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Stimulation of the nucleus basalis of Meynert increases acetylcholine release in the cerebral cortex in rats
Neuroscience Letters, 98 (1989) 45 50
45
Elsevier Scientific Publishers Ireland Ltd.
NSL 05914
Stimulation of the nucleus basalis of Meynert increases acetylcholine release in the cerebral cortex in rats M i e k o K u r o s a w a , A k i o Sato and Y u k o Sato D~7~arlment o/'Physioh~gy, Tol~-yoMetropolitan Institute q/'Gerontolo~v, Tokyo (Japan/ (Received 21 October I988; Accepted 31 October 1988) Key word~:
Acetylcholinc; Release ofacetylcholine: Nucleus basalis of Meynert; Focal slimulation: Cerebral cortex; Parietal lobe; In vivo microdialysis; Rat
The effect of focal slimulation of Ihe magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM) on acetylcholine (ACh) release in the cerebral cortex in the parietal lobc v~as examined m halothane-anesthctizcd rats. A C h was measured using a microdialysis method. Focal elcctrical stimula tion of the unilateral NBM increased ACh release in the ipsilateral cerebral cortex in stimulus intensiL~ and frequent? dcpendently. Microinjection of L-glutamate (I00 nmol) into the unilateral NBM also increased A('fi release in the ipsilateral cerebral cortex. The ACh release in the contralateral cerebral cortex ~ a s not affected by these unilateral stimulations of the NBM. It was concluded that fi)cal stimulatiola of the N BM releases ACh from cortical terminals of cholinergic fibers originating in the N BM.
In the preceding paper [1], we demonstrated that a focal stimulation of the unilateral nucleus basalis of Meynert (NBM), either electrically or chemically, produced an increase in cerebral cortical blood flow in the parietal lobe. Further, we suggested that the majority of the increased response of the cerebral cortical blood flow could be attributed to the increased release of acetylcholine (ACh) from the cortical nerve terminals of cholinergic nerve fibers originating in the NBM, since these responses were almost nonexistent after intravenous injections of muscarinic and nicotinic cholinergic blocking agents (i.e. atropine and mecamylamine). It has been reported that the NBM sends its cholinergic fiber projections to the cortex in a specific topographical arrangement [11]. However, the preceding study [1] did not determine whether focal stimulation of the NBM actually releases ACh from the nerve terminals of those chofinergic projecting fibers within the cerebral cortex. Recently, Ungerstedt et al. [10] developed an in vivo microdialysis technique to measure chemical substances within the brain using a small probe made of a dialyzing membrane. ACh and catecholamine released in vivo in the brain and measured using this dialysis techCorre.~pondence. A. Sato, Department of Physiology, Tokyo Metropolitan Institute of Gerontology. 35-2 Sakaecfio, ltabashiku, Tokyo 173, Japan. 0304-3940,89S 03.50 i': 1989 Elsevier Scientific Publishers Ireland Ltd.
46 nique have been r e p o r t e d to d e p e n d o n the release o f these chemicals f r o m nerve terminals in the b r a i n [2, 5-7], T h e p r e s e n t e x p e r i m e n t was c a r r i e d o u t to d e t e r m i n e w h e t h e r the e x t r a c e l l u l a r A C h release in the cerebral cortex o f the p a r i e t a l lobe is increased by focal s t i m u l a t i o n o f the N B M . The e x p e r i m e n t s were p e r f o r m e d o n 21 a d u l t Fischer-344 rats (350-440 g) anesthetized with 1.0% h a l o t h a n e ( T a k e d a ) . All o t h e r general e x p e r i m e n t a l p r o c e d u r e s with r e g a r d to m a i n t e n a n c e o f r e s p i r a t i o n , b o d y t e m p e r a t u r e a n d m o n i t o r i n g o f b l o o d pressure were similar to those d e s c r i b e d in the p r e c e d i n g p a p e r [1]. T h e a n i m a l s were m o u n t e d in a p r o n e p o s i t i o n on a stereotaxic i n s t r u m e n t (SR-5, Narishige), a n d the skull was o p e n e d . E x t r a c e l l u l a r A C h was collected using the m i c r o d i a l y s i s technique o f U n g e r s t e d t et al. [10]. A c o a x i a l m i c r o d i a l y s i s p r o b e c o v e r e d b y a d i a l y z i n g m e m b r a n e 3.0 m m in length a n d 0.5 m m in d i a m e t e r ( C M A / 1 0 , C a r n e g i e M e d i c i n ) with b o t h an inlet a n d a n o u t l e t was inserted into the p a r i e t a l c o r t e x with a lateral inclination o f 30 ° to a d e p t h o f 3.5 m m f r o m the cortical surface at 0.2 m m a n t e r i o r to the EHC 0.1nA
inj. 0.2mm anterior to bregma
I
I
I
I
I
0
2
4
6
8 10min
electrical stim. e
C
C
2 0 0 I J A , 5 0 H z , 10min
F=
glutamate
o o
100nmol (100nl)
1.0
1.0
--•
E 0.5 ¢.-
L~ ,<
I
m
0.5
0 !
-30
I
I
!
0
I
30 time
I
I
I
60 min
I
-30
!
I
I
0
I
I
30 min time
Fig. 1. A: diagram of the experiment demonstrating the microdialysis probe in the cerebral parietal cortex (left), and the chromatogram of 20/zl of mixed solution containing sample and internal standard (right). The ACh and EHC peaks represent 0.7 pmol and 1,3 pmot measured by ECD after separation by HPLC. B, C: effects of focal stimulation of the unilateral NBM, either electrically (B) or chemically (C), on extracellular ACh release in the parietal cortex ipsilateral to the stimulation. The amount of ACh release in the perfusate every 10 min, measured by the mierodialysis technique, is plotted on the ordinate. Onset of either stimulation to the NBM expressed as time zero on the abscissa. B: electrical stimulation (200 /~A, 0.5 ms, 50 Hz) for 10 min, as indicated by the upper horizontal bar, in one rat. C: microinjection of L-glutamate (100 nmol in 100 nl) for 1 min as indicated by the upper triangle, in another rat.
47
bregma and 4 mm lateral from the midline according to the rat brain stereotaxic coordinates outlined in the atlas of Paxinos and Watson [8] as shown in Fig. 1A (left). The coaxial probe was perfused at a rate of 2/~l/min with saline containing physostigmine (100 ¢tM) to inhibit acetylcholinesterase through the inlet using a microinjection pump (CMA/100, Carnegie Medicin). The recovery rate of ACh by individual microdialysis probe varied between 15 and 23%. Thus, the recovery rates of ACh obtained using different probes were adjusted to 20% by calculation after comparing the ACh concentrations in different animals. Once stabilization of ACh concentration was attained about 1 h after the insertion of the probe into the cortex and the initiation of the perfusion, the perfusate was collected every 10 rain into a sample tube kept in an ice-cooled bath. Each sample (20 Itl) was mixed with 10 Itl of 0.02 M citrate phosphate buffer (pH; 3.5) containing 2 pmol of ethylhomocholine (EHC) [9] as an internal standard. ACh was principally measured by the method of high-performance liquid chromatography (HPLC) using electrochemical detection described by Potter et al. [9]. A mobile phase (pH =8.3) composed of 50 mM disodium phosphate, 1 mM EDTA.2Na and 50/~M octyl sodium sulfate (Kodak) was delivered by the pump (L6200, Hitachi) at a flow rate of 0.8 ml/min. First, ACh was separated using a polymeric reversed-phase column (4 x 60 ram, 51-0564, BAS, Japan) and then converted to hydrogen peroxide and betaine by immobilized acetylcholinesterase and choline oxidase packed into a column (4 x 5 ram, 51-0554, BAS, Japan [3]). Then, hydrogen peroxide was measured using an electrochemical detector (ECD: LC-4B, Bioanalytical Systems). The platinum working electrode of the ECD was held at 500 mV vs Ag/AgCI. Both the separation column and enzyme column were maintained at 3 7 C using a column heater (LC-22A/23B, Bioanalytical Systems). Twenty/11 out of the total 30/4 of mixed solutions was injected into the HPLC system. Sample chromatogram measuring ACh was shown in Fig. 1A (right). The coefficients of variation for determination of the amount of perfusate containing 2 pmol ACh were 4.6%. The minimum detection limit for ACh was approximately 0.1 pmol. The ACh release was calculated using the concentration of ACh in the perfusate and the perfusion rate. The unilateral NBM was focally stimulated, either electrically or chemically, by the same method described in the preceding paper [!]. After the experiments, the brains were dissected and the positions of the tips of both the stimulating electrode (or microinjection needle) and the microdialysis probe were examined histologically via brain slice sections [1]. Extracellular ACh release in the cerebral cortex in the parietal lobe measured every 10 rain under resting conditions in all the animals tested ranged from 0.5 to 1.0 pmol/ 10 min. In each animal amount of ACh release under resting conditions was quite stable. Fig. I B demonstrates a typical response of extracellular ACh release in the cerebral cortex in the parietal lobe ipsilateral to the focal electrical stimulation of the unilateral NBM with an intensity of 200/~A at 50 Hz (0.5 ms pulse duration) for 10 rain. In this example, the ACh release under resting conditions was approximately 0.64).7 pmol/10 min. During the stimulation, the ACh release reached about 1.3 pmol/10 rain. After cessation of the stimulation, the ACh decreased to about 1.1 pmol/10 rain for the next 10 rain, and then returned to the prestimulus control level.
48
Similar results were observed in 5 additional rats. The same stimulation of the unilateral NBM did not produce any response of the extracellular ACh in the contralateral parietal cortex. Fig. IC demonstrates typical response in one rat of the extracelluli~r ACh release in the ipsilateral parietal cortex following a microinjection of L-glutamate (100 nmol in 100 nl, Wako Pure Chemicals) for 1 rain into the unilateral NBM. In this rat, the ACh release under the resting conditions was between 0.6 and 0.7 pmol/10 rain. which increased to about 0.9 pmol/10 min during the first 10 min following the microinjection of L-glutamate into the NBM. The ACh release then returned to the prestimulus control level for the next 10 min. Increased responses of ACh release simihtr to this were obtained in two additional rats. Stimulation using L-glutamate is considered to excite only the cell bodies within the NBM. As it is generally accepted that this amount of L-glutamate is able to excite enough cells around the tip of the microinjection needle [4], we did not attempt to use larger doses of L-glutamate. Fig. 2A summarizes the responses of the extracellular ACh release in the parietal cortex to the focal electrical stimulation of the NBM with different stimulus intensities (at constant frequency of 50 Hz) tested in 6 rats. In all 6 rats, no increase in the ACh release was evident during 20/IA stimulation. In 3 of the 6 rats, 50/tA was effective in producing an increase in ACh release, but the total responses in all 6 rats were insignificant. A stimulus intensity of 100/~A produced an increase in ACh release in all 6 rats tested (P<0.05). With further increases in intensity to 200 and 500 ItA, larger increases in ACh release were produced (P < 0.01 ).
1.5
A
o
E
1.0 o.5
o)
g
o
<
control
2O
50 100 stimulus intensity
500 ,uA
200 :¢
E ~ 1.5
o
+
B
E
~ 1.0 o.s g
0 control
1
2
5 10 20 stimulus f r e q u e n c y
50
lOO Hz
Fig. 2. Effects of various intensities in A ( n = 6) and frequencies in B (n = 6) of electrical stimulation of the unilateral NBM on extracellular ACh release in the parietal cortex ipsilateral to the stimulation. A: stimulus frequency, 50 ttz. B: stimulus intensity, 200/tA. ACh release in the perfusate during stimulation for 10 min was summarized as the mean±S.E.M. ACh responses to the stimulation were compared with ACh in rcsling control condition using thc paired/-test. *P<0.05, **P<0,01.
49
Fig. 2B summarizes the responses of the extracellular ACh release in the parietal cortex to the electrical stimulation of the N B M with various stimulus frequencies at a constant intensity of 200 #A in a second group of 6 rats. The ACh release was significantly increased by stimulation with frequencies above 5 Hz and finally to 100 Hz. At frequencies between 20 and 50 Hz, the responses were the largest and reached approximately two times the prestimulus control ACh release. At 100 Hz, the increased responses were slightly reduced. As it was necessary in the present in vivo experiments to use physostigmine, an inhibitor of acetylcholinesterase, the absolute value of the present cortical ACh may differ from the physiological value of ACh. In fact, D a m s m a et al. [2] reported that the ACh in the striata measured by the microdialysis method was increased in a dosedependent manner by a concentration of neostigmine in the perfusate. In the physiological condition without application ofphysostigmine (or neostigmine), extracellular ACh must be destroyed very rapidly by acetylcholinesterase. Nevertheless, it should still be emphasized that the extracellular ACh release increased significantly in response to local stimulation of the N B M in anesthetized, artificially ventilated animals. It is evident that the increase in the ACh release to the NBM stimulation found in the present study is attributed to increased secretion from the cortical terminals of the cholinergic nerve fibers originating in the NBM. Considering the preceding evidence [1] that the cerebral cortical blood flow increases following focal stimulation of the N BM, and that the majority of the increased response is abolished by intravenous administration of cholinergic blocking agents, it is suggested that the increased release of ACh t¥om the cholinergic nerve terminals in the cerebral cortex following the stimulation of the NBM is an important factor in producing the increase in cerebral cortical blood flow. It is most likely that ACh released from the cholinergic terminals acts as a potent vasodilator substance in the cortical vasodilating system. The present cholinergic nerve does not belong to the traditional autonomic nerves, however it behaves as the traditional autonomic nerves do with respect to the regulation of blood vessels. This work was partly supported by a Grant-in-Aid for Scientific Research (B) No. 63480102 from the Ministry of Education, Scientific and Culture, and research grants from the Ministry of Health and Welfare of Japan. I Biesold, D., lnanalni, O., Sato, A. and Sato, Y., Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats, Neurosci. Lett., 98 (1989) 41 46. 2 lOanlsma, G., Westerink, B.H.C.. de Vries, J.B., Vail den Berg, C.J. and Horn, A.S., Measurement of acelylcholine release in freely moving rats by means of automated inlraccrebral dial,,sis..1. Ncurochem., 48 (I987) 1523 1528. 3 l:ujimori, K. and Y a m a m o l o , K., l)elermination of acctylcholine and choline in perchloratc extracts of brain lissue using liquid chromatography-electrochemistry with an immobilized-enzyme reactor. J. Chronlatogr., 414 (1987) 167 173. 4 Goodchild, A.K., Dampney, R . A i . and Bandler, R., A method [klr evoking physiological responses by stimulation of cell bodies, but no! axons of passage, within localized regions of the central llervotls system, J. Ncurosci. Methods, 6(1982)351 363. 5 hnpcrato. A. and Di Chiara, G., Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with clectrochemical detection: at new method for the study of the m viw~ release of endogenous dopamine and metabolites, J. Neurosci., 4 (1984) 966 977.
50 6 L'Heureux, R., Dennis, T., Curet, O. and Scatton, B., Measurement of endogenous noradrenaline release in the rat cerebral cortex in vivo by transcortical dialysis: effects of drugs affecting noradrenergic transmission, J. Neurochem., 46 (1986) 1794-1801. 7 Maysinger, D., Herrera-Marschitz, M., Carlsson, A., Garofalo, L., CueUo, A.C. and Ungerstedt, U., Striatal and cortical acetylchotine release in vivo in rats with unilateral decortication: effects of trealment with monosialoganglioside GM1, Brain Res., 461 (1988) 355-360. 8 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic, Sydney, 1986. 9 Potter, P.E., Meek, J.L. and Neff, N.H., Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection, J. Neurochem., 41 (1983) 188 194. 10 Ungerstedt, U., Herrera-Marschitz, M,, Jungnelius, U., Stfi,hle, L., Tossmann, U., and Zetterstr6m, T., Dopamine synaptic mechanisms reflected in studies combining behavioural recordings and brain dialysis. In M. Kohsaka, T. Shohmori, Y. Tsukada and G.N. Woodruff(Eds.), Advances in Dopamine Research, Advances in the Biosciences, Vol. 37, Pergamon, Oxford, 1982, pp. 219--231. 11 Wenk, H., Bigl, V. and Meyer, U., Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Rev., 2 (1980) 295-316.
45
Elsevier Scientific Publishers Ireland Ltd.
NSL 05914
Stimulation of the nucleus basalis of Meynert increases acetylcholine release in the cerebral cortex in rats M i e k o K u r o s a w a , A k i o Sato and Y u k o Sato D~7~arlment o/'Physioh~gy, Tol~-yoMetropolitan Institute q/'Gerontolo~v, Tokyo (Japan/ (Received 21 October I988; Accepted 31 October 1988) Key word~:
Acetylcholinc; Release ofacetylcholine: Nucleus basalis of Meynert; Focal slimulation: Cerebral cortex; Parietal lobe; In vivo microdialysis; Rat
The effect of focal slimulation of Ihe magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM) on acetylcholine (ACh) release in the cerebral cortex in the parietal lobc v~as examined m halothane-anesthctizcd rats. A C h was measured using a microdialysis method. Focal elcctrical stimula tion of the unilateral NBM increased ACh release in the ipsilateral cerebral cortex in stimulus intensiL~ and frequent? dcpendently. Microinjection of L-glutamate (I00 nmol) into the unilateral NBM also increased A('fi release in the ipsilateral cerebral cortex. The ACh release in the contralateral cerebral cortex ~ a s not affected by these unilateral stimulations of the NBM. It was concluded that fi)cal stimulatiola of the N BM releases ACh from cortical terminals of cholinergic fibers originating in the N BM.
In the preceding paper [1], we demonstrated that a focal stimulation of the unilateral nucleus basalis of Meynert (NBM), either electrically or chemically, produced an increase in cerebral cortical blood flow in the parietal lobe. Further, we suggested that the majority of the increased response of the cerebral cortical blood flow could be attributed to the increased release of acetylcholine (ACh) from the cortical nerve terminals of cholinergic nerve fibers originating in the NBM, since these responses were almost nonexistent after intravenous injections of muscarinic and nicotinic cholinergic blocking agents (i.e. atropine and mecamylamine). It has been reported that the NBM sends its cholinergic fiber projections to the cortex in a specific topographical arrangement [11]. However, the preceding study [1] did not determine whether focal stimulation of the NBM actually releases ACh from the nerve terminals of those chofinergic projecting fibers within the cerebral cortex. Recently, Ungerstedt et al. [10] developed an in vivo microdialysis technique to measure chemical substances within the brain using a small probe made of a dialyzing membrane. ACh and catecholamine released in vivo in the brain and measured using this dialysis techCorre.~pondence. A. Sato, Department of Physiology, Tokyo Metropolitan Institute of Gerontology. 35-2 Sakaecfio, ltabashiku, Tokyo 173, Japan. 0304-3940,89S 03.50 i': 1989 Elsevier Scientific Publishers Ireland Ltd.
46 nique have been r e p o r t e d to d e p e n d o n the release o f these chemicals f r o m nerve terminals in the b r a i n [2, 5-7], T h e p r e s e n t e x p e r i m e n t was c a r r i e d o u t to d e t e r m i n e w h e t h e r the e x t r a c e l l u l a r A C h release in the cerebral cortex o f the p a r i e t a l lobe is increased by focal s t i m u l a t i o n o f the N B M . The e x p e r i m e n t s were p e r f o r m e d o n 21 a d u l t Fischer-344 rats (350-440 g) anesthetized with 1.0% h a l o t h a n e ( T a k e d a ) . All o t h e r general e x p e r i m e n t a l p r o c e d u r e s with r e g a r d to m a i n t e n a n c e o f r e s p i r a t i o n , b o d y t e m p e r a t u r e a n d m o n i t o r i n g o f b l o o d pressure were similar to those d e s c r i b e d in the p r e c e d i n g p a p e r [1]. T h e a n i m a l s were m o u n t e d in a p r o n e p o s i t i o n on a stereotaxic i n s t r u m e n t (SR-5, Narishige), a n d the skull was o p e n e d . E x t r a c e l l u l a r A C h was collected using the m i c r o d i a l y s i s technique o f U n g e r s t e d t et al. [10]. A c o a x i a l m i c r o d i a l y s i s p r o b e c o v e r e d b y a d i a l y z i n g m e m b r a n e 3.0 m m in length a n d 0.5 m m in d i a m e t e r ( C M A / 1 0 , C a r n e g i e M e d i c i n ) with b o t h an inlet a n d a n o u t l e t was inserted into the p a r i e t a l c o r t e x with a lateral inclination o f 30 ° to a d e p t h o f 3.5 m m f r o m the cortical surface at 0.2 m m a n t e r i o r to the EHC 0.1nA
inj. 0.2mm anterior to bregma
I
I
I
I
I
0
2
4
6
8 10min
electrical stim. e
C
C
2 0 0 I J A , 5 0 H z , 10min
F=
glutamate
o o
100nmol (100nl)
1.0
1.0
--•
E 0.5 ¢.-
L~ ,<
I
m
0.5
0 !
-30
I
I
!
0
I
30 time
I
I
I
60 min
I
-30
!
I
I
0
I
I
30 min time
Fig. 1. A: diagram of the experiment demonstrating the microdialysis probe in the cerebral parietal cortex (left), and the chromatogram of 20/zl of mixed solution containing sample and internal standard (right). The ACh and EHC peaks represent 0.7 pmol and 1,3 pmot measured by ECD after separation by HPLC. B, C: effects of focal stimulation of the unilateral NBM, either electrically (B) or chemically (C), on extracellular ACh release in the parietal cortex ipsilateral to the stimulation. The amount of ACh release in the perfusate every 10 min, measured by the mierodialysis technique, is plotted on the ordinate. Onset of either stimulation to the NBM expressed as time zero on the abscissa. B: electrical stimulation (200 /~A, 0.5 ms, 50 Hz) for 10 min, as indicated by the upper horizontal bar, in one rat. C: microinjection of L-glutamate (100 nmol in 100 nl) for 1 min as indicated by the upper triangle, in another rat.
47
bregma and 4 mm lateral from the midline according to the rat brain stereotaxic coordinates outlined in the atlas of Paxinos and Watson [8] as shown in Fig. 1A (left). The coaxial probe was perfused at a rate of 2/~l/min with saline containing physostigmine (100 ¢tM) to inhibit acetylcholinesterase through the inlet using a microinjection pump (CMA/100, Carnegie Medicin). The recovery rate of ACh by individual microdialysis probe varied between 15 and 23%. Thus, the recovery rates of ACh obtained using different probes were adjusted to 20% by calculation after comparing the ACh concentrations in different animals. Once stabilization of ACh concentration was attained about 1 h after the insertion of the probe into the cortex and the initiation of the perfusion, the perfusate was collected every 10 rain into a sample tube kept in an ice-cooled bath. Each sample (20 Itl) was mixed with 10 Itl of 0.02 M citrate phosphate buffer (pH; 3.5) containing 2 pmol of ethylhomocholine (EHC) [9] as an internal standard. ACh was principally measured by the method of high-performance liquid chromatography (HPLC) using electrochemical detection described by Potter et al. [9]. A mobile phase (pH =8.3) composed of 50 mM disodium phosphate, 1 mM EDTA.2Na and 50/~M octyl sodium sulfate (Kodak) was delivered by the pump (L6200, Hitachi) at a flow rate of 0.8 ml/min. First, ACh was separated using a polymeric reversed-phase column (4 x 60 ram, 51-0564, BAS, Japan) and then converted to hydrogen peroxide and betaine by immobilized acetylcholinesterase and choline oxidase packed into a column (4 x 5 ram, 51-0554, BAS, Japan [3]). Then, hydrogen peroxide was measured using an electrochemical detector (ECD: LC-4B, Bioanalytical Systems). The platinum working electrode of the ECD was held at 500 mV vs Ag/AgCI. Both the separation column and enzyme column were maintained at 3 7 C using a column heater (LC-22A/23B, Bioanalytical Systems). Twenty/11 out of the total 30/4 of mixed solutions was injected into the HPLC system. Sample chromatogram measuring ACh was shown in Fig. 1A (right). The coefficients of variation for determination of the amount of perfusate containing 2 pmol ACh were 4.6%. The minimum detection limit for ACh was approximately 0.1 pmol. The ACh release was calculated using the concentration of ACh in the perfusate and the perfusion rate. The unilateral NBM was focally stimulated, either electrically or chemically, by the same method described in the preceding paper [!]. After the experiments, the brains were dissected and the positions of the tips of both the stimulating electrode (or microinjection needle) and the microdialysis probe were examined histologically via brain slice sections [1]. Extracellular ACh release in the cerebral cortex in the parietal lobe measured every 10 rain under resting conditions in all the animals tested ranged from 0.5 to 1.0 pmol/ 10 min. In each animal amount of ACh release under resting conditions was quite stable. Fig. I B demonstrates a typical response of extracellular ACh release in the cerebral cortex in the parietal lobe ipsilateral to the focal electrical stimulation of the unilateral NBM with an intensity of 200/~A at 50 Hz (0.5 ms pulse duration) for 10 rain. In this example, the ACh release under resting conditions was approximately 0.64).7 pmol/10 min. During the stimulation, the ACh release reached about 1.3 pmol/10 rain. After cessation of the stimulation, the ACh decreased to about 1.1 pmol/10 rain for the next 10 rain, and then returned to the prestimulus control level.
48
Similar results were observed in 5 additional rats. The same stimulation of the unilateral NBM did not produce any response of the extracellular ACh in the contralateral parietal cortex. Fig. IC demonstrates typical response in one rat of the extracelluli~r ACh release in the ipsilateral parietal cortex following a microinjection of L-glutamate (100 nmol in 100 nl, Wako Pure Chemicals) for 1 rain into the unilateral NBM. In this rat, the ACh release under the resting conditions was between 0.6 and 0.7 pmol/10 rain. which increased to about 0.9 pmol/10 min during the first 10 min following the microinjection of L-glutamate into the NBM. The ACh release then returned to the prestimulus control level for the next 10 min. Increased responses of ACh release simihtr to this were obtained in two additional rats. Stimulation using L-glutamate is considered to excite only the cell bodies within the NBM. As it is generally accepted that this amount of L-glutamate is able to excite enough cells around the tip of the microinjection needle [4], we did not attempt to use larger doses of L-glutamate. Fig. 2A summarizes the responses of the extracellular ACh release in the parietal cortex to the focal electrical stimulation of the NBM with different stimulus intensities (at constant frequency of 50 Hz) tested in 6 rats. In all 6 rats, no increase in the ACh release was evident during 20/IA stimulation. In 3 of the 6 rats, 50/tA was effective in producing an increase in ACh release, but the total responses in all 6 rats were insignificant. A stimulus intensity of 100/~A produced an increase in ACh release in all 6 rats tested (P<0.05). With further increases in intensity to 200 and 500 ItA, larger increases in ACh release were produced (P < 0.01 ).
1.5
A
o
E
1.0 o.5
o)
g
o
<
control
2O
50 100 stimulus intensity
500 ,uA
200 :¢
E ~ 1.5
o
+
B
E
~ 1.0 o.s g
0 control
1
2
5 10 20 stimulus f r e q u e n c y
50
lOO Hz
Fig. 2. Effects of various intensities in A ( n = 6) and frequencies in B (n = 6) of electrical stimulation of the unilateral NBM on extracellular ACh release in the parietal cortex ipsilateral to the stimulation. A: stimulus frequency, 50 ttz. B: stimulus intensity, 200/tA. ACh release in the perfusate during stimulation for 10 min was summarized as the mean±S.E.M. ACh responses to the stimulation were compared with ACh in rcsling control condition using thc paired/-test. *P<0.05, **P<0,01.
49
Fig. 2B summarizes the responses of the extracellular ACh release in the parietal cortex to the electrical stimulation of the N B M with various stimulus frequencies at a constant intensity of 200 #A in a second group of 6 rats. The ACh release was significantly increased by stimulation with frequencies above 5 Hz and finally to 100 Hz. At frequencies between 20 and 50 Hz, the responses were the largest and reached approximately two times the prestimulus control ACh release. At 100 Hz, the increased responses were slightly reduced. As it was necessary in the present in vivo experiments to use physostigmine, an inhibitor of acetylcholinesterase, the absolute value of the present cortical ACh may differ from the physiological value of ACh. In fact, D a m s m a et al. [2] reported that the ACh in the striata measured by the microdialysis method was increased in a dosedependent manner by a concentration of neostigmine in the perfusate. In the physiological condition without application ofphysostigmine (or neostigmine), extracellular ACh must be destroyed very rapidly by acetylcholinesterase. Nevertheless, it should still be emphasized that the extracellular ACh release increased significantly in response to local stimulation of the N B M in anesthetized, artificially ventilated animals. It is evident that the increase in the ACh release to the NBM stimulation found in the present study is attributed to increased secretion from the cortical terminals of the cholinergic nerve fibers originating in the NBM. Considering the preceding evidence [1] that the cerebral cortical blood flow increases following focal stimulation of the N BM, and that the majority of the increased response is abolished by intravenous administration of cholinergic blocking agents, it is suggested that the increased release of ACh t¥om the cholinergic nerve terminals in the cerebral cortex following the stimulation of the NBM is an important factor in producing the increase in cerebral cortical blood flow. It is most likely that ACh released from the cholinergic terminals acts as a potent vasodilator substance in the cortical vasodilating system. The present cholinergic nerve does not belong to the traditional autonomic nerves, however it behaves as the traditional autonomic nerves do with respect to the regulation of blood vessels. This work was partly supported by a Grant-in-Aid for Scientific Research (B) No. 63480102 from the Ministry of Education, Scientific and Culture, and research grants from the Ministry of Health and Welfare of Japan. I Biesold, D., lnanalni, O., Sato, A. and Sato, Y., Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats, Neurosci. Lett., 98 (1989) 41 46. 2 lOanlsma, G., Westerink, B.H.C.. de Vries, J.B., Vail den Berg, C.J. and Horn, A.S., Measurement of acelylcholine release in freely moving rats by means of automated inlraccrebral dial,,sis..1. Ncurochem., 48 (I987) 1523 1528. 3 l:ujimori, K. and Y a m a m o l o , K., l)elermination of acctylcholine and choline in perchloratc extracts of brain lissue using liquid chromatography-electrochemistry with an immobilized-enzyme reactor. J. Chronlatogr., 414 (1987) 167 173. 4 Goodchild, A.K., Dampney, R . A i . and Bandler, R., A method [klr evoking physiological responses by stimulation of cell bodies, but no! axons of passage, within localized regions of the central llervotls system, J. Ncurosci. Methods, 6(1982)351 363. 5 hnpcrato. A. and Di Chiara, G., Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with clectrochemical detection: at new method for the study of the m viw~ release of endogenous dopamine and metabolites, J. Neurosci., 4 (1984) 966 977.
50 6 L'Heureux, R., Dennis, T., Curet, O. and Scatton, B., Measurement of endogenous noradrenaline release in the rat cerebral cortex in vivo by transcortical dialysis: effects of drugs affecting noradrenergic transmission, J. Neurochem., 46 (1986) 1794-1801. 7 Maysinger, D., Herrera-Marschitz, M., Carlsson, A., Garofalo, L., CueUo, A.C. and Ungerstedt, U., Striatal and cortical acetylchotine release in vivo in rats with unilateral decortication: effects of trealment with monosialoganglioside GM1, Brain Res., 461 (1988) 355-360. 8 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic, Sydney, 1986. 9 Potter, P.E., Meek, J.L. and Neff, N.H., Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection, J. Neurochem., 41 (1983) 188 194. 10 Ungerstedt, U., Herrera-Marschitz, M,, Jungnelius, U., Stfi,hle, L., Tossmann, U., and Zetterstr6m, T., Dopamine synaptic mechanisms reflected in studies combining behavioural recordings and brain dialysis. In M. Kohsaka, T. Shohmori, Y. Tsukada and G.N. Woodruff(Eds.), Advances in Dopamine Research, Advances in the Biosciences, Vol. 37, Pergamon, Oxford, 1982, pp. 219--231. 11 Wenk, H., Bigl, V. and Meyer, U., Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Rev., 2 (1980) 295-316.