Effects of electrical stimulation on acetylcholine synthesis in cat caudate nucleus

BWi/i Rcwwrc~li B~/iirtlrr. Vol. IO, pp. 437-440. 1983. “’ Ankho Intemation;rt. Printed in the U.S.A

Effects of Electrical Stimulation on Acetylcholine Synthesis in Cat Caudate Nucleus

Received 27 October

1982

S. G. AND E. GARCIA-RILL. ,[email protected] cd’ c~l~ciricrtl .~~itF~~~i~~t~o~? ou nc,r,t~l{,h~llitti, .~ynthrG in tierccrudtrrt RES BULL IO(4) 437-440, 1983.-The concentration ofendo~enous- and deuterium-labeled ac~tyl~holine (ACh) in the cat caudate nucleus was determined after stimulation of either the substantia nigra or the precruciate cortex. In this procedure the caudate nucleus is exposed surgically, and a coring device is used to obtain biopsy specimens which are immediately frozen in liquid nitrogen. Samples are collected at rest, 5 min after stimulation. and again 5 min after a resting period. An infusion of?H,,-choline is maintained during these manipulations to provide a label for ACh synthesis. Electrical stimulation of the substantia nigra, which increases the release of dopamine, produced a decrease in endogenous ACh and the newly synthesized deuterium-labeled ACh. Stimulation of the precruciatr cortex produced no significant effect on the levels or synthesis of ACh. but attenuated the effect of subsequent nigral stimulation. These preliminary results indicate that stimulation of the substantia nigra has a net excitatory effect on ACh synthesis in the caudate. This stimulation apparently is modulated by input from the cortex. HOWARD,

t~rct~/ct~.s. BRAIN

Acetylcholine synthesis

Caudatc nucleus

Substantia nigra

cat

neurons in the caudate nucleus monitored by determining levels of endogenous and labeled ACh in biopsy specimens obtained from the caudate nucleus. As a control, the effects of stimulation of cortical afferents on caudate ACh were also measured.

THE interaction between dopamine (DA} and acetylcholine (ACh) in the caudate nucleus is well documented and has been studied extensively in recent years (for review, see [S]). However the results of experiments examining this interaction have been interpreted to indicate both an excitatory and an inhibitory coupling. The interaction has been examined in several ways: (I) by examining the effects of dopaminergic agonists and antagonists on the turnover rate of ACh in the caudate nucleus [ 10,25]; (2) by measuring the release of ACh by means of a push-pull cannula after injection of drugs thought to affect dopaminergic mechanisms 113); (3) by measuring levels of ACh in the caudate nucleus after administration of dopaminergic agonists or antagonists 16, I I, 12, IS]; and (4) by studying the effect of lesions of the substantia nigra on the levels and synthesis of endogenous and dcuterium-labeled ACh and choline (Ch) in the caudate nucleus [X]. Electrophysiological studies also have added to this “inhibitory-excitatory” controversy. Iontophoretic application of DA has been reported to produce both excitation and inhibition in caudate neurons [3, IO. 17, 21, 241. Furthermore, intracellular recording experiments have shown that stimulation of the nigrostriatal pathway almost always produces an initial excitation in caudate neurons [ 14,171, indirectly suggesting that the DA pathway may be excitatory. This report describes an intact preparation in which the substantia nigra can be stimulated and the activity of ACh ‘Present address: Department of Anatomy,

Electrical stimulation

METHOD Adult cats (3-5 kg) were anesthetized with sodium methohexital, a short-acting barbiturate. The femoral vein was cannulated and the trachea was intubated. The cranium was exposed and the bone overlying the caudate nucleus and precruciate cortex was removed. A four-pronged stimulating comb (exposed tips 0.5 mm. intertip distance I mm) was stereotaxicaIly introduced into the left substantia nigra (placements were verified histologically). On the right side of the brain, double stimulating wires were implanted into the white matter immediately below the precruciate cortex. The caudate nuclei were exposed bilaterally by aspirating the overlying cortex. The cat was paralyzed with gallamine triethiodide and artificially respired. All wound margins and pressure points were infiltrated with procaine. The experiment was started 2 hr after gallamine treatment. This preparation was chosen for study due to the effects of anesthesia in decreasing Ch uptake and ACh synthesis, which are well established [Z, 22, 231. An IV infusion of deuterium-labeled Ch (YH,,-Ch, 0.5 ~mol/k~min) was started and continued

University of Arkansas, Little Rock. AR

437

throughout the experimental procedure. lo minimize postmortem changes in the concentration of ACh, tissue samples were withdrawn into a syringe containing IS% I N formic acid/acetone (15:X5 v/v) and injected into liquid nitrogen within 3-2 sec. The rapid ~ntr~~duction of the tis\uc into liquid nitrogen is critical for absoiute level measurements. The specific activity ~neasurements control for small variations since the hydolysis of labeled and unlabeled ACh are assumed to be equal. After 5 min of infusion. biopsies were removed from both caudate nuclei using a coring device dcscribed previously [I]. The first pair of biopsies from the left (1 L) and right (IR) caudate nuclei served as control samples. This procedure was followed by stimulation of the left substantia nigra for 5 min. After 5 min of stimulation. a second pair of biopsies was removed from the left (2L) and right (2R) caudate nuclei. This was followed by stimulation of the right precruciate cortex for 5 min, after which time a third pair of biopsies (3L and 3R) was removed. After a final 5 min period during which no stimulation occurred. the last pair of’ biopsies was withdrawn (4L and 4R). The last pair of samples served as a control for unspecific effects due to the coring procedure. This procedure was carried out in four animals. In a second group of four animals. the order of stimulation was reversed, i.e., the right precruciate cortex was stimulated preceding the second pair of biopsies (2L and 2R) and the left substantia nigra was stimulated preceding the third pair of biopsies. The biopsies ranged in weight from 5 to If mg. The parameters of stimulation were IC V pulses. 0.2 ms in duration each, delivered at 20 Hz for the nigral and cortical stimulating sites. The frozen biopsy samples were weighed and homogenized in a 2 ml volume of IS% formic acid/acetone to which 2 nmol of 2H,-ACh and 2H,-Ch were added as internal standards. Following hom~~genizat~l)n. the samples were centrifuged at 15,000 g for 20 min and analyzed by a gas chrol~~tographic/mass spectrometric method [ Ih]. Brains were removed after perfusion with 1%X formal saline for histofogical verification of the stimulating and sampling sites.

RESULTS

The results from the first series of experiments, in which the substantia nigra was stimulated first, are shown in Table. I. Each set of values represents the mean specific activity of ACh and the standard error of the mean. The specific activity of ACh is defined as the mole fraction of the labeled compound 2H,, -- ACh i.e., 2H,, + 2H,, - AC% ! ’ ( The results show that, after 5 min of unilateral stimulation of the substantia nigra, the specific activity and endogenous level of ACh decreased significantly on the stimulated side from prestimulation controls (Protocol A: compare IL vs. 2L), but not on the nonstimulated side. Cortical stimulation applied during the following 5 min period produced no statistically significant increase in specific activity of ACh on the stimulated side compared to previous levels (compare 2R vs. 3R). On the nonstimulated side (3L), there was a statistically signi~cant increase in the specific activity of ACh after cortical stimulation. This effect may be rebound from the previously decreased specific activity produced by nigral stimulation (2L). A similar “rebound” effect has been noted in the peripheral nervous system after intense stimulation of the superior cervical ganglia of the cat [4] and has also been

Protocol B. n- 4 Pre-Stim.

IL

0.077 ‘. 0.017

IR

0.w

Control Stim. Right

2L

0.063 i- 0.014

1R

0.114 ?I O.Olh

Precr. Cortex Stim. Left

3L

0.050 .+. 0.015

?K

0.072 -Y0.008

Subs.

No

L 0.038

Nigra 4L ON9 f 0.032

JR 0.093 r 0.025

Slim. ‘~~T+O.OS. An analysis of variance was used to assess the effect\ of repeated measures on each group of animals. When statistical sipnificance occurred. the pool error variance was used and the data iubjeeted to post-hoc tests (Dunnet‘s).

reported in the rat caudate nucleus after DFP treatment j7j. The fourth pair of biopsies, taken 5 min poststimulation. showed that the specific activity of ACh had returned to near control levels after 5 min of no stimulation. The results from a second protocol in which the precruciate cortex was stimulated first are also shown in Table I. Stimulation of the right cortex produced a slight. although not statistically significant increase in the specitic activity of ACh on the stimulated side (compare IR vs. 2R). Following stimulation of the left substantia nigra, the decrease in the specific activity of ACh was not statistically significant in the left caudate nucleus (3L) compared to previous values (I I_ and 2L). The effects of stimulation of’the substantia nigra on the specific activity of ACh appears to have been attenuated by the previously occurring contralateral cortical stimulation. The fourth pair of biopsies showed a return to control levels after 5 min without stimulation on the right side (4R). There was an increase, although not significant, in the specific activity of ACh on the left side (4L), again suggesting a rebound effect from previous stimulation of the substantia nigra. The effects of nigral and cortical stimulation on the concentration of labeled ACh (“H,-ACh) are shown in Table 2. Stimulation of the Left substantia nigra produced a marked decrease in the concentration of %I,-ACh in the left caudate nucleus (Table 2, Protocol A; compare IL. vs. 2L). Although there was a tendency for the concentration of ‘H,-ACh to increase after stimulation of the right precruciate cortex, this increase was not statistically significant (3RI. Stimulation of the right cortex (Table 2, Protocol B, 2R) had no effect on the

ACh

SYNTHESIS

AFTER

NIGRAL

TABLE CONCENTRATION

439

2

OF LABELLED ACh (*H,-ACh) PH,,-ACh nmolesi&t) Left Caudate Mean t S.E.

Protocol

STIPULATION

Right Caudate Mean t S.E.

A. n =4

Pre-Stim.

iL

3.063

+ 0.45

1R

2.938

t

2L

0.397

t

2R

2.405

k 0.56

3L

3.581

ct 1.02

3R

4.713

-c 1.81

4L

3.530

i: 0.61

4R

3.354

ir 0.63

IL

2.611 + 0.22

IR

3.543

i

1.04

2L

2,004

ir 0.39

2R

3.007

t

0.65

31~

1.135 r 0.31

3R

2.422

t

0.68

4L

3.674

4R

2.935

t

0.81

0.61

Control Stim.

Left

0.20”

Subs. Nipra

Sum. Right Precr. Cortex N0 Slim. Protocol

B. n-4

Pre-Stim.

Control Slim. Right Subs. Nigra Stim. Left Subs. Nigra No

it 0.87

Slim. “/>~~0.025. An analysis of variance was used to assess the effects of repeated measures on each group of animals. When statistical significance occurred. the pool error variance was used and the data

subjected to post-hoc tests (Dunn~t’s).

concentration of ‘H,,-ACh in the right caudate nucleus. In contrast to results in Table 2A. stimulation of the left substantia nigra was less effective in decreasing the concentration of ‘H,-ACh on the left side (Table 2, Protocol B, 3Lf when it was preceded by cortical stimulation.

The results of this study indicate that: (I) stimulation of the substantia nigra produces a decrease in the specific activity of ACh and the concentration of labeled ACh; (2) these effects are attenuated by previous stimulation of the contralateral cortex; and (3) a rebound increase in both specific activity and labeled ACh occurs after 5 min of nigral stimulation.

The decrease in the concentrations of both endogenous and labeled ACh following stimulation of the substantia nigra suggests that there is either an increase in release of newly synthesized ACh or a decrease in its synthesis. A decrease in synthesis and release would be predicted if DA formed an inhibitory synapse on cholinergic neurons. This would also produce an increase in endogenous levels of ACh. Analogously. an increase in neuronal activity in accordance with an excitatory synapse would be coupled with an increase in Ch uptake. CH acetylation. and release of ACh (see [IS] for discussion). The predicted outcome would then be either a decrease in endogenous ACh levels and an increase in labeled Ch and ACh, or a decrease in both endogenous and newly synthesized ACh. These results are consistent with an excitatory nigrostriatal input to the cholinergic interneuron. In a study examining the effects of a unilateral lesion in the substantia nigra on ACh levels and synthesis fg]. decreases in synthesis were observed also, but levels of endogenous ACh were unaltered. Thus, the decline in endogenous ACh levels observed here suggest that the prolonged stimulation can decrease total ACh stores. Stimulation of the substantia nigra. however, not only produces stimulation of the nigrostriatal pathway. but also antidromic activation of the striatonigral pathway. This latter pathway contains both excitatory and inhibitory neurotransmitters, so that this antidromic stimulation may alter the actual response somewhat. The direct effects of cortical stimulation on caudate ACh activity are not pronounced, but there are indications of an interaction with the nigrostriatal pathway. When cortical stimulation preceded nigral stimulation. the decrease in specific activity and the concentration of labeled ACh induced by stimulation of the substantia nigra was attenuated. This suggests that the response of the cholinergic neuron in the caudate nucleus was altered by the cortical input. In a related study, Nieoullon r’r (I/. [?I] have demonstrated release of DA in the contralateral caudate nucleus after cortical stimulation. These findings may explain the lack of a more pronounced effect on the cholinergic parameters measured in the second series of experiments (Table I B and 2B). Because of the prior release of DA by contralateral cortical stimulation, the additional release of DA produced by ipsilateral nigral stimulatj(~n may no longer be as effective in altering cholinergic activity. Although the effects of nigral stimulation cannot be considered without some qualifications. the results of these experiments do suggest that the nigral input to the caudate nucleus has an excitatory effect on ACh synthesis and release. More interesting, perhaps. is the attenuation of the nigral-induced response by cortical stimulation, suggesting a subtle, yet important. control mechanism.

REFERENCES 1. Anchors, J. M. and E. Garcia-Kill. Dopamine, a modulator of carbohydrate metabolism in the caudate nucleus. Bvcrirz Rcs 133: 183-189, 1977. 2. Ayuilonius. S. M., F. Flentge, J. Schuberth, B. Sparf and A. Sundwall. Synthesis of acetylcholine in different compartments of brain nerve terminals in \,ivo as studied by the incorporation of choline from plasma and the effect of pentobarbitol on this process. .I Nertrr~hrni 20: 1SO9- 152 I, i 973.

3. Bloom. F. E.. E. Costa and G. C. SaJmoiraghi. Anesthesia and the responsiveness of individual neurons of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine administered by microelectrophoresis. .I Phcrr~t~r~ol E.rp 7’/1~r 2: 244-252, 1965. 4 Bourdois. P. S.. D. L. McCandless and R. C. Macintosh. A prolonged after-effect of intense synaptic activity on acetylcholine in a sympathetic ganglion. S’rrfr .I Plrysiol Phrtrmrrc oi 53: i5s-16.5, 1975.

HOWARD

440

5. Butcher.

L. L., editor. C‘/lolirlcrRic,-Monouminrrgic. Infcrtrc.San Francisco: Academic Press. 1978. 6. Butcher. S. H. Effects of dopaminergic and serotonergic agonists and antagonists on acetylcholine levels and synthesis in the neostriatum. In: C‘Aoli,lc,rRic,-MonournirlcrKic, fnf~~r.trc.tiom i/r the Bruin, edited by L. LA.Butcher. San Francisco: Academic Press. 1978, pp. 247-253. 7. Butcher. S. H. and L. L. Butcher. Effects of DFP on neostriatal acetylcholinesterase (ACHE, EC 2. I. 17) and on levels and synthesis of acetylcholine (ACh) and CH (Ch) in the caudate putamen nucleus. Feel Prc>c, 36: 978, 1977. 8. Butcher. S. H.. L. L. Butcher and A. K. Cho. Modulation of neostriatal acetylcholine in the rat by dopamine and S-hydroxytryptamine afferents. L*, Sd 18: 733-744. 1976. 9. Butcher, S. H., M. S. Levine, N. A. Buchwald and C. D. Hull. Interaction of dopamine and acetylcholine in the caudate nucleus of the developing kitten. In: Fourth fn~~~rntr/i~mtr/ (‘arcc~holurnir~c~ Symposium. edited by E. Usdin. New York: Pergamon Press. 1978, pp. 808-802. 10. Connor. J. D. Caudate nucleus neurons: Correlation of the effects of substantia nigra stimulation with iontophoretic dopamine. J Physiol (Land) 209: 691-704, 1970. 11. Consolo, S., H. Ladinsky and S. Garrattini. Effect of several dopaminergic drugs and trihexyphenizine on cholinergic parameters in the rat striatum. J Phurrtz Phurtnuc,o/ 26: 275-277. 1974. 12. Consolo, S.. H. Ladinsky, G. Peri and S. Garrattini. Effect of central stimulants and depressants on mouse brain acetylcholine and choline levels. Eur .I Pharrnucd 18: 25 l-255. 1972. 13. Guyenet, P. Cl.. Y. Agid, F. Janey, J. C. Beaujouan. J. Rossier and J. Glowinski. Effects of dopaminergic receptor agonists and antagonists on the activity of the neo-striatal cholinergic system. Bruin Rtjs 84: 227-244, 1975. 14. Hull, C. D., G. A. Bernardi and N. A. Buchwald. Intracellular responses of caudate neurons of brain stem stimulation. Brtrirl Kc.3 22: 163-179. 1970. tions

in the Bruirl.

AND (J.L\RC’IA-RII.1,

IS. Hull. C. D.. M. S. Levine, N. A. Buchwald, A. lieller 2nd K. A

Browning. The spontaneous tiring patterns of forehraiil neurons. I. The effects of dopamine and nondopamine depleting lesions of caudate unit firing patterns. Urtrrji li(,.\ 73: 241--X?. 1974. 16. Jenden, D. J.. M. Roth and K. A. Booth. Stimultancous ureasurements of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicolmole quantities by gas chromatography/mass spectrometry. Atrul Hitr~,hcr,r 55: 43% 448. 1973. 17. Kitai. S. T.. M. Sugimori and J. D. Kocsis. h~citatory nature of dopamine in the nigrocaudate pathway. f’L\p Hrtrirl Rc.\ 24: 35 I363. 1976. 18. Macintosh. F. C. and B. Collier. Neurochemistry ofcholinergic terminals. In: Harrtlhool, c!f’~.rpc,rir,lc,rrtlrl P/rtrrnlccc,o/~,R~. vol. 42. edited by E. Faimis. New York: Springer-Verlag. 1976. pp. 99-228. 19. McGeer. P. L., D. S. Grewaal and 6. G. McGeer. Extrapyramidalamine interactions. Bruin Rcs 80: 27-48. 1974. 20. McLennan. H. and D. H. York. The action of dopamine on neurons of the caudate nucleus. .I Physird (f.r~ml) 189: 393-402. 1967. 21. Nieoullon, A., A. Cheramy and J. Glowinski. Release ot’dopamine evoked by electrical stimulation of the motor and visual areas of the cerebral cortex in both caudate nuclei and in the substantia nigra in the cat. &trio Rc.v 145: 69-83, 1978. 22. Nordberg, A. Effect of oxotremorine and sodium pentobarbitol on the pharmacokinetics of intravenous tracer doses of rddioactive choline. J Phurrn Phtrrmrrc~rd 29: 9fw98. 1977. 23. Nordberg. A. and A. Sundwall. Effect of sodium pcntobarbitol on the apparent turnover of acetylcholine in different brain regions. A(,ttr Physiol S~rrrtf 99: 336-344, 1976. 24. Siggins, G. R., G. J. Hoffer and U. Ungerstedt. Electrophysiological evidence for involvement of cyclic adenosinc monophosphate in dopamine responses for caudate neurons. Lifi, Sci 15: 779-792. 1974. 2.5, Trabucchi, M.. D. L. Cheney. G. Racagni and E. Costa. f/r ,ii,o inhibition of striatal acetylcholine turnover by L-DOPA. apomorpine and (+)-amphetamine. Bruin Rc\ 85: 13%134. IY7F