Regional 5-hydroxytryptamine following selective midbrain raphe lesions in the rat

Brain Research, 78 (1974) 45-56

45

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

REGIONAL 5-HYDROXYTRYPTAMINE FOLLOWING SELECTIVE MIDBRAIN RAPHE LESIONS IN THE RAT

STANLEY A. LORENS AND HANS C. G U L D B E R G

Institute of Psychology and Department of Pharmacology, University of Bergen, Bergen (Norway) (Accepted May 1st, 1974)

SUMMARY

Lesions were produced in either the dorsal or median raphe nucleus and regional 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and norepinephrine (NE) determined 26--30 days post-operatively. Only lesions in the dorsal raphe nucleus produced a fall (54 ~o) in striatal 5-HT, while only lesions in the median raphe nucleus reduced (62 ~o) hippocampal 5-HT. Reductions in the 5-HT and 5-HIAA contents of the remaining portion of the telencephalon and of the diencephalon following dorsal raphe lesions were twice as great as after median raphe lesions. The lowered 5-HT concentration (50 ~) of the telencephalon (excluding hippocampus and striatum) after dorsal raphe lesions, furthermore, was twice as large as that of the diencephalon (24 ~). Only dorsal raphe lesions produced a fall in brain stem 5-HT content. Neither lesion affected spinal (cervical-thoracic) 5-HT, nor NE in any of the brain areas assayed. It would appear that the dorsal and median raphe nuclei project 5-HT fibers into the forebrain but not to the spinal cord. The larger number of 5-HT fibers seems to originate in the dorsal raphe nucleus, which also seems to send a greater number of its 5-HT fibers to telencephalic than diencephalic structures. And, lastly, hippocampal and striatal 5-HT inputs apparently originate chiefly in the median and dorsal raphe nuclei, respectively.

INTRODUCTION

Considerable attention has been focussed recently on the midbrain dorsal and median (also known as the central superior nucleus of Bechterew) raphe nuclei. These nuclei were designated some time ago as the 5-hydroxytryptamine (5-HT) containing B7 and B8 cell groups, respectively, by Dahlstr6m and Fuxe 14. Subsequently, the results from a number of stimulation1,30, 31 and lesiong,1z,15,17-25,29 studies have provided supporting evidence for the existence of 5-HT neurons in these cell groups.

46

S. A. LORENS A N D H. C. G U L l ) B E R G

Large brain stem lesions involving both the dorsal and median raphe nuclei have been found to reduce by up to 80~, forebrain 5-HT, 5-hydroxyindole acetic acid (5-HIAA), and tryptophan hydroxylase 9,12,15,17,19 ?l,?a-z.5. Smaller lesions, reported to involve the median but not the dorsal raphe nucleus, also produce lowered forebrain 5-HT (33-74~o) and 5-HIAA (41-79 ~)1s,22,24,25,29. Surprisingly, there have been no reports on the 5-HT effects of lesions restricted to the dorsal raphe nucleus. Furthermore, little is known about the differential projection of these cell groups, although it appears well-established that their fibers project via the medial forebrain bundle to a variety of forebrain areas a,~,s,lv,~6,2s. The present study was undertaken to determine if the 5-HT fibers originating in the dorsal and median raphe nuclei have a differential projection. Thus, lesions were placed in either the dorsal or median raphe nucleus and the resultant effect on regional central 5-HT observed 26-30 days post-operatively. METHOD

Animals' Seventy-five male albino rats (Wistar) weighing 267-359 g at the time of surgery were used. The animals were housed individually in conventional rat cages located in a temperature (22 ~: 2 °C) controlled room with a 12 h dark-light cycle. Food and water were available ad libitum.

Surgery and histology Electrolytic lesions were produced under sodium pentobarbital anesthesia (50 mg/kg, i.p.) by passing 2 m A for 10 sec (dorsal nucleus) or 15 sec (median nucleus) through an intracranial cathode and an anode clipped to the wound edge. The cathode was a 0.25 m m diameter tungsten wire insulated with Epoxylite except for 0.5 mm at its tip. A K o p f stereotaxic instrument was employed with the incisor bar set 3.2 m m above the inter-aural plane. The cathode was inserted through the cerebellum at an angle of 47 ° to the vertical plane. Lesions were produced in the mid-sagittal plane in either the dorsal (n = 29) or median (n = 30) raphe nucleus. Lesion coordinates were based on measurements taken from the midline 1.0 mm anterior to lambda: 6.4 mm caudal and 9.5 mm ventral to the surface of the skull for the dorsal nucleus, and 8.4 m m caudal and 12.3 mm ventral for the median nucleus. Operated control animals (n == 16) were treated in the same manner as the lesion rats except that the electrode was not lowered into the brain. Brain stems used in the histological analysis were kept in 1 0 ~ formalin for at least 8 days. Frozen sections were cut at 30/zm and every tenth section saved and stained by the thionin technique. Lesion cavitation and glial scarring were determined by microscopic examination of the histological material and plotted on pre-prepared diagrams.

Determinations of 5-HT, 5-HIAA and norepinephrine ( NE) 5-HT was determined spectrophotofluorometrically by the method of Bertler 4

REGIONAL

5-HT

AFTER RAPHE LESIONS

47

with the modification of Ahtee et al. z. 5-HT and 5-HIAA were estimated in the same. tissue sample by a combination of previously described methodsL N E was determined as described by Crawford and Yates 13. Recoveries of compounds added to the homogenates were about 6 0 ~ for 5-HT, about 8 0 ~ for 5-HIAA, and 80-90 ~ for NE. Results have not been corrected for recovery.

Procedure On the 26-30th post-operative day, the animals were sacrificed by decapitation and the brain and upper 5 cm (cervical-thoracic region) of the spinal cord rapidly removed. The brain was dissected on a cold glass plate which covered a tray of dry ice. The olfactory tract was sectioned at the level of the frontal pole, the pineal gland removed, and both discarded. After a callosal section, the telencephalon was peeled forward and dissected from the diencephalon by means of sections made rostral (through the preoptic area) and lateral (through the internal capsule) to the thalamus. The hippocampi (including the subiculum, hippocampus proper, and dentate gyrus, but excluding the parasubiculum and entorhinal cortex) were peeled free. The corpus striatum (caudate-putamen and globus pallidus) was then extracted bilaterally using scissors. The remaining portion of the telencephalon (including such structures as the amygdala, septal area, entorhinal area, parasubiculum, and cerebral cortex) was also saved for analysis. The diencephalon (excluding the pineal gland) was freed by a dorsal-ventral section made rostral to the superior colliculi and caudal to the mammillary bodies. The brain stem was placed in formalin for histological analysis (dorsal lesion group, n = 19; median lesion group, n ---- 20), or the cerebellum was removed and the remaining portion (midbrain, pons, and medulla) used for biochemical analysis (controls, n = 16; and n = 10 from each lesion group). The tissue samples were wrapped in aluminum foil, frozen in dry ice, then stored (--20 °C) for a maximum of 3 weeks prior to biochemical analysis. The time which elapsed from the moment o f decapitation until all samples had been frozen was 8-12 min. 5-HT analysis was performed on all samples, whereas N E and 5-HIAA analysis was conducted on only a few. Also, two of the 5-HT samples, one of the 5-HIAA, and three o f the NE samples were lost due to technical errors. RESULTS

General observations All animals survived the surgery. No gross neurological or behavioral abnormalities were observed. The animals gained weight post-operatively and at the time of sacrifice the group body weights (mean ± S.D.) were: controls, 345 + 31 g; dorsal lesion group, 339 -4- 26 g; and, median lesion group, 341 q- 29 g. Lesion placement Median raphe lesions. Two of the 20 median raphe lesion brains histologically examined were rejected from further analysis as the damage was caudal, unilateral and did not involve the median raphe nucleus. The 18 acceptable lesions are shown dia-

HEDIANliAPHELESIONS k~

d t g B6

dr ~f~ ~8c vtg J r rf

115

87

~

51

95

59

107

69

.

_

REGIONAL 5-HT AFTER RAPHE LESIONS

49

grammatically in Fig. 1. As can be seen, 30-70 ~ of the median raphe nucleus was destroyed. The medial aspect of the tectospinal tracts, both the dorsal and deep (ventral) tegmental nuclei of Gudden, and the medial longitudinal fasciculus were damaged either unilaterally or bilaterally in all animals. The lesion involved the area B6 as defined by Dahlstr6m and Fuxe 14 in 8 rats. The decussation of the brachium conjunctivum was invaded in only 3 rats (Nos. 84, 107, and 115) while the lesion involved the interpeduncular nucleus in only one animal (No. 38). In 5 animals (Nos. 51, 59, 64, 69 and 79) the lesion did not extend rostrally to the level of the decussation of the brachium conjunctivum (see lower right portion of Fig. 1). These animals comprised the subgroup with 'caudal' median raphe lesions. The remaining 13 rats made up the 'rostrai' median raphe lesion group. Dorsal raphe lesion. All 19 dorsal raphe lesions (presented schematically in Fig. 2) were considered acceptable. The lesions were well localized and ablated 30-50 ~o of the dorsal raphe nucleus. In fact, in 8 animals (Nos. 45, 63, 76, 86, 88, 106, 108 and 111) the lesion was restricted to the dorsal raphe nucleus and immediately surrounding ventromedial aspect of the periaqueductal gray matter. The medial longitudinal fasciculus was damaged either uni- or bilaterally in the remaining 11 animals. In 2 rats (Nos. 42 and 83) the lesion extended caudally and unilaterally into the dorsal tegmental nucleus of Gudden. The lesion invaded the decussation of the brachium conjunctivum in only one rat (No. 42). The animals comprising the dorsal raphe lesion group were subdivided on the basis of the rostral extent o f the lesion. The lesions in the animals making up the 'rostral' dorsal raphe lesion group (n = 9) are depicted in the left-hand column o f Fig. 2, while those in the 'mid-caudal' dorsal raphe lesion group (n ---- 10) are shown in the right-hand column. The major distinction between these subgroups was that the lesion in the 'rostral' group seriously damaged the dorsal raphe nucleus at the level of the trochlear nuclei.

Biochemical analysis The wet weights of the regional samples were quite consistent, and no group differences were found (Table I). Median raphe lesion. As seen in Table II, lesions in the median raphe nucleus produced significant reductions in the 5-HT content of the diencephalon, hippocampus and remaining telencephalon (excluding the striatum). The 5-HT concentrations o f

Fig. 1. Reproductions of median raphe lesions (blackened area) on 4 coronal planes separated by approximately 0.6 mm. Numbers identify individual animals. Abbreviations: c, dorsal tegmental nucleus, pars centralis27; dr, dorsal raphe nucleus; dtg, dorsal tegmental nucleus of Gudden; ipn, interpeduncular nucleus; lc, locus coeruleus; li, intermediate linear nucleus; m, median raphe nucleus, medium-sizedcell parta4; mr, median raphe (or, central superior) nucleus; p, median raphe nucleus, small-sized cell regiona4; rf, reticular formation; rtp, pontine tegmental reticular nucleus; sn, substantia nigra; vtg, ventral (or deep) tegmental nucleus of Gudden; B6, cell group B6 of Dahlstr6m and Fuxe14; caudal extension of dorsal raphe nucleus34; BC, brachium conjunctivum (superior cerebellar peduncle); DBC, decussation of brachium conjunctivum; IV, trochlear nucleus; ML, medial lemniscus; MLF, medial longitudinal fasciculus; TTS, tecto-spinal tract.

DORSALRAPHI LESIONS F / ~TTS

rf

vtg

z

78

/~\

8e ~

/N\

89

.,

76

/ ~_ ",

111

/~_~\

/ ~",

lo8.,~,~ /~

Fig. 2. Dorsal rapbe lesions (blackened area) shown on 3 transverse sections (separated by about 0.6 mm). Numbers and abbreviations as in Fig. 1.

51

REGIONAL 5 - H T AFTER RAPHE LESIONS TABLE I (mg)

REGIONAL SAMPLE WEIGHTS

Control

Region

Telencephalon Hippocampus Striatum Diencephalon Brain stem Spinal cord

Dorsal raphe lesion

Median raphe lesion

n

Mean ± S.D.

n

Mean -- S.D.

n

Mean ± S.D.

16 16 16 16 16 16

734 142 127 160 391 319

27 27 26 27 8 27

725 137 127 166 377 323

27 28 28 28 10 28

739 139 125 166 379 324

± ± ± ± ± ±

36 8 15 14 22 47

± ± ± ± ± --

47 14 13 14 18 33

4± ± ± ± ±

45 14 12 12 16 33

the striatum and spinal cord were not affected. The lowered (11 ~) 5-HT content of the brain stem was not statistically significant. The 19 ~ telencephalic reduction in 5-HT was accompanied by a significant 23 ~ decrease in 5-HIAA concentration (Table II). The mean 25 ~ reduction in diencephalic 5-HIAA (corresponding to a significant 14 decrease in 5-HT) did not reach significance (0.05 < P < 0.10). NE content was not affected in the regions assayed (Table III). Lesions which involved the 'rostral' median raphe nucleus were more effective in reducing forebrain 5-HT (Table IV). In fact, apart from the 27 ~ decrease in hippocampal 5-HT, the 'caudal' median raphe lesions did not produce any significant regional changes in 5-HT. It should be noted, however, that the 16~ reduction in diencephalic 5-HT, although not statistically significant, was of equal magnitude to TABLE II REGIONAL 5-HYDROXYTRYPTAMINE AND 5-HYDROXYINDOLE ACETIC ACID CONCENTRATIONS FOLLOWING SELECTIVE MIDBRAIN RAPHE LESIONS

Region

Control n

Dorsal raphe lesion Mean 5_ S.D.

n

Mean ± S.D.

± 022 4- 039 ± 042 ± 070 ± 055 ± 046

27 27 26 27 8 27

137 221 151 291 300 254

5-tIydroxyindole acetic acid (ng/g) Telencephalon 4 269 ± 032 Diencephalon 3 522 ± 075

4 4

5-Hydro xytryptamine ( ng/g ) Telencephalon 16 275 Hippocampus 16 233 Striatum 16 326 Diencephalon 16 385 Brain stem 16 378 Spinal cord 16 271

± ± ± ± ± ±

Median rophe lesion %

n

Mean dz S.D.

223 088 337 333 337 257

038 042 058 043 054 043

--50zz -- 5 --54zz --24% --21y -- 6

27 28 28 28 10 28

141 4- 030 318 ± 041

~8Yy --39x

5 5

± 033 ± 060 2_ 042 ± 063 ± 077 ± 048

208 ± 013 389 ± 060

%

--19z --62zz q- 3 --14 x --11 -- 5 --23" --25

Letters above and below percentage figures indicate significant (x:p < 0.02; y:p < 0.01 ; z:p < 0.001) differences between lesion and control (above) or the two lesion (below) groups. Probabilities determined by Student's t-test, two-tailed.

52

S.A.

L O R E N S A N D H. ('. G U L I ) B E R ( J

T A B L E Ill REGIONAL NOREPINEPHRINE CONCENTRATIONS FOLLOWING SELECTIVE MIDBRAIN RAPHE LESIONS

Region

Norepinephrine (ng/g) Control

Telencephalon Diencephalon Brain stem

Dorsal raphe lesion

Median raphe leskm

n

Mean ± S.D.

n

Mean 7.: S.D.

%

;l

Mean ± S.D.

%

4 4 4

227 ± 048 455 ± 148 282 ± 034

3 3 2

253 ± 035 458 -- 107 256 ± 087

±11 + 1 -- 9

5 5 3

233 ± 035 473 ± 055 260 ~ 031

~3 ~i 4 --8

that following 'rostral' median raphe lesions. Other attempts to correlate the biochemical data with lesion locus and size did not produce any significant results. Dorsal raphe lesion. The dorsal raphe lesions produced significant reductions (Table II) in the 5-HT content of the brain stem, diencephalon, and remaining telencephalon (excluding the hippocampus and striatum). In contrast to the effect of median raphe lesions, the dorsal raphe lesions also reduced striatal 5-HT, but failed to affect hippocampal 5-HT. In addition, the di- and telencephalic 5-HT falls were significantly greater after dorsal than median raphe lesions. The 5-HT content of the spinal cord was not affected. The 5-HT falls in the di- and telencephalon were accompanied by significant reductions in 5-HIAA (Table II). NE, however, was unaffected in the areas assayed (Table Ill). As seen in Table V, lesions in the 'rostral' dorsal raphe produced a greater reduction in striatal 5-HT content (66 ~) than lesions in the 'mid-caudal' dorsal raphe TABLE

IV

REGIONAL 5-HYDROXYTRYPTAMINE

(ng/g) FOLLOWING MEDIAN RAPHE LESIONS; EFFECT OF ROSTRAL-

CAUDAL POSITION

Region

Rostral

Caudal

n

Mean ± S.D.

o/,

p**

n

Mean ± S.D.

,. %*

p**

Telencephalon

12

21l ± 021

--23

5

252 ± 039

-- 8

NS

Hippocampus

13

055 ± 022

--76

< 0.001 ( < 0.02) -< 0.001

5

170 :£ 058

--27

~ 0.001

5

343 :~: 047

F 5

NS

5

322 ± 073

--16

NS

5

277 ± 048

-Y 2

NS

(< o.ool) Striatum

13

343 ± 030

+ 5

Diencephalon

13

318 ± 040

--17

Spinal cord

13

249 ± 034

-- 8

NS (NS) < 0.001 (NS) NS (NS)

* ~ m e a n differences f r o m the control values (cf. Table II). * * P values (determined by Student's t-test, two-tailed) are given for m e a n differences from controls a n d between lesion groups (in parentheses).

REGIONAL

5-HT

53

AFTER RAPHE LESIONS

TABLE V REGIONAL 5-HYDROXYTRYPTAMINE ( r i g / g ) FOLLOWING DORSAL RAPHE LESIONS." EFFECT OF ROSTRALCAUDAL POSITION

Region

Telencephalon

Rostral

Mid-caudal

n

Mean q- S.D.

%*

9

131 ± 046

--52

P** < 0.001

n

Mean 4- S.D.

%*

P**

10

134 ± 035

--51

10

233 ± 052

0

9

206 4- 046

--37

< 0.001

10

301 4- 047

--22

< 0.01

10

258 -- 033

-- 5

NS

< 0.001

(NS) Hippocampus

9

230 4- 031

-- 1

NS

NS

(NS) Striatum

9

112 ± 040

--66

Diencephalon

9

264 -- 027

--31

< 0.001 ( < 0.001) < 0.001

(NS) Spinal cord

9

255 4- 032

-- 6

NS

(NS) * ~ m e a n difference f r o m control (cf. Table I). ** D e t e r m i n e d by Student's t-test, two-tailed; P values in parentheses refer to differences between lesion groups.

(37 ~). Otherwise, the 5-HT effects of these two types of dorsal raphe lesions were quite similar. Lastly, two of the dorsal raphe lesion animals were dropped from the study although their lesions were not histologically verified. These animals were eliminated because their striatal 5-HT concentrations were within normal range. The striatal 5-HT concentrations of all histologically verified lesions (n = 19, with no rejections), in contrast, were all below normal range. DISCUSSION

Striking differences were observed between the effects of dorsal and median raphe lesions on central regional 5-HT concentrations. (1) Only lesions in the median raphe nucleus produced a fall in hippocampal 5-HT. In contrast, only dorsal raphe lesions affected striatal 5-HT. (2) Although both lesions produced reductions in the 5-HT and 5-HIAA concentrations of the remaining portion of the telencephalon and of the diencephalon, the decreases observed following dorsal raphe lesions were significantly greater than those seen after median raphe lesions. The greatest number of ascending 5-HT fibers, therefore, appear to originate in the dorsal raphe nucleus. The striatal and hippocampal 5-HT projections, however, seem to arise chiefly in the dorsal and median raphe nuclei, respectively. Only the dorsal raphe lesion group showed a significant reduction (21 ~) in brain stem 5-HT content. The dorsal raphe lesions also produced significantly greater falls in regional forebrain (except hippocampus) 5-HT than median raphe lesions. This suggests that the number of 5-HT neurons in the dorsal raphe nucleus is greater than that of the median raphe nucleus. This observation supports the view of Bj6rklund

54

s.A.I.ORENS

A N D |1. ( ' . G U L I ) B I : R ( I

who have reported that 5-HT (C-type) neurons predominate in the dorsal raphe nucleus, whereas B-type (possibly 5-methoxytryptam ine) cell bodies are more numerous in the median raphe nucleus. It is not surprising, furthermore, that lesions restricted to either the dorsal or median raphe nucleus should not greatly affect brain stem 5-HT content as these are hut two of the many 5-HT cell groups in the brain stem 1'~. It seems reasonably clear that the dorsal raphe nucleus supplies a substantial portion of forebrain 5-HT fibers. In addition, the 5-HT input to the striatum appears to originate principally in this nucleus. It is interesting in this respect that the position of the dorsal raphe lesions (all of which were approximately the same size) was important in determining the degree of reduction in striatal 5-HT (Table V). Dorsal lesion position, however, did not differentially affect 5-HT content in other forebrain regions. This suggests the possibility that the course taken by the dorsal raphe-striatal projection is different from that to other forebrain areas. Whereas the latter may well descend through the brachium conjunctivum before ascending in the medial forebrain bundle, striatolopetal fibers may follow a course rostrally through the nucleus itself and then turn ventrally at a more anterior level. Bj6rklund et al. 6 have recently described a dorsal indoleamine pathway arising in the B5 and 6 cell groups and coursing through the medial longitudinal fasciculus before abruptly descending near the fasciculus retroflexus. Perhaps the striatolopetal projection from B7 follows a similar course. Of interest in this regard is the recent report of Jalowiec et al. '6 who failed to observe a fall in cat striatal 5-HT after lesions in the ventral tegmental area of Tsai which transected a number of other ascending 5-HT fibers. It appears that a greater number of ascending 5-HT fibers arising in the dorsal raphe nucleus bypass the diencephalon to reach telencephalic structures. As seen in Table II, the dorsal lesions produced a fall (50 ~o) in telencephalic (excluding striatum and hippocampus) 5-HT which was twice as great as the fall (24~) in diencephalic 5-HT. Since the median raphe nucleus itself does not seem to contribute an enormous number of diencephalic 5-HT fibers, the 5-HT input to this region is clearly reinforced by fibers originating in other cell groups (B9, for example). The effects on forebrain 5-HT concentration following median raphe lesions present several difficulties in interpretation. In the first place, the di- and telencephalic (excluding hippocampus) falls in 5-HT are not great (Table II). How much these falls are due to interruption of fibers leaving the dorsal nucleus is unclear. Of particular importance in this respect is the observation that lesions in the 'caudal' median raphe (Table IV) only affected hippocampal 5-HT. Furthermore, Jalowiec et al. t6 have reported that lesions in the tegmental nuclei of Gudden produced a reduction in telencephalic (but not hypothalamic) 5-HT. It is well known that these nuclei not only send fibers to but en p a s s a g e through the median raphe nucleus v,2v. In addition, the medial longitudinal fasciculus was damaged either unilaterally or bilaterally by all, and area B6 was invaded by 8 of the histologically verified median raphe lesions. Thus, the number of 5-HT fibers originating in the median raphe nucleus which project to the forebrain (excluding hippocampus) remains a matter for further study. The potential origin of a large number of hippocampal 5-HT neurons in the median raphe nucleus is of great interest. In this respect, it should be pointed out that et al. a

REGIONAL 5-HT AFTER RAPHE LESIONS

55

hippocampal 5-HT almost certainly derives from fibers originating outside this area3Z,3L Furthermore, although damage to fibers originating in the medullary and pontine 5-HT cell groups, such as B2 s and B56, cannot be ruled out, certainly fibers en passage from the dorsal raphe nucleus can, as lesions in this nucleus failed to affect hippocampal 5-HT. However, Jalowiec et al. 1° observed reductions in posterior pyriform (including hippocampus) 5-HT after lesions in the region o f Gudden's nuclei in the cat, suggesting that some hippocampal 5-HT fibers arise in these nuclei. Thus, although the data suggest that hippocampal 5-HT derives largely from fibers originating in the median raphe nucleus, the possibility of such 5-HT fibers arising in other cell groups cannot be ruled out. Brodal et al. s described retrograde degeneration following cervical hemisection only in the pontine and medullary raphe nuclei. Histochemical studies 1°,11,14 have suggested that descending 5-HT fibers originate primarily in these nuclei. The present data can be considered to support the view that spinalopetal 5-HT fibers have their origin in the raphe nuclei of the pons and medulla, as our midbrain raphe lesions failed to affect spinal 5-HT content. Lastly, it should be noted that lesions well restricted to the midbrain raphe nuclei did net result in any significant effect on central NE concentration. ACKNOWLEDGEMENTS

The authors wish to thank Mrs. S. A. Lorens, Miss Randi Soraas and Mr. H. Bergesen for their invaluable technical assistance. Dr. Charles Marsden is to be acknowledged for his helpful comments on the dissection procedure. This research was supported in part by the Norwegian Research Council for Science and the Humanities, and by National Institute on Drug Abuse Grant DA 00568-01 (to S.L.). REFERENCES 1 AGHAJANIAN,G. K., ROSECRANS,J. A., ANDSHEARD,M. H., Serotonin: release in the forebrain by stimulation of midbrain raph6, Science, 156 (1967) 402-403. 2 AHTEE, L., SHARMAN, O. F., AND VOGT, M., Acid metabolites of monoamines in avian brain; effects of probenecid and reserpine, Brit. J. Pkarmacol., 38 (1970) 72-85. 3 ANDI~N,N.-E., DAHLSTROM,A., FUXE,K., LARSSON,K., OLSON,L., ANDUNGERSTEDT,U., Ascending monoamine neurons to the telencephalon and diencephalon, ,4cta physiol, scand., 67 (1966) 313-326. 4 BERTLER, ,~., Effect of reserpine on the storage of catecholamines in brain and other tissues, Acta physiol, seand., 51 (1961) 75-83. 5 BJ~RKLUND,m., FALCK,B., AND STENEVI,U., Classification of monoamine neurons in the rat mesencephalon: distribution of a new monoamine neuron system, Brain Research, 32 (1971) 1-17. 6 BJORKLUND,A., NOBIN, A., AND STENEVI,U., The use of neurotoxic dihydroxytryptaminesas tools for morphological studies and localized lesioning of central indoleamine neurons, Z. Zellforsch., 145 (1973) 479-501. 7 BRinGS,T. L., ANDKAELBER,W. W., Efferent fiber connections of the dorsal and deep tegmental nuclei of Gudden. An experimental study in the cat, Brain Research, 29 (1971) 17-29 8 BRODAL,A., TABER,E., AND WALBERG, F., The raph6 nuclei of the brain stem in the cat. II. Efferent connections, J. comp. Neurol., 114 (1960) 239-260. 9 BUXaAUM,D. M., YARaROUGH,G. G., ANDCARTER,M. E., Biogenic amines and narcotic effects. I. Modification of morphine-induced analgesia and motor activity after alteration of cerebral amine levels, J. Pharmacol. exp. Ther., 185 (1973) 317-327.

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S. A. I_ORENS AND H. C. GULI)BIRG

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