Gabaergic interneurons in the dorsal raphe mediate the effects of apomorphine on serotonergic system

Brain Research Bulletin, Vol. 18, pp. 345-353.D PcrgamonJournalsLtd., 1987.Rinted in the U.S.A.

0361~9230/87 $3.00 + .OO

Gabaergic Interneurons in the Dorsal Raphe Mediate the Effects of Apomorphine on Serotonergic System EMINY

H. Y. LEE,’

FENG

B. WANG,

YU P. TANG

AND

MARK

A. GEYER*

Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, The Republic of China and *Department of Psychiatry, University of California at San Diego, La Jolla, CA 92093 Received

27 May 1986

LEE, E. H. Y., F. B. WANG, Y. P. TANG AND M. A. GEYER. Gabaergic interneurons in the dorsal raphe mediate the effects ofapomorphine on serotonergic system. BRAIN RES BULL 18(3) 345-353, 1987.-Apomorphine (APO) has been shown to elevate the concentrations of serotonin (5HT) and its major metabolite 5-hydroxyindoleacetic acid (J-HIAA) in

the mesostriatal but not the mesolimbic serotonergic systems. We have previously demonstrated that the serotonergic actions of APO were secondary to dopamine (DA) autoreceptor stimulation in the substantia nigra. Using picrotoxin as a pharmacological tool, we have presently found that these effects of APO were also indirectly mediated through gammaaminobutyric acid (GABA) neurons. In examination of the exact anatomical locus of GABA neurons responsible for the

observed effects of APO, the results indicate that bilateral lateral habenular lesions did not block the effects of APO on 5-HT neurons, while direct picrotoxin infusion to the dorsal raphe, at a dose having no significant influence by itself, antagonized APO’s actions. Together with the anatomical, biochemical and histofluorescent findings, it is suggested that APO influences dorsal raphe 5-HT by stimulation of DA autoreceptors in the substantia nigra; therefore, inhibition of DA neuron activity and the nigro-raphe pathway. Normally, DA probably exerts an excitatory influence on gabaergic interneurons in the dorsal raphe, and these inhibitory interneurons then synapse on 5-HT neurons in the same area. Activation of 5-HT neurons were explained by a disinhibitory effect as a result of reduced release of GABA due to feedback inhibition of DA neuron firing following APO activation of DA autoreceptors in the substantia nigra. The striatal presynaptic and postsynaptic DA receptors, however, do not appear to mediate the above effects of APO. Apomorphine Serotonin Lateral habenular lesion

Dopamine Gamma-aminobutyric acid Dorsal raphe Picrotoxin infusion High performance liquid chromatography

APOMORPHINE (APO) is believed to be a specific dopaminergic agonist in the central nervous system [3, 6, 71. However, biochemical studies have suggested that APO also influenced central serotonergic neurons in rat and mouse [ 15,303. Anatomical [4] and histological [lo] evidence has demonstrated that the dorsal and median raphe nuclei in rat midbrain provide two distinct but somehow overlapping serotonergic systems: the pathway originating in the dorsal raphe primarily innervates the striatum and thalamus; it also projects to the ventral hippocampus and amygdala. Another pathway derived from the median raphe mainly innervates the hippocampus and septal area. Since the striatum and most of the hippocampus are the terminal regions of the dorsal and median raphe nuclei, respectively, Geyer et al. [ 10,l l] have used the terms “mesostriatal” and “mesolimbit” serotonergic pathways to distinguish these two systems. Various pharmacological and behavioral studies have proven that these two projections are functionally differentiated as well [8,9, 111. Using fluorescence histochemical,

‘Requests for reprints should be addressed Taiwan, The Republic of China.

Median raphe Rat

biochemical and pharmacological methods, we have previously reported that APO selectively elevated serotonin (5HT) as well as 5hydroxyindoleacetic acid (5-HIAA), the major metabolite of S-III’, levels in the dorsal raphe and striatum. Conversely, 5-HT neurons in the median raphe and hippocampus were not significantly influenced by APO [ 191. Further anatomical, biochemical and histological studies have suggested that the effects of APO on the mesostriatal serotonergic system were indirectly mediated through dopamine (DA) neurons in the substantia nigra and the nigro-raphe pathway [20,22]. Moreover, using 6-hydroxydopamine as a pharmacological tool [21] and direct APO infusion technique [21,221, we have demonstrated that DA autoreceptors in the substantia nigra mediate APO’s effects on 5-HT. The postsynaptic DA receptors were not responsible for the serotonergic actions of APO [21]. Anatomical evidence has demonstrated the co-existence of DA, 5-HT and GABA in several brain regions; for example, the striatum, the substantia nigra and possibly the dorsal

to Dr. Eminy H. Y. Lee, Institute of Biomedical Sciences, Academia Sinica, Taipei (11529)

345

FIG. 1. ~monst~tion of electric tesion in the lateral h~~benu~~nuclei. A learn-thjck section through the lateral h~~be~tit~I> \ho~r~. 1he lateral habenula was lesioned bilaterally with passage of a current of 2 mA for 10 sec. H: hippocampus. LH: lateral hahenula. M H: medial hahenula.

raphe nucleus [2_5]. Therefore, GABA may be a neurotransmitter at some step in a multisynaptic pathway by which APO affects 5-HT activity in the dorsal raphe. One major gabaergic projection is a polysynaptic pathway efferent from the striatum to the entopeduncular nucIeus, it then follows the projection from the ento~duncu~ar nucleus to the lateral habenula and feedback influence 5-HT neurons in the dorsal raphe [ 1,24,32]. This suggestion is based on a few findings emphasizing the important role of the habenular complex as a major conduction system of info~ation between the forebrain and midbrain [34,35]. Alternatively, interactions among DA, GABA and 5-NT could occur within the dorsal raphe nucleus since ~munohistochemi~~ studies have suggested the coexistence of GABA and 5-m neurons in this area [ZS], and a dopaminergic projection from the substantia nigra to the dorsal raphe was also proposed 1221. The present study was designed to examine the role of GABA and the exact anatomical locus of GABA neurons in mediating the effects of APO on the mesostriatal serotonergic system. The possible involvement of striatal presynaptic DA receptors was also studied. Results suggest that GABA, but not presynaptic DA receptors, is involved in the serotonergic actions of APO and that the dorsal raphe nucleus is the area where neurotransmitter interactions occurred. The possible synaptic relationships and neurochemical events are discussed.

METHOD Subjects

Animals were experimentally naive male Sprague-Dawley rats weighing 17.5-225 g. Upon receipt from the supplier (Animal Resource Center, National Taiwan University, Taiwan, The Republic of China), animals were housed in pairs in a temperature-regulated animal room (25~2°C) on a 12112 hr 1ightJdark cycle with free access to food and water. At least a week was allowed prior to any expe~m~ntation, Animals were sacrificed by decapitation at appropriate time after stereotaxic surgery and drug administration. The dorsal raphe, median raphe, striatum and hippocampus were dissected according to the method of Knapp et tri. [19J and were collected for biochemical assays. Drugs

Apomorphine hydrochloride and picrotoxin (Sigma) were used. Doses refer to the salt form. Drugs were dissolved in 0.9% isotonic saline immediately before use. Due to rapid decomposition, a fresh APO sofution was replaced every 2 hr. Both drugs were given either intr~~tone~~y (iP) or by direct intracerebral infusions. The systemic injection volume was 1 mb’kg. The dose of APO was determined from a previous dose-response study which produced maximum effects on 5-HT neurons [ 191.

347

FIG. 2. Distribution of infused dye in the dorsal raphe. A 16qm-thick section through the antero-dorsal raphe is shown. The dorsal raphe was infused with methylene blue dye (3 mg/ml) at a rate of 0.4 pl/min for 3 min. The area of the infused dye is indicated by the arrows. DR: dorsal raphe. MR: median raphe.

Infusions

Lesion

Animals receiving drug infusions were anesthetized with Nembutal(40 mg/lcg, IP). Picrotoxin was infused to the dorsal raphe at a rate of 0.8 &0.8 pU3 mm through a 30 gauge stainless steel needle attached by polyethylene tubing to a ten ~1 Hamilton syringe. The coordinates of needle placement were AP+ 1.0 mm; L&O.0 mm; DV-6.6 mm from lambda. Apomorphine was infused to the striatum bilaterally at the following coordinates: AP+0.4 mm; Lk2.7 mm; DV -5.4 mm from Bregma. The tooth bar was at -2.4 mm. One minute was ahowed before removing the needle at the end of the infusion to miniznize drug backflow.

Electric lesions were made in the lateral habenula bilaterally at the following coordinates: AP-3.1 mm; LkO.7 mm; DV - 5 mm from Bregma with tooth bar at - 2.4 mm. Animals were anesthetized with Nembutal (40 mg/kg, IP), and the electrode (insulated except at the tip) was mserted stereotaxically into the nuclei. Lesions were made by passage of DC current of 2 mA for 10 set from a Grass Model S8 stimulator. Histology For histological

verification

of needle placement

in the

FIG. 3. Localization of infused dye in the striatum. A 16-pm-thick section through the striatum is shown. The striarum w:l\ mfused bilaterally with methylene blue dye (3 mglmf) at a rate of 0.5 &‘min for a total of 3 ~1. RV: right ventricle.

dorsal raphe and striatum, methylene blue dye (3 mg/ml) was infused using the same coordinates as picrotoxin and APO infusions in separate animals. The dye-infused brains were frozen-sectioned in a cryostat. For histological examination of electrode placement in the lateral habenula, the iesioned brains were also frozen-sectioned in a cryostat and checked individually. Sixteen-micron thick sections taken at 50 Frn intervals through the dorsal raphe, striatum and habenula were mounted on slides and stained with thionine blue. Those animals whose needle and electrode placement was not in the correct area were deleted from the study. Figures I,2 and 3 illustrate the placement of the electrode and needle positions in the lateral habenula, dorsal raphe and striatum. respectively, under microscopic examination.

The chromatographic system used was a 5 pm Ultrasphere QDS reverse phase column (4.6 mm by 15 cm, Altex) with an Altex pump and Shimadzu RF-530 spectromonitor; excitation and emission wavelengths were at 290 and 330 nm, respectively. The flow was maintained at 2 ml/min; the sensitivity set at 2 nAfV. The mobile phase consisted of 0.02 M potassium phosphate monobasic containing 1 g/liter of I-h~ptanesulfonic acid sodium salt (pH 3.3) and a mixture of methanol: Hz0 (3:2). Output was recorded with a Shimadzu C-RlB Integrator. Tryptophan, S-HT and 5-HIAA were estimated according to the method of Peat and Gibb (271 with some modifications.

Tissue was weighed while still frozen and homogenized in 10 vol. of 0.1 M perchloric acid containing 4 mM sodium metabisulfite. The homogenate was then centrifuged at 15,000 rpm for 15 min using a refrigerated centrifuge, and the clear supernatant was injected directly into the chromatographic system.

The data were analyzed using the one-way analysis of variance (ANOVA). Specific comparisons between each treatment group and a common control group were made with Dunnett’s method.

To examine the role of GABA in mediation of APO’s actions, thirty-four rats were randomly divided into four equal groups. Group I (N=7) received saline/saline injections; Group 2 (N=7) received saline/APO (I mgikg) injections: Group 3 (N=7) received picrotoxin (4 mg/kg)/saline injections; Group 4 (N=7) received picrotoxin/APO injections. The time interval between the first and second injections was 1.5min. Animals were sacrificed 30 min after the second injection.

This experiment was designed to investigate the possibility of lateral habenula involvement in the mediation of the effects of APO on S-HT neurons. Thirty-two rats were

349

TABLE 1 EFF%CTS OF A~~O~~E

(1 trig/kg,IF) AND FICROTOXIN (4B&& W) ON TRYl'TOpHAN,5-m AND S-HIAALEVRLS IN THE DORSAL RAFHR AND STRIATUhi

Dorsal Raphe Treatment

N

SAL APO PIG PIG + APO

7 7 7 7

Trp 2445 3584 2377 2650

Striatum

5-HT

k 85 * 11l3t + 158 k 138

3075 3966 3424 3320

r t c rt

5-HIAA

334 243* 2% 250

10462 74 1573 r 97t 1102 r 98 1205 ‘, 108

5-HT

Trp 3334 4283 3473 3566

f 84 + 463* + 143 f 148

932 1157 958 962

4 2 + +

5-HIAA 22 477 58 55

952 1131 999 994

rfr34 z!z712 t 56 St 43

*p
TABLE 2 EFFECtXOF A~MO~HINE

(1 mg&, IP) AND PICR~O~N (4n&kg.IP) ON TRYWOPHAN, THE MEDIAN RAPHE AND BONUS Median Raphe

Treatment

N

SAL APO PIC PIG + APO

7 7 7 7

5-HT

Trp 2217 2262 1829 1820

rt + r i

273 220 129 169

1697 1578 1469 1587

r + r 2

Hippocampus 5-HIAA

105 113 109 t71

SHT AND 5-~&+ LEVELS IN

1Ow-c83 894 2 83 1208 z!z 59 885 r 154

Trp 3646 3772 3829 3709

T’_226 k 245 * 361 + 289

S-HT 1014 967 1153 975

r 74 r 62 z!z60 s 75

5-HIAA 868+ 953 f 8682 970 zk

90 51 110 95

Data are expressed as in Table 1.

randomly divided into four groups. Group 1 (N=7) had sham operation and a single IP injection of saline; Group 2 (N=7) had sham operation and an IP injection of 1 mg/kg APO; Group 3 (N=9) had bilateral lateral habenular lesion and an IP injection of saline; Group 4 fN=9) had bilateral lateral habenular lesion followed by an IP injection of 1 mg/kg APO. Apomorphine and saline injections were given 7 days after the surgery. Animals were sacrificed 30 min after injections. Experiment 3

This experiment.aimed to examine the role of GABA in the dorsal raphe in the ‘mediation of the serotonergic actions of APO. Thirty rats were randomly assigned to four groups. Group 1 (N =6) had saline infusion into the dorsal raphe and an IP injection of saline; Group 2 (N=6) had saline intusion to the dorsal raphe and an IP injection of 1 mg/kg APO, Group 3 (N=8) had picrotoxin infusion to the dorsal raphe (0.8 &0.8 $/3 min) and an IP injection of saline; Group 4 (N=8) had picrotoxin infusion to the dorsal raphe at the same infusion rate used above and an IP injection of 1 mg/kg APO. The time interval between surgery and injection was 15 min. Animals were sacrificed 30 min after injections. Experiment 4

We have previously demonstrated that the striatal postsynaptic DA receptors are not responsible for the serotonergic actions of APO [20]. To examine the possible importance of presynaptic DA receptors in the same area in

mediating APO’s effects, twenty-four rats were assigned to four groups of six rats each. Group 1 received bilateral intrastriatal saline infusion at a rate of 2.5 pi/S mitt/per side; Group 2 received bilateral intrastriatal APO infusion at a rate of 0.05 ~812.5 pi/S mm/per side for a total of 0.05 fig; Group 3 received bilateral intrastriatal APO infusion at a rate of 0.8 pgl2.5 @l/Smitt/per side for a total of 0.8 pg; Group 4 received the same APO infusion at a rate of 5 pg/S mitt/per side for a total of 5 pg. Animals were sacrificed 20 min after drug infusions. RESULTS

Experiment I

Consistent with previous reports 113,161,Tables I and 2 indicate that APO preferentially elevated tryptophau, 5-HT and MIIAA levels in both the dorsal raphe, t(3,24)=6.49, ~~0.01; t(3,24)=2.19, ~~0.05; and t(3,24)=6.35, pCO.01; respectively, and striatum, t(3,24?=2.57, ~~0.05; t(3,24)= 3.36, pO.O5, ns.).

350

LEE.

WANG.

TANG

AND

GEYER

TABLE 3 EFFECTS OF AFOMORPHINE

(I mg/kg) AND LATERAL HABENULAR LESION ON TRYFTOPHAN, DORSAL RAPHE AND STRlATUM

5-W

AND 5-HIAA

I_.FVFLS

IN THF

“_

Dorsal Raphe

striatum

Treatment

N

Trp

5-HT

SHAM

7

3273 rt 193

3010 t 57

it05 i-

46

4205 zk 77

1050 2

62

706 i so

SHAM

7

3762 tt 250*

3435 -e t99*

1231 i-

57

4656 I 14.5”

1301 i

72*

X61 ir 73”

+ APO NAB. LES

8

3584 t 295

3204 2 198

1208 -t

86

4349 rf- 106

1092 +

8t

830 t 41*

+ SAL HAB. LES

8

4109 t t79t

3360 rt 100”

1301 i

1i5*

4680 + 148’

1320 ir 152*

S-HIAA

S-HT

Trp

5-HIAA

t SAL

1012 rt 64f

+ APO *p
I.

TABLE 4 EFFECTSOF APOMORPHINE

(I mg&gj AND PICROTOXIN INFUSION TO THE DORSAL RAPHE (0.8 &O.S $1 ON TRYPTOPHAN, S-HIAA LEVELS IN THE DORSAL RAPHE AND STRIATUM

Striatum

Dorsal Raphe Treatment

N

Trp

SHAM + SAL SHAM + APO PIC + SAL PIC + APO

6 6 6 7

30062 4060 r 2792 + 2768 -e

S-HIAA

5”HT 85 94i 198 198

2970 3428 3162 2928

i t r r

192 11s* 186 100

5-HT AND

1150 f 1,221 -+ l200 t 1126 +

48 122; 30 41

Trp 4500% 81 4773 r 108 4511 2 197 4621 c 410

S-HT 1186 t356 1169 1223

L 58 -+:50* rt 75 iz 74

5-HIAA 740 908 848 784

2 t + 2

54 58* I6* 22

*p
TABLE 5 EFFECTS OF A~MORPH~N~

INFUSION

TO THE STRATUS ON TRY~OPHAN, RAPHE AND STRIATUM

S-HT AND 5-HIAA LEVELS IN THE DORSAL

Median Raphe

Dorsal Raphe Treatment

N

SAL APO (0.05 pg)

6 6 6 6

APO (0.8 & APO (5 ,ud Data are

Experiment

5-NT

Trp 2614 2711 2379 2509

“+ I t

185 291 82 136

2903 3105 3166 2965

I 94 c 327 L 234 xh 114

S-HIAA 996 k 42 949 t 95 1067+82 974 + 48

3911 4024 3959 3603

5 2 2 2

5-HfAA

S-HT

Trp 122 401 190 219

1308 1348 1242 1241

-t t rt it

68 74 60 54

770 775 761 787

if “I r

65 99 29 47

expressed as in Table 1

2

Effects of APO and lateral habenular lesion on 5-HT neurons in the mesostriatal and mesoiimbic systems are summarized in Table 3. As revealed in this table, APO significantly increased tryptophan and S-HT concentrations in the dorsal raphe; tD(4,26)=2.21, p
tD(4,26)=2.15,p<0.05; and tD(4,26)=2.23,p<0.05; respectively. As the results in the dorsal raphe. APQ consistently augmented tryptophan, _S-HT as well as 5-H&AA levels in the striatum, rD(4,26)=2.42, ~~0.05; tD(4,26)=2.27, ~~0.05; and tD(4,26)=2.28, ~~0.05; respectivety. Lateral habenuiar iesion also increased striatal S-HIAA content, tDf4,26)=2.19, ~~0.05, but it did not antagqnize the serotonergic actions of APO in this area, tD(4,26)=2.7, p-c&OS; tD(4,26)=2.16,p<0.05; and rD(4,26)==4.46, p
pcO.01;

A~MO~HINE

AND SEROTONIN

addition, effects of APO and habenular lesion on 5-HIAA appeared to be additive in the striatum, rD(4,26)=4.46, pCO.01. Similar to the results in Table 2, neither APG and habenular lesion alone nor the ambition of these two treatments markedly altered any biochemical measures examined in the median mphe and hippocampus (all p>O.O5, n.s.), and data are not shown. Experiment 3

Table 4 summarizes the effects of APO and picrotoxin infusion to the dorsal raphe on the mesostriatal serotonergic system. As shown in this table, APO consistently increased t~ptoph~, 5-HT and 5-HIAA contents in the dorsaI raphe, rD(4,21)=4.86, ~~0.01; tD(4,21)=2.1, pcO.05; and tM4,21)=11.57, pO.O5, n.s.), and data are not shown. Experiment 4

Effects of striatal APO infusion on tryptophan, 5-HT and 5-HIAA levels in the same regions examined above are indicated in Table 5. As shown in this table, APO at both the low and high doses did not significantly influence any of these measures in both the dorsal raphe and striatum. Serotonin neurons in the median raphe and hippocampus were not altered by APO either (all ~~0.05, n.s.) and data are not shown. DISCUSSION The present results are consistent with previous histofluorescent and biochemical findings that APO selectively increased S-HT and 5-HIAA concentrations in the dorsal raphe and striatum without markedly altering the same measures in the median raphe and hippocampus [19,20]. Furthermore, APO was found to augment tryptophan, the synthetic precursor of S-HT, in the same areas as well. Tryptophan hydroxylase is known as the rate-limiting enzyme in the process of S-HT biosynthesis, and brain tryptophan concentrations are subsaturating under normal conditions. Therefore, the increment of 5-HT and 5-HIAA in both regions may be due to the eievation of tryptophan levels in these nuclei. We have demonstrated and discussed the same results in another study 1241.Apomorphine probably facilitated the uptake of tryptophan into 5-I-IT neurons. It may also influence other neurotransmitter systems and indirectly modulate the activity of 5-HT neurons in the dorsal raphe. However, more studies are needed to clarify the pharmacological and molecular mechanisms of APO on 5-HT system. In assessing the role of GABA in mediating the serotonergic actions of APO, the present results revealed that gabaergic neurons in the dorsal raphe, but not the lateral habenula, are responsible for the observed effects of APO. Lack of an effect of lateral habenular lesion also suggests

351 that the proposed polysynaptic gabaetgic pathway Originating from the striatum, course through the entopeduncular nucleus and lateral habenula to the dorsal raphe is not involved in APO’s actions. This suggestion is also consistent with the results of a previous APO time-course study since effects on 5-HT neurons wefe observed as soon as 10 min after systemic APO ~m~ist~tion 1191.In addition, lateral habenular lesion a1one augmented S-HIAA level in the striatum. This observation is congruent with a previous report showing an accelerated rate of 5-HT metabolism following bilateral habenular lesion [30,31]. This result is explained by a disinhibition effect of 5-HT neurons since electrical stimulation of the lateral habenula was shown to inhibit dorsal raphe neuron firing [32], and GABA was suggested to be the inhibitor ne~t~s~tter utilized in this pathway [35]. Furthermore, picrotoxin given at a relatively higher dose (8 mglkg) produced similar effects as lateral habenular lesion; it elevated tryptophan, 5-HT and 5-HIAA levels in the dorsal raphe (unpublished observations). Moreover, the habenula was also shown to contain other neurotransmitters, for example, acetylcholine (Ach), substance P (SP) and possibly histamine [S]. The present results also rule out the possibilities of these neurotransmitters in mediation of APO’s effects on 5-HT in this area. In the present study we have also found that picrotoxin infusion to the dorsal raphe, at a dose without producing a significant influence on 5-I-IT by itself, antagonized the effects of APO on tryptophan, 5-HT as well as 5-HIAA in the mesostriatal serotonergic system. These results suggest that the dorsal raphe nucleus may be the anatomical locus for neurotransmitter interactions to occur. Evidence from immunohistoch~mic~ studies has demonstrated the coexistence of GABA and 5-HT neurons in the dorsat raphe 1251. Using neuronal tracing technique, Aghajanian and Wang have observed gabaergic terminals in the arca immediately surrounding the clusters of 5-HT cells in this nucleus [2]. Other investigators have also supported the notion of gabaergic interneurons in the same region [13,23]. Together with our previous finding of a dopaminergic afferent from the substantia nigra to the dorsal raphe [22], the synaptic relationships might be that the excitatory dopaminergic inputs terminate on gabaergic intemeurons in the dorsal raphe, these inhibitory inteme~ons then synapse on 5-HT neurons in the same nucleus. Elevations of tryptophan, 5-HT and 5-HIAA levels in the dorsal raphe and striatum after APO may be due to a reduced release of DA in the nigro-raphe pathway since electrical stimulation of the substantia nigra has been shown to inhibit dorsal raphe neuron firing 132,331 and APO was demonstrated to inhibit DA neuron firing in the substantia nigra via DA autoreceptor mediation [ 161.Therefore, the mesostriatal serotonergic system was activated through a disinhibition mechanism. This suggestion is further supported by a recent study showing that DA directly stimulated GABA release from the goldfish retina [26]. This proposed short pathway and synaptic connections are also consistent with the fast-acting effects of APO on the mesostriatal serotonergic system [19]. Moreover, the differential influence of APO on the mesostriatal and the mesolimbic serotonergic systems further confirms previous anatomical, behavioral and pha~acologic~ eviden :e that the dorsal and the median raphe nuclei constitute the origins of two distinct serotonergic projections f4, 8, 111. Similar to the results of habenular lesion, picrotoxin infusion to the dorsal raphe also augmented 5-HIAA level in the striatum. This finding further supports the concept of disin-

hibition of S-HT neurons; therefore, increased rate of S-H? release and turnover. Furthermore, there are other types of containing different neurotransmitters and neurons neuropeptides have been found in the dorsal raphe: for example, Ach, SP, enkephalin and possibly thyrotropinreleasing hormone 112, 17, IS]. Whether these neurotransmitters and peptides also indirectly mediate or modulate the serotonergic actions of APO requires further investigation. Finally, using direct APO infusion technique, we have found that striatal presynaptic and postsynaptic DA receptors do not appear to mediate the effects of APO on S-HT neurons. Together with the results of a previous study 1221. DA autoreceptors were demonstrated to be the only type of DA receptors mediating the pharmacological actions of APO. In summary, using biochemical and pharmacological methods, we have previously reported that APO preferentially augmented 5-HT and 5-HIAA levels in the mesostriatal serotonergic system without markedly altering 5-HT neurons in the mesolimbic system. Other than dopaminergic trans-

mission, we have presently demonstrated &hat APO’s ef’feezt~ were also indirectly mediated through GABA neurons. Using direct drug infusion and lesion techniques together, with biochemical assays. we have found that the dorsal raphe nucleus is the exact anatomical locus for DA, GABA and 5-HT interactions to occur. The lateral habenuIa, though is an important station conveying information between the forebrain and midbrain and is also rich in GABA. was found not to be responsible for the serotonergic actions of APO. Furthermore, the striatal presynaptic DA receptors. like the postsynaptic receptors. do not appear to kc involved in the above

effects

of APO.

This work was supported by a Grant from the National Science

Council (NSC75-0412-BOOl-01) ofTaiwan and partly by the research fund from the Institute of Biomedical Sciences, Academia Sinica. Taiwan, The Republic of China. Feng B. Wang was supported partly by the Institute of Biomedical Sciences, Academia Sinica.

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