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Stimulation of the rat dorsal raphe in vivo releases labeled serotonin from the parietal cortex
Brain Research, 216 (1981) 351-360 O Elsevier/North-Holland Biomedical Press
351
S T I M U L A T I O N OF T H E RAT D O R S A L R A P H E IN VIVO RELEASES LABELED S E R O T O N I N F R O M T H E P A R I E T A L C O R T E X
HIROSHI FUJIWARA, MASAHARU UEMOTO and CHIKAKO TANAKA* Department of Pharmacology, Kobe University School of Medicine, Kobe 650 (Japan)
(Accepted November 27th, 1980) Key words: in vivo 5-HT release -- rat parietal cortex - - dorsal raphe stimulation
SUMMARY In vivo release of labeled serotonin ([aH]5-HT) from the parietal cortex was investigated by cortical cup technique and electrical stimulation of midbrain raphe in rats anesthetized with pentobarbital sodium. The spontaneous efflux of tritium from the parietal cortex preloaded with [3H]5-HT followed a multiphasic exponential course. After 120 min, the rate of etttux appeared to fit the single exponential function (slow phase). Imipramine (10-6-10 -8 M) produced a dose-dependent increase in the spontaneous release. When pargyline in concentrations ranging from 10-4 to 10-3 M were added to the medium in the cup, the unchanged [aH]5-HT significantly increased in a dose-dependent manner and the slow declining coefficient of tritium efflux significantly decreased in the presence of 10-4 pargyline. Stimulation of the rostral two-thirds of the dorsal raphe and the lateral 5-HT bundle originating from the dorsal raphe significantly increased the release of [aH]5-HT and its metabolites while stimulation of the caudal one-third of the dorsal raphe did not produce a significant increase in the release of [3H]5-HT and its metabolites. Stimulation of the median raphe produced no or only a slight increase in the release of [3H]5-HT and its metabolites. These findings are a direct demonstration of the in vivo release of [3H]5H T from the parietal cortex with stimulation of the dorsal raphe, particularly the rostral two-thirds of the nucleus and provide the neurochemical evidence for the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle. INTRODUCTION Fluorescence histochemical studies demonstrated that the perikarya of serotonin (5-hydroxytryptamine; 5-HT) containing neurons are present almost exclusively in * To whom correspondence should be addressed.
352 the raphe nuclei 10 and that the 5-HT neuron in the midbrain raphe projects into the forebrain2,14. Aghajanian et al. 1,1s suggested that 5-HT can be released via a specific neural pathway, projecting into the forebrain, as based on neurochemical findings that electrical stimulation of the midbrain raphe in the rat produced an increase in the levels of 5-hydroxyindole acetic acid (5-HIAA) and a decrease in 5-HT content in the forebrain 1,24. This concept was also supported by reports that selective destruction of the midbrain raphe produced a decrease in 5-HT levels 7,1s-20,2z, histofluorescence intensity of 5-HT 18, tryptophan hydroxylase activity18,19 and synaptosomal uptake of 5-HT 1s,19. However, these findings are indirect evidence for the release of forebrain 5HT on stimulation of the midbrain raphe. The in vivo release of central transmitters can be estimated in perfusates of the lateral ventricle as obtained from superfusates from the push-pull cannula inserted into the deep structures and from a cup placed on superficial areas such as the cerebral cortex. The in vivo experiments on 5-HT release were performed mainly in the cat 3,4,12,13,16 and in the monkey6, 22 and the rat 26. As direct estimation of the cortical 5-HT release on stimulation of the midbrair, raphe has apparently not been done in the rat, we carried out such experiments using the cortical cup technique. MATERIALS AND METHODS Male Wistar rats, weighing 250-350 g were anesthetized with pentobarbital sodium 30 mg/kg i.p. and fixed in a stereotaxic apparatus. The skull was removed from 2 mm posterior to bregma and 4 m m lateral to midline. The overlying dura was reflected and the parietal cortex was exposed. A cylindrical plastic cup (4.5 m m in internal diameter, 8 m m in height) was placed on the exposed cortex and sealed with surgical adhesive. The cup was filled with modified Krebs-phosphate solution containing E D T A (disodium salt, 10 mg/1) and ascorbic acid (20 rag/l). This solution was gassed with 95 ~o 02-5 ~/o CO2 at 37 °C before addition to the cup. The parietal cortex was incubated for 1 h with [1,2-aH]serotonin creatinine sulfate ([aH]5-HT; 21.9 Ci/mmol) dissolved in modified Krebs-phosphate solution. After incubation, the medium was removed and the cortex in the cup was washed 5 times with fresh Krebs-phosphate solution. The cup was then filled with 0.05 ml of Krebs-phosphate solution. Samples were collected at 10-rain intervals. Total radioactivity was counted in a Packard Tri-carb liquid scintillation spectrometer and the radioactivity was expressed as the number of count per rain (cpm).
Separation of unchanged [3H]5-HTfrom tritium efflux Six samples collected at 10-min intervals for a period of 5 h were pooled. Half of the volume of the samples was used to separate unchanged [3H]5-HT and the remaining sample was prepared to estimate the total radioactivity. To the sample was added 0.5 #g of unlabeled 5-HT as the carrier and 0.1 N HC1 containing 0.1 ~ ascorbic acid and the preparation was then frozen until the separation. 5-HT was removed from the sample by extraction into n-butanol at a pH 10. n-Heptane was added to the butanol extract and the 5-HT returned to 0.1 N HC1. The radioactivity of [ZH]5-HT in 0.1 N HC1 was counted.
353
Electrical stimulation For stimulation of the dorsal raphe, the median raphe and the ascending lateral 5-HT bundle, a bipolar stainless steel electrode (tip separation 0.3 mm, insulated to 0.3 mm of the tips) was inserted stereotaxically thl ough a hole drilled in the skull into raphe region. The coordinates according to the atlas of K6nig and Klippe117 were the dorsal raphe (anterior 0.62 ~ posterior 0.48, lateral --0.6 ~0.6 and vertical - - 0 . 2 ~ - - 2 . 0 mm), the median raphe (anterior 0.62,,~ posterior 0.48, lateral ---0.5 ~0.5 and vertical --0.2 ~--3.1) and the lateral 5-HT bundle (anterior 0.62 ~0.35, lateral 0.6,,~ 1.2 and vertical --1.2 ~--1.6). The site of electrode tip was marked by small lesions made by passing a direct current of 1 mA for 10 sec after each experiment. Serial cryostat sections (20 #m) were stained with toluidine blue. The position of electrode tip in the section was examined under a light microscope. The histological examination was performed in all experiments. The stimuli composed of monophasic pulses of 2 msec and 4 V were applied to the midbrain raphe. Frequency and stimulation time varied between 5 and 20 Hz and 2-5 min. During the experiments, body temperature was maintained 37.5 °C. RESULTS
Spontaneous release The spontaneous efflux of tritium from the parietal cortex preloaded with [3H]5HT followed a multiphasic exponential course. The efflux rate rapidly declined in the initial 30 min, but after 120 min gradually slowed (Fig. 1). After 120 min, the rate of efflux appeared to fit a single exponential function (slow phase). The declining coefficient of slow phase was 0.0071 dz 0.0003 rain -1. During the 3rd-5th h, the release of [aH]5-HT was stable. When imipramine in concentrations ranging from 10-6 to 10-a M were added to the medium in the cup, the efflux of tritium significantly increased, in a dose-dependent fashion. Twenty to 30 min after replacement with a
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Fig. 1. Spontaneous efflux of [3H]5-HT and its metabolites from the parietal cortex in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. 0--(3, control medium; 0 - - 0 , medium containing pargyline (10 -4 M).
354 TABLE I
Effect of pargyline on the quantity of unchanged [3H /5-hydroxytryptamine in spontaneously released total radioactive substances from the parietal cortex Samples were obtained during the 1st, 2nd, 3rd, 4th and 5th h at 10-rain intervals. Per cent is expressed as proportion of unchanged [3H]5-HT to total radioactive substances. Each value is the mean _-t=_S.E. of 4 experiments.
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34 ± 2* 50 ± 1"** 65 ± 5***
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35 =k 2* 53 i 3*** 77 ± 6***
31 ± 1" 49 ± 3*** 69 £- 4***
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Control Pargyline 10- 4 M 5 x 10- 4 M 10-a M
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Significantly different from control values at each time: * P < 0.05, ** P < 0.01, *** P < 0.005 (Student's t-test).
fresh medium, the effiux was restored to the level before treatment with imipramine. The declining coefficient of tritium efflux did not significantly change in the presence or absence of 10-4 M imipramine. In each sample obtained during the 1st, 2nd, 3rd, 4th and 5th h, total radioactivity was estimated and the 5-HT was separated by the acid-butanol-heptane system. The unchanged [ZH]5-HT with about 25 o/,,of the total radioactivity was found
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Fig. 2. A: effect of imipramine on the release of [3H]5-HT and its metabolites from the parietal cortex following electrical stimulation of the dorsal raphe in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. The horizontal bars indicate the addition of imipramine to the medium. Arrows indicate electrical stimulation (4 V, 2 msec, 20 Hz, 2 min). B : placement site of stimulating bipolar electrode (anterior 0.16, lateral 0, vertical --0.5 ram) indicated by coagulative lesion made immediately before sacrifice. A cryostat section in frontal plane. Toluidine blue staining, x 2.6.
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Fig. 3. Relationship between the frequencies of stimulation and the ratio of the dorsal raphestimulated release (Rst) to spontaneous release (Rsp) of [3H]5-HT and its metabolites from the parietal cortex. Each point represents the mean 4- S.E. of 12 experiments. in each sample from the control cup. When concentrations of pargyline ranging from 10-4 to 10-3 M were added to the medium in the cup, the unchanged [3H]5-HT significantly increased, in a dose-dependent manner (Table I). When 10-3 M pargyline was added to the medium, the unchanged [3H]5-HT increased to 75 ~ of total radioactivity and the slow declining coefficient of tritium efltux significantly decreased in the presence of 10-4 M pargyline (Fig. 1).
Release with stimulation Electrical stimulation of the midbrain raphe and the lateral 5-HT bundle was given during the slow phase of the spontaneous tritium efflux. In all these experiments, 10-4 M pargyline was added to the medium in the cup. Electrical stimulation of the dorsal raphe region (4 V, 2 msec, 20 Hz, 2 rain) produced a significant increase in release of [3H]5-HT and its metabolites (Fig. 2). The stimulated release was enhanced by addition of 10-4 M imipramine to the medium and was suppressed in cases of exclusion of imipramine from the medium, as shown in Fig. 2. The dorsal raphe stimulated release of [3H]5-HT and its metabolites from the parietal cortex was frequency dependent. Even with a frequency of 5 Hz, stimulation resulted in a significant increase of [3H]5-HT and its metabolites, and when the frequency was increased 5-20 Hz, there was an increase in the release of [3H]5-HT and its metabolites which was an asymptotic relationship (Fig. 3). On the other hand, stimulation of the median raphe region (4 V, 2 msec, 20 Hz, 2 min) did not significantly affect the release of [3H]5-HT and its metabolites from the parietal cortex. Stimulation of the lateral 5-HT bundle originating from the dorsal raphe also produced a frequencydependent increase in release of [SH]5-HT and its metabolites (Fig. 4). Even.with a frequency of 5 Hz, the stimulation of the lateral bundle resulted in an increase of 140 of spontaneous [3H]5-HT release and in a maximal increase with 10 Hz.
Tracking experiment The dorsal raphe in the rat is located anterior 0.62 mm ~ posterior 0.48 mm 17. As
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Fig. 4. Release of [aH]5-HT and its metabolites from the ipsilateral parietal cortex following unilateral electrical stimulation of the lateral 5-HT bundle (anterior 0.62, lateral 0.8, vertical --1.2 mm) in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. Arrows indicate electrical stimulation (4 V, 2 msec, 5-20 Hz, 2 min). shown in Fig. 5, tracking experiments of the dorsal raphe were done in 6 frontal planes (anterior 0.62, 0.35, 0.16, posterior 0.1,0.29, 0.48 mm) along the dorsolateral-medioventral axis with an angle of 70 ° to the horizontal plane. Stimulation with frequencies of 10 and 20 Hz just ventral to the anterior pole of the dorsal raphe produced a slight frequency-dependent increase of [aH]5-HT and its metabolites from the parietal cortex (Fig. 5A). As shown in Fig. 5B, C and D the most effective stimulation sites were located in a limited areawithin the dorsal raphe, the stereotaxic coordinates being: anterior 0.35 m m ~ posterior 0.1 mm, lateral 0.5 m m from the midline, vertical --0.8 ~ - - 1 . 2 m m from the horizontal zero plane. Histological examinations of the stimulation sites indicate that these corresponded to a densely packed subgroup dorsomedial of the fasciculus longitudinalis medialis and a bilateral group, located in the rostral two-thirds of the dorsal raphe. [3H]5-HT and its metabolites were undetectable after stimulation of the caudal one-third of the dorsal raphe and the area between the fasciculus longitudinalis medialis (Fig. 5E and F). DISCUSSION Our findings indicate that [aH]5-HT is taken up by the parietal cortex and is released spontaneously in a multiphasic time course in the rat anesthetized with pentobarbital sodium. The multiphasic decline in the tritium efflux presumably represents the efflux of [aH]5-HT and its metabolites from the multi-uptake sites. The initial rapid efflux of tritium may reflect a loss from the extracellular and extraneuronal areas. After 120 min, the eiflux followed a single exponential declining curve, presumably reflecting a release of [aH]5-HT and its metabolites from cortical serotonergic nerve terminals. After 120 rain, the unchanged [aH]5-HT was only 25 ~o of the total radioactivity, thereby showing the rapid degradation of 5-HT released
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Fig. 5. Release of [3H]5-HT and its metabolites from the parietal cortex following electrical stimulation of various sites of the dorsal raphe region in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. Arrows indicate electrical stimulation (4 V, 2 msec, 10-20 Hz, 2 min). Stereotaxic coordinates of stimulation sites shown in bottom of diagram, A, anterior; P, posterior; L, lateral; V, vertical. Ther stimulation sites (blackened area) in each tracking experiment shown on 6 drawings of the frontal section (A, B, C, D, E and F). Abbreviations: DR, nucleus dorsalis raphes; FLM, tasciculus longitudinalis medialis; MR, nucleus medianus raphes; nlV, nucleus n. trochlearis; pill, nucleus principalis n. oculomotorii, PCS, pedunculus cerebellaris superior.
358 from serotonergic nerve terminals during leakage through the brain tissue into the cup medium. These findings can be explained by the fact that unchanged [3H]5-HT is increased dose-dependently by the inhibition of MAO. We demonstrated that the in vivo release of [3H]5-HT from the parietal cortex was increased in response to electrical stimulation of the dorsal raphe and of the lateral 5-HT bundle. The stimulation-induced release of [3H]5-HT was frequency-dependent and was increased by inhibiting the neuronal re-uptake pump. Inhibition of the 5-HT pump by imipramine produced a significant increase in spontaneous release of cortical 5-HT and also an increase in the stimulation-induced release which was adequate for measurement of the 5-HT, even in the absence ofimipramine and pargyline in the cup medium. Chase et al. 8 reported that in brain slices preloaded with [14C]5-HIAA such metabolites are not concentrated in nerve terminals and are not liberated by electrical stimulation. Thus, total radioactivities of sample collections from the parietal cortex following stimulation of the dorsal raphe in the presence of pargyline and imipramine are considered to be a good reflection of the radioactivity of unchanged 5-HT. As we found in early studies 25, [3H]NA release from the parietal cortex was not increased by inhibiting the NA pump and [3H]NA release on stimulation of the locus coeruleus was unmeasurable when the NA pump was not inhibited by desipramine. The finding that in the rat under pentobarbital anesthesia, inhibition of the neuronal re-uptake pump increases spontaneous 5-HT release but not NA release indicates that barbiturates more intensely inhibit NA neuron than 5-HT neuron activities. On the other hand, stimulation-induced release of NA was increased considerably by inhibiting the NA pump as compared with the increment of 5-HT release by inhibiting the 5-HT pump. The tracking experiments demonstrated the increase of cortical 5-HT release on stimulation of the discrete raphe region. When the electrode was placed on the rostral two-thirds of the dorsal raphe, cortical 5-HT release was effectively increased, while the caudal one-third of the dorsal raphe and the region between the fasciculus longitudinalis medialis were non-effective sites. Electrical stimulation of the median raphe produced none or only a slight increase in the cortical 5-HT release. These findings indicate that the major 5-HT projection into the parietal cortex originates mainly from the rostral two-thirds of the dorsal raphe. Histochemical studies using neurotoxic dihydroxytryptaminev,11,14 and autoradiography 5,9,15,21 have demonstrated the existence of the lateral 5-HT bundle and the medial 5-HT bundle in the rat brain. Tracking experiments of the 5-HT bundle were also done in our study. When the lateral 5-HT bundle, located in the dorsomedial mesencephalic tegmentum ventromedial of the dorsal NA bundle, was stimulated electrically, the cortical 5-HT release was significantly increased, while stimulation on the medial ascending 5-HT bundle did not produce a significant response on cortical 5-HT release. Our results provide direct evidence for the existence of the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle. These findings are supported by histochemical data 14 that the dorsal raphe-cortical 5-HT pathway passes through the lateral 5-HT bundle and by neurochemical data 23 that 5-HT content in the parietal cortex is markedly reduced after lesioning the dorsal raphe.
359 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t by research grants f r o m the Scientific Research F u n d o f the Ministry o f E d u c a t i o n , Science an d Culture, Japan. W e t h a n k M. O h a r a o f K y u s h u U n i v e r s i t y for critical reading o f the manuscript.
REFERENCES 1 Aghajanian, G. K., Rosecrans, J. A. and Sheard, M. H., Serotonin: release in the forebrain by stimulation of midbrain raph6, Science, 156 (1967) 402-403. 2 And6n, N.-E., Dahlstr6m, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physiol, scand., 67 (1966) 313-326. 3 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters on stimulation of the nucleus linearis raphe in the cat, J. Physiol. (Lond.), 223 (1972) 255-259. 4 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters into the perfused third cerebral ventricle of the cat, J. Physiol. (Lond.), 233 (1973) 195-209. 5 Azmitia, E. C. and Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. comp. Neurol., 179 (1978) 641-668. 6 Beleslin, D. B. and Myers, R. D., The release of acetylcholine and 5-hydroxytryptamine from the mesencephalon of the unanesthetized rhesus monkey, Brafll Research, 23 (1970) 437-442. 7 Bjbrklund, A., Nobin, A. and Stenevi, U., The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indolamine neurons, Z. Zellforsch, 145 (1973) 479-501. 8 Chase, T. N., Katz, R. I. and Kopin, I. J., Release of [3H]serotonin from brain slices, J. Neurochem., 16 (1969) 607-615. 9 Conrad, L. C. A., Leonard, C. M. and Peaff, D. W., Connections of the median and dorsal raphe nuclei in the rat: an autoradiographic and degeneration study, J. comp. Neurol., 156 (1974) 179-206. 10 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurones in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurones, Acta Physiol. scand., 62 (1964) 1-55. 11 Daly, J., Fuxe, K. and Jonsson, G., Effects of intracerebral injections of 5,6-dihydroxytryptamine on central monoamine neurons: evidence for selective degeneration of central 5-hydroxytryptamine neurons, Brain Research, 49 (1973) 476-482. 12 Eccleston, D., Randic, M., Roberts, M. H. T. and Straughan, D. W., Release of amines and amine metabolites from brain by neural stimulation. In G. Hopper (Ed.), Metabolism of Amines in the Brain, Proceedings of a Symposium, British and Scandinavian Pharmacological Societies, Edinburgh, July, 1968, Macmillan, London, 1968, pp. 29-33. 13 Feldberg, W. and Myers, R. D., Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles, J. Physiol. (Lond.), 184 (1966) 837-855. 14 Fuxe, K. and Johnsson, G., Further mapping of central 5-hydroxytryptamine neurons: Studies with the neurotoxic dihydroxytryptamines, Advanc. Biochem. Psychopharmacol., 10 (1974) 1-12. 15 Halaris, A. E., Jones, B. E. and Moore, R. Y., Axonal transport in serotonin neurons of the midbrain raphe, Brain Research, 107 (1976) 555-574. 16 Holman, R. B. and Vogt, M., Release of 5-hydroxytryptamine from caudate nucleus and septum, J. Physiol. (Lond.), 223 (1972) 243-254. 17 Kbnig, J. F. R. and Klippel, R. A., The Rat Brain: a stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Robert E. Krieger, 1967. 18 Kuhar, M. J., Aghajanian, G. K. and Roth, R. H., Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions: correlations with serotonin levels and histochemical fluorescence, Brain Research, 44 (1972) 165-176. 19 Kuhar, M. J., Roth, R. H. and Aghajanian, G. K., Synaptosomes from forebrains of rats with midbrain lesions: selective reduction of serotonin uptake, J. Pharmacol. exp. Ther., 181 (1972) 36-45.
360 20 Lorens, S. A. and Guldberg, H. C., Regional 5-hydroxytryptamine following selective midbrain raphe lesions in the rat, Brain Research, 78 (1974) 45-56. 21 Moore, R. Y., Halaris, A. E. and Jones, B. E., Serotonin neurons of the midbrain raphe: Ascending projections, J. comp. NeuroL, 180 (1978) 417438. 22 Myers, R. D. and Beleslin, D. B., The spontaneous release of 5-hydroxytryptamine and acetylcholine within the diencephalon of the unanesthetized rhesus monkey, Exp. Brain Res., 11 (1970) 539-552. 23 Palkovits, M., Saavedra, J. M., Jacobowitz, D. M., Kizer, J. S., Zaborszky, L. and Brownstein, M. J., Serotonergic innervation of the forebrain: effect of lesions on serotonin and tryptophan hydroxylase levels, Brain Research, 130 (1977) 121-134. 24 Sheard, M. H. and Aghajanian, G. K., Stimulation of the midbrain raphe: effect on serotonin metabolism, J. Pharmacol. exp. Ther., 163 (1968) 425-430. 25 Tanaka, C., Inagaki, C. and Fujiwara, H., Labeled noradrenaline release from rat cerebral cortex following electrical stimulation of locus coeruleus, Brain Research, 106 (1976) 384--389. 26 Ternaux, J. P., Boireau, A., Bourgoin, S., Hamon, M., Hery, F. and Glowinski, J., In vivo release of 5-HT in the lateral ventricle of the rat: effects of 5-hydroxytryptophan and tryptophan, Brain Research, 101 (1976) 533-548.
351
S T I M U L A T I O N OF T H E RAT D O R S A L R A P H E IN VIVO RELEASES LABELED S E R O T O N I N F R O M T H E P A R I E T A L C O R T E X
HIROSHI FUJIWARA, MASAHARU UEMOTO and CHIKAKO TANAKA* Department of Pharmacology, Kobe University School of Medicine, Kobe 650 (Japan)
(Accepted November 27th, 1980) Key words: in vivo 5-HT release -- rat parietal cortex - - dorsal raphe stimulation
SUMMARY In vivo release of labeled serotonin ([aH]5-HT) from the parietal cortex was investigated by cortical cup technique and electrical stimulation of midbrain raphe in rats anesthetized with pentobarbital sodium. The spontaneous efflux of tritium from the parietal cortex preloaded with [3H]5-HT followed a multiphasic exponential course. After 120 min, the rate of etttux appeared to fit the single exponential function (slow phase). Imipramine (10-6-10 -8 M) produced a dose-dependent increase in the spontaneous release. When pargyline in concentrations ranging from 10-4 to 10-3 M were added to the medium in the cup, the unchanged [aH]5-HT significantly increased in a dose-dependent manner and the slow declining coefficient of tritium efflux significantly decreased in the presence of 10-4 pargyline. Stimulation of the rostral two-thirds of the dorsal raphe and the lateral 5-HT bundle originating from the dorsal raphe significantly increased the release of [aH]5-HT and its metabolites while stimulation of the caudal one-third of the dorsal raphe did not produce a significant increase in the release of [3H]5-HT and its metabolites. Stimulation of the median raphe produced no or only a slight increase in the release of [3H]5-HT and its metabolites. These findings are a direct demonstration of the in vivo release of [3H]5H T from the parietal cortex with stimulation of the dorsal raphe, particularly the rostral two-thirds of the nucleus and provide the neurochemical evidence for the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle. INTRODUCTION Fluorescence histochemical studies demonstrated that the perikarya of serotonin (5-hydroxytryptamine; 5-HT) containing neurons are present almost exclusively in * To whom correspondence should be addressed.
352 the raphe nuclei 10 and that the 5-HT neuron in the midbrain raphe projects into the forebrain2,14. Aghajanian et al. 1,1s suggested that 5-HT can be released via a specific neural pathway, projecting into the forebrain, as based on neurochemical findings that electrical stimulation of the midbrain raphe in the rat produced an increase in the levels of 5-hydroxyindole acetic acid (5-HIAA) and a decrease in 5-HT content in the forebrain 1,24. This concept was also supported by reports that selective destruction of the midbrain raphe produced a decrease in 5-HT levels 7,1s-20,2z, histofluorescence intensity of 5-HT 18, tryptophan hydroxylase activity18,19 and synaptosomal uptake of 5-HT 1s,19. However, these findings are indirect evidence for the release of forebrain 5HT on stimulation of the midbrain raphe. The in vivo release of central transmitters can be estimated in perfusates of the lateral ventricle as obtained from superfusates from the push-pull cannula inserted into the deep structures and from a cup placed on superficial areas such as the cerebral cortex. The in vivo experiments on 5-HT release were performed mainly in the cat 3,4,12,13,16 and in the monkey6, 22 and the rat 26. As direct estimation of the cortical 5-HT release on stimulation of the midbrair, raphe has apparently not been done in the rat, we carried out such experiments using the cortical cup technique. MATERIALS AND METHODS Male Wistar rats, weighing 250-350 g were anesthetized with pentobarbital sodium 30 mg/kg i.p. and fixed in a stereotaxic apparatus. The skull was removed from 2 mm posterior to bregma and 4 m m lateral to midline. The overlying dura was reflected and the parietal cortex was exposed. A cylindrical plastic cup (4.5 m m in internal diameter, 8 m m in height) was placed on the exposed cortex and sealed with surgical adhesive. The cup was filled with modified Krebs-phosphate solution containing E D T A (disodium salt, 10 mg/1) and ascorbic acid (20 rag/l). This solution was gassed with 95 ~o 02-5 ~/o CO2 at 37 °C before addition to the cup. The parietal cortex was incubated for 1 h with [1,2-aH]serotonin creatinine sulfate ([aH]5-HT; 21.9 Ci/mmol) dissolved in modified Krebs-phosphate solution. After incubation, the medium was removed and the cortex in the cup was washed 5 times with fresh Krebs-phosphate solution. The cup was then filled with 0.05 ml of Krebs-phosphate solution. Samples were collected at 10-rain intervals. Total radioactivity was counted in a Packard Tri-carb liquid scintillation spectrometer and the radioactivity was expressed as the number of count per rain (cpm).
Separation of unchanged [3H]5-HTfrom tritium efflux Six samples collected at 10-min intervals for a period of 5 h were pooled. Half of the volume of the samples was used to separate unchanged [3H]5-HT and the remaining sample was prepared to estimate the total radioactivity. To the sample was added 0.5 #g of unlabeled 5-HT as the carrier and 0.1 N HC1 containing 0.1 ~ ascorbic acid and the preparation was then frozen until the separation. 5-HT was removed from the sample by extraction into n-butanol at a pH 10. n-Heptane was added to the butanol extract and the 5-HT returned to 0.1 N HC1. The radioactivity of [ZH]5-HT in 0.1 N HC1 was counted.
353
Electrical stimulation For stimulation of the dorsal raphe, the median raphe and the ascending lateral 5-HT bundle, a bipolar stainless steel electrode (tip separation 0.3 mm, insulated to 0.3 mm of the tips) was inserted stereotaxically thl ough a hole drilled in the skull into raphe region. The coordinates according to the atlas of K6nig and Klippe117 were the dorsal raphe (anterior 0.62 ~ posterior 0.48, lateral --0.6 ~0.6 and vertical - - 0 . 2 ~ - - 2 . 0 mm), the median raphe (anterior 0.62,,~ posterior 0.48, lateral ---0.5 ~0.5 and vertical --0.2 ~--3.1) and the lateral 5-HT bundle (anterior 0.62 ~0.35, lateral 0.6,,~ 1.2 and vertical --1.2 ~--1.6). The site of electrode tip was marked by small lesions made by passing a direct current of 1 mA for 10 sec after each experiment. Serial cryostat sections (20 #m) were stained with toluidine blue. The position of electrode tip in the section was examined under a light microscope. The histological examination was performed in all experiments. The stimuli composed of monophasic pulses of 2 msec and 4 V were applied to the midbrain raphe. Frequency and stimulation time varied between 5 and 20 Hz and 2-5 min. During the experiments, body temperature was maintained 37.5 °C. RESULTS
Spontaneous release The spontaneous efflux of tritium from the parietal cortex preloaded with [3H]5HT followed a multiphasic exponential course. The efflux rate rapidly declined in the initial 30 min, but after 120 min gradually slowed (Fig. 1). After 120 min, the rate of efflux appeared to fit a single exponential function (slow phase). The declining coefficient of slow phase was 0.0071 dz 0.0003 rain -1. During the 3rd-5th h, the release of [aH]5-HT was stable. When imipramine in concentrations ranging from 10-6 to 10-a M were added to the medium in the cup, the efflux of tritium significantly increased, in a dose-dependent fashion. Twenty to 30 min after replacement with a
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Fig. 1. Spontaneous efflux of [3H]5-HT and its metabolites from the parietal cortex in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. 0--(3, control medium; 0 - - 0 , medium containing pargyline (10 -4 M).
354 TABLE I
Effect of pargyline on the quantity of unchanged [3H /5-hydroxytryptamine in spontaneously released total radioactive substances from the parietal cortex Samples were obtained during the 1st, 2nd, 3rd, 4th and 5th h at 10-rain intervals. Per cent is expressed as proportion of unchanged [3H]5-HT to total radioactive substances. Each value is the mean _-t=_S.E. of 4 experiments.
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Significantly different from control values at each time: * P < 0.05, ** P < 0.01, *** P < 0.005 (Student's t-test).
fresh medium, the effiux was restored to the level before treatment with imipramine. The declining coefficient of tritium efflux did not significantly change in the presence or absence of 10-4 M imipramine. In each sample obtained during the 1st, 2nd, 3rd, 4th and 5th h, total radioactivity was estimated and the 5-HT was separated by the acid-butanol-heptane system. The unchanged [ZH]5-HT with about 25 o/,,of the total radioactivity was found
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Fig. 2. A: effect of imipramine on the release of [3H]5-HT and its metabolites from the parietal cortex following electrical stimulation of the dorsal raphe in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. The horizontal bars indicate the addition of imipramine to the medium. Arrows indicate electrical stimulation (4 V, 2 msec, 20 Hz, 2 min). B : placement site of stimulating bipolar electrode (anterior 0.16, lateral 0, vertical --0.5 ram) indicated by coagulative lesion made immediately before sacrifice. A cryostat section in frontal plane. Toluidine blue staining, x 2.6.
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Fig. 3. Relationship between the frequencies of stimulation and the ratio of the dorsal raphestimulated release (Rst) to spontaneous release (Rsp) of [3H]5-HT and its metabolites from the parietal cortex. Each point represents the mean 4- S.E. of 12 experiments. in each sample from the control cup. When concentrations of pargyline ranging from 10-4 to 10-3 M were added to the medium in the cup, the unchanged [3H]5-HT significantly increased, in a dose-dependent manner (Table I). When 10-3 M pargyline was added to the medium, the unchanged [3H]5-HT increased to 75 ~ of total radioactivity and the slow declining coefficient of tritium efltux significantly decreased in the presence of 10-4 M pargyline (Fig. 1).
Release with stimulation Electrical stimulation of the midbrain raphe and the lateral 5-HT bundle was given during the slow phase of the spontaneous tritium efflux. In all these experiments, 10-4 M pargyline was added to the medium in the cup. Electrical stimulation of the dorsal raphe region (4 V, 2 msec, 20 Hz, 2 rain) produced a significant increase in release of [3H]5-HT and its metabolites (Fig. 2). The stimulated release was enhanced by addition of 10-4 M imipramine to the medium and was suppressed in cases of exclusion of imipramine from the medium, as shown in Fig. 2. The dorsal raphe stimulated release of [3H]5-HT and its metabolites from the parietal cortex was frequency dependent. Even with a frequency of 5 Hz, stimulation resulted in a significant increase of [3H]5-HT and its metabolites, and when the frequency was increased 5-20 Hz, there was an increase in the release of [3H]5-HT and its metabolites which was an asymptotic relationship (Fig. 3). On the other hand, stimulation of the median raphe region (4 V, 2 msec, 20 Hz, 2 min) did not significantly affect the release of [3H]5-HT and its metabolites from the parietal cortex. Stimulation of the lateral 5-HT bundle originating from the dorsal raphe also produced a frequencydependent increase in release of [SH]5-HT and its metabolites (Fig. 4). Even.with a frequency of 5 Hz, the stimulation of the lateral bundle resulted in an increase of 140 of spontaneous [3H]5-HT release and in a maximal increase with 10 Hz.
Tracking experiment The dorsal raphe in the rat is located anterior 0.62 mm ~ posterior 0.48 mm 17. As
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Fig. 4. Release of [aH]5-HT and its metabolites from the ipsilateral parietal cortex following unilateral electrical stimulation of the lateral 5-HT bundle (anterior 0.62, lateral 0.8, vertical --1.2 mm) in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. Arrows indicate electrical stimulation (4 V, 2 msec, 5-20 Hz, 2 min). shown in Fig. 5, tracking experiments of the dorsal raphe were done in 6 frontal planes (anterior 0.62, 0.35, 0.16, posterior 0.1,0.29, 0.48 mm) along the dorsolateral-medioventral axis with an angle of 70 ° to the horizontal plane. Stimulation with frequencies of 10 and 20 Hz just ventral to the anterior pole of the dorsal raphe produced a slight frequency-dependent increase of [aH]5-HT and its metabolites from the parietal cortex (Fig. 5A). As shown in Fig. 5B, C and D the most effective stimulation sites were located in a limited areawithin the dorsal raphe, the stereotaxic coordinates being: anterior 0.35 m m ~ posterior 0.1 mm, lateral 0.5 m m from the midline, vertical --0.8 ~ - - 1 . 2 m m from the horizontal zero plane. Histological examinations of the stimulation sites indicate that these corresponded to a densely packed subgroup dorsomedial of the fasciculus longitudinalis medialis and a bilateral group, located in the rostral two-thirds of the dorsal raphe. [3H]5-HT and its metabolites were undetectable after stimulation of the caudal one-third of the dorsal raphe and the area between the fasciculus longitudinalis medialis (Fig. 5E and F). DISCUSSION Our findings indicate that [aH]5-HT is taken up by the parietal cortex and is released spontaneously in a multiphasic time course in the rat anesthetized with pentobarbital sodium. The multiphasic decline in the tritium efflux presumably represents the efflux of [aH]5-HT and its metabolites from the multi-uptake sites. The initial rapid efflux of tritium may reflect a loss from the extracellular and extraneuronal areas. After 120 min, the eiflux followed a single exponential declining curve, presumably reflecting a release of [aH]5-HT and its metabolites from cortical serotonergic nerve terminals. After 120 rain, the unchanged [aH]5-HT was only 25 ~o of the total radioactivity, thereby showing the rapid degradation of 5-HT released
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Fig. 5. Release of [3H]5-HT and its metabolites from the parietal cortex following electrical stimulation of various sites of the dorsal raphe region in rats anesthetized with pentobarbital sodium 30 mg/kg i.p. Arrows indicate electrical stimulation (4 V, 2 msec, 10-20 Hz, 2 min). Stereotaxic coordinates of stimulation sites shown in bottom of diagram, A, anterior; P, posterior; L, lateral; V, vertical. Ther stimulation sites (blackened area) in each tracking experiment shown on 6 drawings of the frontal section (A, B, C, D, E and F). Abbreviations: DR, nucleus dorsalis raphes; FLM, tasciculus longitudinalis medialis; MR, nucleus medianus raphes; nlV, nucleus n. trochlearis; pill, nucleus principalis n. oculomotorii, PCS, pedunculus cerebellaris superior.
358 from serotonergic nerve terminals during leakage through the brain tissue into the cup medium. These findings can be explained by the fact that unchanged [3H]5-HT is increased dose-dependently by the inhibition of MAO. We demonstrated that the in vivo release of [3H]5-HT from the parietal cortex was increased in response to electrical stimulation of the dorsal raphe and of the lateral 5-HT bundle. The stimulation-induced release of [3H]5-HT was frequency-dependent and was increased by inhibiting the neuronal re-uptake pump. Inhibition of the 5-HT pump by imipramine produced a significant increase in spontaneous release of cortical 5-HT and also an increase in the stimulation-induced release which was adequate for measurement of the 5-HT, even in the absence ofimipramine and pargyline in the cup medium. Chase et al. 8 reported that in brain slices preloaded with [14C]5-HIAA such metabolites are not concentrated in nerve terminals and are not liberated by electrical stimulation. Thus, total radioactivities of sample collections from the parietal cortex following stimulation of the dorsal raphe in the presence of pargyline and imipramine are considered to be a good reflection of the radioactivity of unchanged 5-HT. As we found in early studies 25, [3H]NA release from the parietal cortex was not increased by inhibiting the NA pump and [3H]NA release on stimulation of the locus coeruleus was unmeasurable when the NA pump was not inhibited by desipramine. The finding that in the rat under pentobarbital anesthesia, inhibition of the neuronal re-uptake pump increases spontaneous 5-HT release but not NA release indicates that barbiturates more intensely inhibit NA neuron than 5-HT neuron activities. On the other hand, stimulation-induced release of NA was increased considerably by inhibiting the NA pump as compared with the increment of 5-HT release by inhibiting the 5-HT pump. The tracking experiments demonstrated the increase of cortical 5-HT release on stimulation of the discrete raphe region. When the electrode was placed on the rostral two-thirds of the dorsal raphe, cortical 5-HT release was effectively increased, while the caudal one-third of the dorsal raphe and the region between the fasciculus longitudinalis medialis were non-effective sites. Electrical stimulation of the median raphe produced none or only a slight increase in the cortical 5-HT release. These findings indicate that the major 5-HT projection into the parietal cortex originates mainly from the rostral two-thirds of the dorsal raphe. Histochemical studies using neurotoxic dihydroxytryptaminev,11,14 and autoradiography 5,9,15,21 have demonstrated the existence of the lateral 5-HT bundle and the medial 5-HT bundle in the rat brain. Tracking experiments of the 5-HT bundle were also done in our study. When the lateral 5-HT bundle, located in the dorsomedial mesencephalic tegmentum ventromedial of the dorsal NA bundle, was stimulated electrically, the cortical 5-HT release was significantly increased, while stimulation on the medial ascending 5-HT bundle did not produce a significant response on cortical 5-HT release. Our results provide direct evidence for the existence of the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle. These findings are supported by histochemical data 14 that the dorsal raphe-cortical 5-HT pathway passes through the lateral 5-HT bundle and by neurochemical data 23 that 5-HT content in the parietal cortex is markedly reduced after lesioning the dorsal raphe.
359 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t by research grants f r o m the Scientific Research F u n d o f the Ministry o f E d u c a t i o n , Science an d Culture, Japan. W e t h a n k M. O h a r a o f K y u s h u U n i v e r s i t y for critical reading o f the manuscript.
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