increased release of Ach in cholinergic neuronal terminals in the hippocampus. In experiments with the isolated rat diaphragm, the presence of HC-3 also resulted in a rapid decline of Ach stores during stimulation 20. After intraventricular injection, HC-3 penetrates to the hippocampus which is well bathed with ventricular fluid s. While the precise mechanism of action of HC-3 is not clear, it is undoubtedly involved in blocking the selective high affinity uptake

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of choline to cholinergic neurons and the synthesis of Ach 4,11,r~,2s. In earlier studies, we found that the depleting action of HC-3 was prevented by stopping impulse flow and/or release in the hippocampal afferents z2. Here we show that the rate of HC3-induced depletion is increased by increasing impulse flow. This is supportive of our earlier conclusion that the depleting action of HC-3 is impulse flow-dependent. These results are also in agreement with studies indicating that synthesis rate of Ach is coupled to impulse flow rate 3,1°,',4. Experiments involving stimulation of the superior cervical sympathetic ganglia has revealed that approximately 85 ~o of endogenous stores of Ach can be depleted in the presence of HC-3 (see ref. 1). These investigators therefore described the cholinergic nerve terminals as having two pools of Ach. The depletable pool was called 'depot' acetylcholine which could be released, and the remainder was called 'stationary' Ach. In these experiments we also find a maximal depletion beyond which we cannot deplete any further by either increased stimulation rate or increased doses of HC-3. This would suggest that these neurons also have two similar pools of Ach, one releasable and the other stationary. However, this interpretation requires caution because we may not be effectively stimulating all of the afferents to the hippocampus, HC-3 may not penetrate to all cholinergic terminals throughout all the depths of the hippocampus, and we might have caused greater depletions of Ach if we stimulated for longer times when using higher doses of HC-3. Nevertheless, our findings do not rule out the possibility that 'depot' and 'stationary' pools of Ach exist in these central neurons. It has been known that Ach is present in the septal-hippocampal tract t6-ts. Also it is known that Ach is electrophysiologically active on hippocampal neurons 2,27, and Ach is released from the hippocampus after stimulation of the surface of the septum zS. Our data are in agreement with the latter findings and provide further support for the proposal that Ach is a neurotransmitter in these septal-hippocampal neurons. ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of R. DeHaven. These studies were supported by N.I.M.H. Johns Hopkins Drug Abuse Res. Ctr. Grant D A 00266 and Res. G r a n t M H 25078 and M H 25951.

REFERENCES 1 BIRKS,R., AND MACINTOSH,F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad. J. Biochem., 39 (1961) 787-827. 2 BISCOE,T. J., AND STRAUGHAN, D. W., Microelectrophoretic studies of neurones in the cat hippocampus, J. Physiol. (Lond.), 183 (1966) 341-359. 3 BROWNING,E. T., AND SCHULMAN, P. M., [14C]Acetylcholine synthesis by cortex slices of rat brain, J. Neurochem., 15 (1968) 1391-1395. 4 CHOI, R. L., FREEMAN, J. J., AND JENDEN, D. J., Effect of hemicholinium on kinetics of brain choline and acetylcholine, Fed. Proc., 33 (1974) 505.

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5 DELORES ARNAIZ, G. R., ZIEHER, L. M., AND DERORERTIS,E., Neurochemical and structural studies on the mechanism of action of hemicholinium-3 in central cholinergic synapses, J. Neurochem., 17 (1970) 221-229. 6 DOMINO, E. F., CASSANO,G. B., AND PLACIDI, G., Autoradiographic distribution of 14C-hemicholinium-3 in mouse whole body and dog brain, J. PharmacoL exp. Ther., 188 (1974) 77-85. 7 DOMINO, E. F., MOHRMAN,M. E., WILSON,A. E., AND HAARSTADT,V. B., Acetylseco-hemicholinium-3, a new choline acetyltransferase inhibitor useful in neuropharmacological studies, Neuropharmacol., 12 (1973) 549-561. 8 GIARMAN,N. J., AND PEPEU, G., Drug-induced changes in brain acetylcholine, Brit. J. Pharmacol., 19 (1962) 226-234. 9 GOLDBERG, A. M., AND MCCAMAN, R. E., The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem., 20 (1973) 1-8. 10 GREWAAL,D. S., AND QUASTEL,J. H., Control of synthesis and release of radioactive acetylcholine in brain slices from the rat, Biochem. J., 132 (1973) 1-14. l l GUYENET,P., LEFRESNE,P., ROSSIER,J., BEAUJOUAN,J. C., AND GLOWINSKI,J., Effects of sodium, hemicholinium-3, and antiparkinson drugs on [14C]acetylcholine synthesis and [OH]choline uptake in rat striatal synaptosomes, Brain Research, 62 (1973) 523-529. 12 HAGA, T., AND NODA, H., Choline uptake systems of rat brain synaptosomes, Biochim. biophys. Acta (Amst.), 291 (1973) 564-575. 13 HERB, C. O., LING, G. M., MCGEER, E. G., MCGEER, P. L., AND PERKINS, D., Effect of locally applied hemicholinium on the acetylcholine content of the caudate nucleus, Nature (Lond.), 204 (1964) 1309-1311. 14 JENDEN, D. J., CHOI, L., SILVERMAN, R. W., STEINBORN,J. A., ROCH, M., AND BOOTH, R. A., Acetylcholine turnover estimation in brain by gas chromatography/mass spectrometry, Life Sci., 14 (1974) 55-63. 15 KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain. Williams and Wilkins, Baltimore, Md., 1963. 16 KUHAR, M. J., SETHY, V. H., ROTH, R. H., AND AGHAJANIAN,G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 17 LEwis, P. R., SHUTE,C. C. D., AND SILVER,A., Confirmation from choline acetylase of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. 18 MCGEER, E. G., WADA, J. A., TERAO, A., AND JUNG, E., Amine synthesis in various brain regions with caudate or septal lesions, Exp. NeuroL, 24 (1969) 277-284. 19 PEPEU, G., AND MANTEGAZZINI,P., Midbrain hemisection: effect on cortical acetylcholine in the cat, Science, 145 (1964) 1069-1070. 20 POTTER, L. T., Synthesis, storage and release of [14C]acetylcholine in isolated rat diaphragm muscles, J. Physiol. (Lond.), 206 (1970) 145-166. 2l RICHTER,O., AND CROSSLAND,J., Variation in acetylcholine content of the brain with physiologic state, Amer. J. Physiol., 159 (1949) 247-255. 22 ROMMELSPACHER,H., GOLDBERG,A. M., AND KUHAR, M. J., Action of hemicholinium-3 on cholinergic nerve terminals after alteration of neuronal impulse flow, NeuropharmacoL, in press. 23 SCHUBERTH,J., SPARE, B., AND SUNDWALL,A., A technique for the study of acetylcholine turnover in mouse brain in vivo, J. Neurochem., 16 (1969) 695-700. 24 SHARKAWI, M., AND SCHULMAN, M. P., Relationship between acetylcholine synthesis and its concentration in rat cerebral cortex, Brit. J. PharmacoL, 36 (1969) 373-379. 25 SMITH, C. M., The release of acetylcholine from the rabbit hippocampus, Brit. J. Pharmacol., 45 (1972) 172. 26 SPARE, B., On the turnover of acetylcholine in the brain, Acta physiol, scand., Suppl. 397 (1973). 27 STEINER, F. A., Influence of microelectrophoretically applied acetylcholine on responsiveness of hippocampal and lateral geniculate neurons, Pfliig. Arch. ges. Physiol., 303 (1968) 173-180. 28 YAMAMURA,H. I., AND SNYDER, S. H., High affinity transport of choline into synaptosomes of rat brain, J. Neurochem., 21 (1973) 1355-1374.