SYNAF'SE 10:3443 (1992)

Cholinergic and GABAergic Mediations of the Effects of Apomorphine on Serotonin Neurons H.Y. CHEN, Y.P. LIN, AND E.H.Y. LEE Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China

KEY WORDS

Apomorphine, Dopamine, Acetylcholine, y-Aminobutyric acid, Serotonin, Atropine, Carbachol, Mecamylamine, Pirenzepine, Bicuculline, Saclofen, Dorsal raphe, Median raphe, Striatum, Hippocampus, Rat

ABSTRACT Apomorphine (APO)has been shown to elevate tryptophan, serotonin (5-HT),and 5-hydroxyindoleaceticacid (5-HIM)concentrations in the dorsal raphe (DR) and its corresponding projection site, the striatum, but not in the median raphe (MR)and its terminal area, the hippocampus. We have previously demonstrated that these effects are indirectly mediated through dopamine (DA)autoreceptors in the substantia nigra and possibly y-aminobutyric acid (GABA)neurons in or near the DR. In the present study, we have further found that the effects of APO on 5-HT neurons are also mediated through both nicotinic and MImuscarinic cholinergic receptors as well as GABAAreceptors in the DR. This suggestion is based on the findings that both atropine and mecamylamine antagonized the effects ofAPO, while carbachol at a high dose exerted an effect opposite to that of APO. Besides, pirenzepine and bicuculline at low doses also antagonized, whereas saclofen did not alter the influence ofAPO on 5-HT in the striatum. Bicuculline at a higher dose enhanced tryptophan and 5-HT measures by itself. None of the drugs studied had a significant effect on tryptophan, 5-HT, or 5 - H I M in the hippocampus. These results together suggest that DA, ACh, and GABA neurons are all involved in the actions of APO on 5-HT, while the direct synaptic relationships among these neurotransmitters and the precise anatomical locus for these interactions to occur are still unknown. It is possible that APO, by inhibiting DA neuron firing in the substantia nigra and through the GABA disinhibition mechanism, therefore indirectly activates 5-HT neurons in the DR and the striatum. While the above neuronal firing model well explains the elevation of 5-HIM, the simultaneous increases of tryptophan and 5-HT, especially tryptophan, may be more readily explained by a mechanism of tryptophan uptake upon APO administration. Further anatomical, biochemical, and electrophysiological studies are ongoing to test this hypothesis and to clarify the circuit and the anatomical locus (loci)for these interactions to occur. INTRODUCTION Apomorphine (APO) has been suggested to be a specific dopaminergic agonist in the central nervous system (CNS) (Anden et al., 1967; Ernst, 1967; Ernst and Smelik, 1966).However, biochemical and histochemical studies have demonstrated that AF'O also influences central serotonin (5-HT) neurons in rats and mice (Grabowska et al., 1973; Smialowska, 1975; Kuczenski et al., 1987). Anatomical (Azmitia, 1978)and histological (Geyer et al., 1976a) evidence has demonstrated that the dorsal and median raphe nuclei in rat midbrain constitute the origins of two distinct 5-HT cell groups and provide two differential, although somewhat overlapping, serotonergic projections: the mesostriatal serotonergic path@ 1992 WILEY-LISS,INC.

way originates from the dorsal raphe (DR) and primarily innervates the striatum and the thalamus; the mesolimbic serotonergic pathway is derived from the median raphe (MR) and projects to the hippocampus and the septa1 area (Geyer et al., 1976a,b). Other than the anatomical differentiation, various pharmacological and behavioral studies have demonstrated that these two pathways are functionally differentiated as well (Lee and Geyer, 1980; Geyer et al., 1975,1976b). Using Received December 31,1990; accepted in revised form May 17,1991 Address reprint requests to Dr. Eminy H.Y. Lee, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, The Republic of China. Ms. H.Y. Chen is currently a Ph.D. student at the Department of Physiology, Maryland University, College Park, MD; Y.P. Lin is a Ph.D. student in the Neuroscience Program, University of North Carolina,Chapel Hill, NC.

AF’OMORPHINE, DA, ACh, GABA, AND 5-HT

combined fluorescence histochemical, biochemical, and pharmacological methods, we have previously reported that APO selectively elevates 5-HT as well as 5-hydroxyindoleacetic acid ( 5 - H M ) , the major metabolite of 5-HT, levels in the DR and the striatum. The same treatment does not affect 5-HT and 5-HIAA in the MR and the hippocampus (Lee and Geyer, 1982). Further anatomical, biochemical, and histological studies have shown that the effects of APO on the mesostriatal serotonergic system are indirectly mediated through dopamine (DA) autoreceptors in the substantia nigra and the nigroraphe pathway (Lee and Geyer, 1983, 1984a,b).More recently, we have further found that the observed effects of APO are also indirectly mediated through y-aminobutyric acid (GABA) neurons, since picrotoxin pretreatment antagonizes the effects of APO on 5-HT (Lee et al., 1987). In other studies, DA receptor activation was found to stimulate the release of GABA in the basal ganglia of rat (Floran et al., 1990)and direct DA application was found to stimulate GABA release from the goldfish retina (Nagy et al., 1978). It is therefore possible that the nigral DA neurons probably exert an excitatory influence on GABA neurons; the enhanced GABAergic inhibitory inputs on 5-HT neurons caused by DA neuron activation may result in inactivation of 5-HT neurons in the DR. Apomorphine was shown to inhibit DA neuron firing in the substantia nigra via DA autoreceptor stimulation (Groves et al., 1975).Through a disinhibition mechanism, APO therefore produces an activation of 5-HT neurons and consequently elevates 5-HT and 5 - H I M concentrations in the DR and in its corresponding projection site, the striatum. Anatomical and histochemical evidence has suggested that the medullary raphe nuclei contain acetylcholine (ACh)neurons, which project to the spinal cord (Bowker et al., 1983).This finding implies the possibility that the same 5-HT-ACh interactions may also be present in other raphe nuclei. The cholinergic receptors can be generally divided into two pharmacologically distinguished types: nicotinic receptors, and muscarinic receptors. More recently, several drugs have been identified that can distinguish between muscarinic receptor subtypes (Ml-M5) (Bonner, 1989). Among these drugs, probably the most notable one is pirenzepine, which has formed the basis for the classification of receptors MI vs. M, (Hammer et al., 1980). M, receptors have high affinity for pirenzepine (Watson et al., 1984; Pitschner and Wellstein, 1988), whereas M, receptors have low affinity for the drug. Similarly, GABA receptors in the brain can be generally divided into GABAAand GABAB subtypes, although a third-class ‘‘GABG has also been reported in a few studies (Drew et al., 1984). With most neurons studied in the literature, the synaptic action of GABA is mediated by bicuculline-sensitive GABAAreceptors. However, recent studies have also revealed important physiological (Dutar and Nicoll, 19881,

35

pathological (Lloyd et al., 1985, 1987), and functional roles of GABAB receptors in mammalian brain (for review, see Bowery, 1989). The aims of the present study are to examine the possible involvement ofACh and GABA in the actions of APO on 5-HT neurons as well as to study the receptor subtypes involved in these actions. MATERIALS AND METHODS Subjects Animals were experimentally naive male SpragueDawley rats weighing 200-250 g. Upon receipt from the supplier (Animal Facility, Institute of Biomedical Sciences, Academia Sinica, Taiwan), animals were housed three per cage in a temperature-regulated animal room (22 & 2°C) on a 12112-hr lightldark cycle with food and water continuously available. At least 1 week in the adaptation room was allowed before any experimentation. Animals were sacrificed by decapitation at appropriate times after drug administrations. The DR, MR, striatum, and hippocampus were dissected out according to the method of Knapp et al. (1974) and were collected for biochemical assays of tryptophan, 5-HT, and 5-HIAA. Drugs Apomorphine hydrochloride, atropine, carbachol, mecamylamine, pirenzepine (Sigma), bicuculline, and saclofen (TocrisNeuramin) were used. Doses refer to the salt form. Drugs were dissolved in 0.9% isotonic saline immediately before injection. Because of rapid decomposition, a fresh APO solution was replaced every 2 hr. Apomorphine was given intraperitoneally (ip) at an injection volume of 1 mVkg; all other drugs were directly infused to the DR at a volume of 0.75 ~ 1The . dose of APO was determined from a previous dose-response study that produced the maximum and most consistent effects on 5-HT neurons (Lee and Geyer, 1982; Lee et al., 1987) Intra-DR drug infusions Animals receiving drug infusions were anesthetized with nembutal(40 mgkg, ip). All drugs were infused to the DR a t a rate of 1 pg/~V3min through a 30-gauge stainless steel needle attached by polyethylene tubing to a 10-+1Hamilton syringe. The coordinates of needle placement were AF’ + 1.0 mm, L -t- 0.0 mm, DV - 6.6 mm from lambda. The tooth bar was at -2.4 mm. One minute was allowed before removing the needle at the end of infusion to minimize drug backflow. To examine the possible diffusion of drugs from the DR, in two experiments atropine and bicuculline were also injected to the reticular formation (RF) area near the DR in separate animals.

Histology For histological verification of needle placement in the DR, animals were sacrificed at appropriate times

H.Y.CHEN ET AL.

36

after drug administration. Their brains containing the DR were frozen-sectioned in a cryostat and checked individually; 20-pm-thick sections taken at 40-pm intervals through the DR were mounted on slides and stained with thionine blue. Animals were accepted for data analysis if needle placement was located within the DR according to the atlas of Paxinos and Watson (1986). Figure 1A illustrates the placement of needle position in the DR of a representative rat after infusion of 0.5 pl methylene blue dye (3mg/ml) to the DR at the end of the experiment; Figure 1B illustrates the needle positions and dye distribution (0.5 p1 each side) in the RF in control animals. High-performance liquid chromatography with fluorescence detection of tryptophan, 5-HT, and 5-HIAA The chromatographic system used was a 5-pm Ultrasphere ODS reverse-phase column (4.6 mm x 15 cm, Altex) with an Altex pump and a Shimadzu RF-530 spectromonitor; excitation and emission wavelengths were set at 290 and 330 nm, respectively. The flow was maintained at 1.2 mumin; the sensitivity was set at 2 M.The mobile phase consisted of 0.02 M potassium phosphate monobasic containing 1 g/L of l-heptanesulfonic acid sodium salt (pH 3.3) and a mixture of methanol: H,O (3:2). Output was recorded with a Shimadzu C-RIB Integrator. Tryptophan, 5-HT, and 5-HIAA were estimated according to the method of Peat and Gibb (1983), with some modifications. Tissue was weighed whie still frozen and homogenized in 7 vol of 0.1 M perchloric acid containing 4 mM sodium metabisulfite. The homogenate was then centrifuged at 15,000 rpm (6,000g) for 15 min using a refrigerated centrifuge; the clear supernatant was injected directly into the chromatographic system. 5-HIAA,5-HT, and tryptophan were resolved a t 5.2,17.5, and 22.1 min, respectively. Statistics All the data were analyzed with separate sets of one-way analysis of variance (ANOVA). Specific comparisons between each treatment group and a common control group were made with Dunnett’s method. Comparisons between experimental groups were made by the Tukey’s method (Winer, 1971). Experiment 1 This experiment was designed to examine whether ACh neurons are involved in the actions ofAPO on 5-HT and whether muscarinic or nicotinic receptors are involved. Seventy-two rats were randomly divided into nine groups of eight rats each. Group I received saline + saline injections; Group I1 received saline + APO injections; Group 111 received atropine (0.05 pg/O.75pl) + saline injections; Group IV received atropine (0.1 pg/0.75 pl) + saline injections; Group V received atro-

pine (0.05 ~g/0.75pl) + APO injections; Group VI received atropine (0.1 pg/0.75 pl) + APO injections; Group VII received mecamylamine (0.25 pgI0.75 p1) + saline injections, and Group VIII received mecamylamine (0.25 pg/0.75 pl) + APO injections. In ) infused to the Group IX, atropine (0.1 pg/0.75 ~ 1was RF area near the DR to test for possible diffusion of the drug from the DR. The dose of APO used was 1mgkg. This dose was determined based on a previous doseresponse study that shows the most consistent and significant effect on 5-HT (Lee and Geyer, 1982). The first injection was given to the DR directly, and the second injection was administered intraperitoneally. The time interval between the first and the second injections was 30 min. Animals were sacrificed 30 min after the second injection. Experiment 2 If ACh neurons are involved in the actions of APO on 5-HT, the cholinergic agonist should produce an effect opposite that of atropine or mecamylamine. This experiment was designed to test this hypothesis by examining the dose-response effects of carbachol infused to the DR and the interactive effects of carbachol and APO on 5-HT neurons. Forty rats were assigned to five equal groups. Group I received saline (0.75 p1) + saline injections; Group I1 received saline + APO injections; Group I11 received carbachol (0.03 pg/O.75pl) + saline injections; Group N received carbachol (0.1 pgYO.75 p1) + saline injections, and Group V received carbachol (0.03 pg/0.75 p1) + APO injections. The dose of APO used was 1 mgkg. Other conditions were the same as that in Experiment 1. Experiment 3 This experiment was aimed to examine whether the muscarinic cholinerpc receptors are involved in the effects of APO on 5-HT from Experiment 1 and, if so, which receptor subtype is involved. Forty rats were randomly distributed to five equal groups. Group I received saline (0.75 pl) + saline injections; Group I1 received saline (0.75 p1) + APO injections; Group I11 received pirenzepine (2 pg/0.75pl) + saline injections; Group IV received pirenzepine (5 pg/0.75 J J , ~ ) + saline injections, and Group V received pirenzepine (2 pg/0.75 p1) + APO injections. All injection and sacrifice conditions were the same as that in Experiment 1. Experiment 4 We have previously suggested that GABA neurons are probably also involved in the actions of APO of 5-HT (Lee et al., 1987).This experiment was aimed t o further examine this hypothesis and to study whether GABAA or GABABreceptors are involved in the effects of APO. Sixty-four rats were randomly divided into eight equal groups. Group I received saline (0.75 p1) + saline injections; Group I1 received saline (0.75 ~ 1 +) APO injec-

APOMORPHINE, DA, ACh, GABA, AND 5-HT

Fig.1. A. Typical placement of injectionneedle in the DR of a representativerat. Methylene blue dye 0.5 +1(3mg/ml)was infused to the DR. A 2O-+m-thick section through the DR is shown. B. The needle position and dye distribution in the RF (0.5 ~1 each side) near the DR in control animals is shown.

37

38

H.Y. CHEN ET AL.

TABLE I. Effects of apomorphine (1 mg/kg), atropine, mecamylamine, as well as their interactive effects on 5-HT neurons in the striatum and the hippocampus 1-3 Treatment

+ + +

Sal Sal Sal APO Atr Sal (0.05 pg/0.75 p l ) Atr Sal (0.1 pg/0.75 p l ) Atr APO (0.05 pg/0.75 p l ) Atr APO (0.1 pg/0.75 p l ) Mec Sal (0.25 pgl0.75 p l ) Mec APO (0.25 pg/0.75 pl) Atr(RF) Sal

N

Striatum 5-HT

TrYP

*

Hippocampus 5-HT

5-HIAA

TrYP

962 f 59 1,050 f 93 1,008 f 59

532 f 24 564 f 34 543 f 43

5-HIAA

8 8 7

3,240 178 4,152 f 261* 3,487 372

+

900 f 41 1,553 f 202** 1,084 f 103

836 f 72 1,388 f 154** 936 f 76

2,769 f 299 2,840 f 309 2,870 z t 366

1,164 f 120*

2,851 f 290

1,085 f 71

563 5 27

2,747 f 203

880 f 85

518 f 62

2,814 f 257

1,035 f 68

572 f 42

1,088 f 119

555 f 49

+ + +

8

3,962 f 213*

1,296 f 117*

7

3,365 f 235*74

1,106 f 132**4

7

4,043 f 221*

1,479 5 155*

1,152 f 98*

+

8

2,964 f 162

898 f 97

746 f 62

2,858

+

7

3,081 f 278**j4

1,181 f 117*s4

850 f 28**34

2,718 f 266

972 f 95

472 f 35

8

3.384 f 297

1.010 f 93

885 f 92

2.818 f 279

950 f 90

529 f 46

+

914 f 8 4 * * ~ ~

+ 221

*P < 0.05 and **P< 0.01 as compared with the Sal + Sal controls.

‘Data are expressed as ng/g tissue and are not corrected for recovery. Values are means +SEM. 3AP0, apomorphine;Atr, atropine; Mec, mecamylamine; RF, reticular formation. 4As compared with the Sal +APO group.

tions; Group I11 received bicuculline (1.0 Fg/ 0.75~1)+ saline injections; Group IV received bicuculline (2.0 pg/0.75 p1) + saline injections; Group V received bicuculline (1.0 pg/0.75 p.1) + APO injections; Group VI received saclofen (1.0 pg/0.75 pl) + saline injections; and Group VII received saclofen (1.0 pg/0.75 pl) + APO injections. In Group VIII, bicuculline (2.0 ) infused to the RF near the DR to test for p.g/0.75 ~ 1was possible drug diffusions. Similarly, the first injection was directly given t o the DR and the second injection administered intraperitoneally in all groups. The dose of APO used was also 1mg/kg. All other conditions were the same as that in Experiment 1.

RESULTS Experiment 1 The effects of APO, atropine, mecamylamine, as well as their interactive effects on 5-HT neurons in the striatum and the hippocampus are shown in Table I. As shown in Table I, APO significantly increased tryptophan (tD = 2.13,P < 0.05),5-HT (tD = 3.07,P < 0.01), and 5-HIAA (tD = 2.95, P < 0.01) levels in the striatum. Atropine infused to the DR a t a low dose (0.05 pg/0.75 p1) did not have any significant effect on these measures by itself (all P> 0.051, while completely antagonizing the effects of APO on 5-HT neurons in the striatum (all P> 0.05 as compared with the saline controls). Besides, Tukey’s comparisons revealed that tryptophan, 5-HT and 5-HIAAlevels in the Atr (0.05 p,g/0.75 p-1) + APO group also differed significantly from that of the Sal + APO group (P < 0.05, P < 0.05, and P < 0.01, respectively). However, atropine infused to the DR at a higher dose (0.1 ~g/0.75p1) markedly elevated tryp-

tophan (tD = 2.09,P < 0.05), 5-HT (tD = 1.88,P < 0.05), and 5-HIAA (tD = 1.94,P < 0.05) concentrations in the striatum by itself; it also prevented the effects ofAPO on 5-HT neurons since the effects of the combination of atropine (0.1 ~g/0.75pl) and APO were not different from that of atropine (1.0 p,g/0.75 pl) alone (tD = 1.73, P < 0.05; t D = 1.69, P < 0.05; and tD = 2.18, P < 0.051, for tryptophan, 5-HT and 5-HIAA, respectively, when comparing the effects of atropine (0.1 Fgl 0.75 pl) + APO group with the saline-treated controls). Mecamylamine (0.25 p.g/0.75 pl) infused to the DR, at a dose that did not affect the serotonergic measures alone (all P > 0.051, also blocked the effects of APO on tryptophan, 5-HT, and 5-HIAA in the striatum (all P > 0.05 as compared with the saline-treated controls). Tukey’s tests also indicated that the Mec + APO group also significantly differed from that of the Sal + APO group as tryptophan ( P < 0.01),5-HT ( P < 0.051, and 5-HIAA (P < 0.01) levels are concerned. Atropine injected into the RF did not have a significant effect on any serotonergic measures in the striatum (allP > 0.05, ns). None of the above drugs examined had a marked effect on tryptophan, 5-HT, and 5-HIAA in the hippocampus (all P > 0.05, ns). Experiment 2 The effects of carbachol,APO, as well as their interactive effects on three serotonergic measures in the striaturn and the hippocampus are shown in Table 11. As indicated in Table 11, APO consistently and markedly increased tryptophan (tD = 2.33, P < 0.051, 5-HT (tD = 2.25, P < 0.05), and 5-HIAA (tD = 1.96, P < 0.05) levels in the striatum. Carbachol infused to the DR at a low dose (0.03 pg/0.75 pl) did not affect these measures

39

AF'OMORPHINE, DA, ACh, GABA, AND 5-HT

TABLE II. Effects of apomorphine (1 mg/kg), carbachol, and their interactive effectson 5-HT neurons in the striatum and the hippocampus1.2 Treatment

+ + + +

Sal Sal Sal APO Carb Sal (0.03 pgl0.75 p1) Carb Sal (0.1 pg/0.75 pl) Carb APO (0.03 pg/0.75 pl)

+

N

TWP

Striatum 5-HT

5-HIAA

Trm

8 8 7

2,988 f 257 3,471 f 308* 2,745 f 231

1,092 zk 119 1,362 f 108* 1,067 f 125

820 f 63 1,023 f 90* 716 f 62

2,584 f 212 2,497 f 209 2,603 f 231

845 f 87 861 f 72 772 f 37

654 43 623 f 57 621 f 34

7

2,547

8

3,033 f 282*z3

+ 203*

Hippocampus 5-HT

5-HIM

+

897 f 82*

646 f 53**

2,714 f 246

859 f 80

664 f 70

939 f 87*z3

841 f 75*s3

2,657 f 238

908 f 48

675 f 50

*P < 0.05 and **P< 0.01.

'Data are expressed as in Table I. %arb, carbachol. "s compared with the Sal APO group.

+

(all P > 0.05); whereas at a higher dose (1.0 pd0.75 pl), it significantly decreased tryptophan (tD = 1.82, P < 0.051, 5-HT (tD = 1.71, P < 0.05) and 5-HIAA (tD = 2.02, P < 0.01) contents in the striatum by itself. The combined treatment of carbachol (1.0 pd0.75 ~ 1 and APO brought tryptophan, 5-HT, and 5-HIAA concentrations back to the control level ( P > 0.05) when compared with the saline-treated controls). There were also significant differences in tryptophan, 5-HT, and 5-HIM concentrations between the Carb + APO group and the Sal + APO group, as indicated by individual Tukey's comparisons (all P < 0.05). Similar to the results in Experiment 1,neither APO nor carbachol had any significant effects on tryptophan, 5-HT, or 5-HIAA in the hippocampus (all P > 0.05, ns). Experiment 3 The effects of APO, pirenzepine, as well as their interactive effects on serotonergic measures in the striatum and the hippocampus are shown in Table 111. Similar to the results in Experiments 1 and 2, APO markedly increased tryptophan (tD = 1.95, P < 0.051, 5-HT (tD = 2.46, P < 0.01), and 5-HIM (tD = 1.78, P < 0.05) concentrations in the striatum. Pirenzepine infused to the DR at a low dose (2 pd0.75 pl) did not markedly alter these measures in the striatum (P > 0.051, while antagonizing the effects of APO on tryptophan, 5-HT, and 5-HIAA in the same area (P > 0.05 when comparing the pirenzepine + APO group with the saline + saline group). Tukey's tests also revealed significant differences of 5-HT and 5-HIAA levels between Pir + APO group and Sal + APO group P < 0.01 and P < 0.05, respectively). However, pirenzepine infused to the DR at a higher dose (5 pg/0.75 pl) mimicked the effects of APO. It markedly elevated tryptophan (tD = 1.97, P < 0.05), 5-HT (tD = 1.78, P < 0.051, as well as 5-HIM (tD = 1.72, P < 0.05) concentrations in the striatum. Similarly, neither drug had a significant effect on serotonergic system in the hippocampus (P > 0.05, ns).

Experiment 4 The effects of APO, bicuculline, saclofen, as well as their interactive effects on 5-HT neurons in the striatum and the hippocampus are summarized in Table IV. )As shown in Table IV,APO consistently and markedly increased tryptophan (tD = 1.87,P < 0.05),5-HT(tD = 1.66, P < 0.051, and 5-HIAA (tD = 1.74, P < 0.05) concentrations in the striatum. Bicuculline at 1 pg/0.75 p,1 did not have a significant effect on these serotonergic measures alone ( P > 0.051, while completely blocking the effects ofAPO on these measures in the striatum (all P > 0.05, when comparing the bicuculline (1.0 pg/0.75 p1) + APO group with the control group). There were also significant differences of tryptophan and 5-HT levels between the Bic (1.0 pg/0.75 pl) + APO group and the Sal + APO group (both P < 0.05, Tukey's tests). Bicuculline at a higher dose (2.0 pg/0.75 1.1) examined markedly elevated tryptophan (tD = 2.13, P < 0.05) and 5-HT (tD = 1.97, P < 0.05) levels in the striatum. By contrast, saclofen at a dose (1 pg/0.75 p1) having no significant effect on these measures by itself (all P > 0.05) did not antagonize the effects of APO on tryptophan (tD = 1.94, P < 0.05), 5-HT (tD = 1.77, P < 0.05) and 5-HIAA (tD = 1.75, P < 0.05) in the striatum (when comparing the saclofen + APO group with the control group). Similar to atropine injections, bicuculline (2.0 pg/0.75 p1) injected into the RF did not produce any significant effect on these measures in the striatum either (all P > 0.05, ns). Consistent with the results from previous experiments, none of these drugs had a significant effect on any serotonergic measures in the hippocampus (all P > 0.05, ns). DISCUSSION The present results are consistent with previous histofluorescent and biochemical findings that APO preferentially increased tryptophan, 5-HT, and 5-HIAA concentrations in the DR and the striatum without markedly affecting the same measures in the MR and the hippocampus (Lee and Geyer, 1982,1983; Lee et al.,

40

H.Y. CHEN ET AL.

TABLE III. Effects of apomorphine (1 mg/kg), pirenzepine, as well as their interactive effects on 5-HT neurons in the striatum and the hippocampus's2 Treatment

Striatum 5-HT

Tryp

8 8 7

3,101 f 152 3,623 f 105* 3,208 f 169

1,192 f 68 1,502 f 62** 1,239 f 96

822 38 966 f 78* 917 f 50

2,955 f 197 2,931 rt 52 3,012 f 185

828 f 55 885 f 84 864 f 61

562 f 55 568 f 57 580 i 35

8

3,649 f 177*

1,440 f 81*

988 f 67*

2,887 f 163

803 f 79

541 f 39

7

3.232 f 160

1.179 & 45**a3

853 f 40*g3

2.729 f 288

829 f 52

580 f 70

+ + + +

Sal Sal Sal APO Pir Sal (2 pg/0.75 pl) Pir Sal (5 pg/0.75 pl) Pir APO

+

5-HIAA

*

TrYP

Hippocampus 5-HT

N

5-HIAA

*P< 0.05 and **P< 0.01.

'Data are expressed as in Table I. Wr, pirenzepine. 3Ascompared with the Sal APO group.

+

TABLE IV. Effects of apomorphine (1 mg/kg), bicuculline, and saclofen (1 pg/0.75 pl), as well as their interactive effects on 5-HT neurons in the striatum and the hippocampus1s2

+

+

+

644 f 77 612 f 57 703 f 81

1,053 f 108

2.885 f 270

951 f 78

662 f 59

1,167 $: 49**3

1,080 f 62

2,937 f 170

1,010 f 121

597 f 70

1,151 f 102 1,306 f 59* 1,078 f 93

990 f 37 1,110 f 93* 967 f 88

2,841 f 228 3,031 f 361 3,013 f 267

1,023 f 93 1,076 f 134 965 f 69

650 rt 65 582 f 53 673 62

8 8

3,559 f 318 4,213 f 386* 3,806 f 279

1,153 f 91 1,461 f 122* 971 f 20

930 f 95 1,170 f 130* 998 rf. 39

8

4.134 f 339*

1.296 f 87*

7

3,599 f 335*13

6 8 8

3,488 f 251 4,157 f 195* 3,601 f 298

8

+ + +

994 f 109 944 f 90 1,017 f 195

Tryp

+ Sal Sal + APO

Bic Sal (1 pg/0.75 pl) Bic Sal (2 pgl0.75 p l ) Bic APO (1 pg/0.75 pl) Sac Sal Sac APO Bic (RF) Sal (2 udO.75 ul)

5-HIAA

2,984 f 401 2,823 f 80 2,957 f 341

5-HIAA

N

Sal

Hippo camp u s 5-HT

Striatum 5-HT

Treatment

nYP

*

*P< 0.05. 'Data are expressed as in Table I. 2Bic, bicuculline; Sac, saclofen; RF, reticular formation. 3Ascompared with the Sal APO group.

+

1987). These findings are also congruent with most of the literatures showing the differential mesostriatal and mesolimbic serotonergic projections. These effects ofAPO have been demonstrated to be mediated through DA autoreceptors in the substantia nigra (see Introduction) and possibly GABA neurons in the DR (Lee et al., 1987). Other than confirming the suggestion of a GABAergic mediation, the present results have further demonstrated that ACh neurons are also involved in the actions of APO on 5-HT, since atropine at a low dose prevented, and at a high dose mimicked, the effects of APO. By contrast, carbachol decreased these serotonergic measures. Furthermore, by using specific muscarinic and nicotinic receptor antagonists as well as muscarinic receptor subtype antagonists, we have demonstrated that both nicotinic receptors and muscarinic M, receptors are involved in the actions of APO on 5-HT. Moreover, by using the specific GAJ3AA antagonist bicuculline (Johnston et al., 1972) and specific GABA, antagonist saclofen (Kerr et al., 1988; Lambert et al., 1989;Harrison et al., 19901,we have found that GABAA instead of GABA, receptors in the DR mediate the

effects of APO.Putting all these results together, it is possible that the synaptic relationships among these neurotransmitters are that DA neurons originating from the substantia nigra send an excitatory input to ACh neurons; ACh neurons then make excitatory synapses with GABA neurons, and GABA neurons finally send an inhibitory input upon 5-HT neurons in the DR. By inhibiting DA neuron firing in the substantia nigra through DA autoreceptor regulation (Groves et al., 1975) and the GABA disinhibition mechanism, APO activates 5-HT neurons in the DR and in its corresponding terminal area, the striatum. While the above pharmacological studies and biochemical measures have suggested the possible interrelationships among these neurotransmitter systems, they do not provide direct evidence of the circuit and of where these interactions may occur. However, results from our previous studies suggest that the interactions must occur at the area(s) near the DR, since the effects of APO on 5-HT neurons occur as soon as 20 min after a systemic injection. Furthermore, the time-course study revealed that the effects of APO on 5-HT occur earlier in

AF’OMORPHINE, DA, ACh, GABA, AND 5-HT

the DR than in the striatum (Lee and Geyer, 1982). These latter results suggest that the neurotransmitter interactions should take place earlier in the DR and consequently affect 5-HT terminals in the striatum. However, these results do not eliminate other possibilities involving more complicated diagrams, while still consistent with the current working model. More anatomical and electrophysiological studies are ongoing to examine the precise anatomical locus for these interactions to occur. Although the precise synaptic relationship among these neurotransmitters is unknown, similar neurotransmitter systems and anatomical connections have been found in rat striatum (Graybiel and Ragsdale, 1983; Carlsson and Carlsson, 1990; Smith and Bolam, 1990). For example, direct application of ACh was shown to reduce 5-HT release in the striatum in cats. This effect was demonstrated to be mediated through GABA neurons in the same area (Becquet et al., 1988). The suggested DA and ACh interaction is supported by recent studies showing that activation of D, DA receptors stimulates the release of ACh in the striatum of a freely moving rat (Ajima et al., 1990), and DA agonist induces an elevation of striatal ACh release (Enz et al., 1990) as well as ACh level (Bymaster et al., 1986) in rats. Hensler et al. (1987) and Hensler and Dubocovich (1988) also demonstrated that D1 DA receptor agonist mediates ACh release in rabbit retina. Similar results have also been obtained in the invertebrate that intracellular injection of DA enhances ACh response in Aplysia (Yurchenko et al., 1987). Besides, immunohistochemical (Chang, 1988) and electromyographic (Ellenbroek et al., 1986)studies have also demonstrated a direct DA-ACh interaction in the rat brain. However, there are also studies showing the opposite results. For example, Wedzony et al. (1988) found that D, DA receptor stimulation inhibits the release of ACh in rat nucleus accumbens. Jackson et al. (1988) also reported that DA exerts an inhibitory influence on ACh release from rat striatal slice. Dz DA receptor stimulation was also found t o reduce ACh release in rat nucleus accumbens slices (Stoff et al., 1987). Discrepancies among these results are probably due to the differentiation of DA receptor subtypes involved, since D, DA receptors are suggested to mediate mainly ACh release and D2DA receptors modify the effects of D, DAreceptors (Ajima et al., 1990). It is also possible that the same DA receptor (or even the same receptor subtype) may play different roles in different brain regions. Experiments are undertaken t o study the DA receptor subtypeb) that mediates the effects of APO on 5-HT. Regarding the proposed functional interactions between GABA and 5-HT, it is supported by the findings of Soubrie et al. (1981) that GABA directly inhibits 5-HT release in the cat substantia nigra. Wang and Aghajanian (1977)also reported that the lateral habenula exerts an inhibitory input on 5-HT neurons in the DR and this

41

pathway uses GABA as its neurotransmitter. Recently, using the in vivo microdialysis method, Kalen et al. (1989)also found that striatal5-HTrelease is regulated by the GABAergic synaptic activity in the DR in rats. Moreover, Osborne and Beaton (1986) found that 5-HT may be taken up by a subpopulation of GABA neurons in the rabbit retina. Millhorn et al. (1988) further suggested that 5-HT and GABA neurons may be colocalized in the ventral medulla oblongata in rats. These results suggest that it is likely that the pharmacological interactions between GABA and 5-HT neurons may take place within or near the DR. However, there has been no report directly addressing the synaptic relationships among all these neurotransmitters (DA, ACh, GABA, and 5-HT) yet. The present pharmacological studies combined with biochemical measures may provide some indirect evidence for these interactions. While the proposed neuronal firing model well explains the results of elevated 5-HIAA levels in both the DR and the striatum after APO injections, it does not readily explain the concurrent elevations of tryptophan and 5-HT in both areas. It is possible that APO also accelerates the synthesis rate of 5-HT in either the DR or the striatum, or both. However, it is also likely that APO directly facilitates tryptophan uptake into 5-HT neurons or indirectly facilitates its uptake through other mechanisms. Although no direct evidence regarding this issue has been reported, in a related study, Knapp et al. found that DA could be taken up by 5-HT neurons directly and influences 5-HT biosynthesis by altering tryptophan hydroxylase activity (unpublished observations). However, since the effects of APO on 5-HT neurons have been robust and consistent, the possible mechanism of tryptophan uptake together with a neuronal firing model may better explain the increases of all three measures. In summary, by using pharmacological approaches together with biochemical measures, we have demonstrated that both ACh and GABA (other than DA) neurons mediate the effects of APO on the mesostriatal serotonergic system. Furthermore, using various receptor subtype agonists and antagonists, we have demonstrated that both the nicotinic and muscarinic M, cholinergic receptors as well as GAEiAA receptor are involved in the actions of APO on 5-HT. These results have certainly demonstrated the neurotransmitter interactions among DA, ACh, GABA, and 5-HT, while they do not directly demonstrate the synaptic relationships and anatomical circuits among these transmitter systems. Moreover, these interactions do not readily explain the elevation of tryptophan either. The robust and consistent effects of APO on elevations of tryptophan, 5-HT, and 5-HIAA simultaneously suggest a possible and perhaps more important mechanism of “tryptophan uptake” upon APO administration other than the neuronal firing model. Further anatomical, electrophysiological, and biochemical experiments are

42

H.Y. CHEN ET AL.

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