Selective effects of apomorphine on dorsal raphe neurons: A cytofluorimetric study

Brain Remrch

BuNerin,

Vol. 9, pp. 71%725, 1982.Printed in the U.S.A.

Selective Effects of Apomorphine on Dorsal Raphe Neurons: A Cytofluorimetric Study’ EMINY H. Y. LEE AND MARK A GEYER2 Departments of Neuroscience and Psychiatry, T-004, School of Medicine University of California at San Diego, La Jolla, CA 92093

LEE, E. H. Y. AND M. A. GEYER. Selective effects of apomorphine on dorsal raphe neurons: A cytojluorimetric study. BRAIN RES. BULL. 9(1-6) 719-725, 1982.-Using a quantitative cytofluorimetric method to detect changes in the intracellular levels of serotonin (5-HT) in individual neurons in rat brain, we have found that the dopaminergic agonist apomorphine increases 5-HT content of dorsal raphe cell bodies without affecting cells in the median raphe nucleus. Liquid chromatographic studies revealed that apomorphine also elevated the concentrations of both .5-HT and its metabolite 5-hydroxyindoleacetic acid (S-HIAA) in striatum, the projection site for dorsal raphe neurons. Conversely, the dopaminergic antagonist haloperidol, at a dose of 0.8 m&g, decreased 5-HT levels in dorsal raphe cells. A lower dose of haloperidol (0.4 m&g), which had no significant effect alone, completely blocked the effect of apomorphine in the dorsal raphe. These results support the hypothesis that the effects of apomorphine on serotonergic neurons are secondary to dopamine receptor stimulation. Serotonin Raphe nuclei

Apomorphine Dopamine Transmitter interactions

Haloperidol

THE publication 20 years ago of a sensitive method for the cellular visualization of neuronal monoamines precipitated a remarkable growth in the field of neuroscience. The pioneering advance by Bengt Falck, Nils-Ake Hillarp, and their colleagues provided the foundation for an enormous proliferation of information regarding the anatomy and physiology of catecholaminergic and serotonergic systems in brain, which in turn engendered many of the techniques and models used in the study of other neurotransmitters as well. However, the impact of the Falck-Hillarp histochemical method in pharmacological research has been slower to develop. While the method’s use as an anatomical tool was rapidly accepted, its application to the study of drug-induced changes in neuronal monoamines required additional standardization and considerable improvement in the instrumentation of fluorescence microscopy. Only after the introduction of the Ploem epi-illuminator and stabilized light sources was it possible to demonstrate quantitation of the stable fluorophore derived from the reaction of formaldehyde with the catecholamines (CAs), dopamine and norepinephrine [4, 6,241. The technological and procedural refinements made in the context of quantifying formaldehyde-induced fluorescence at the microscopic level have paved the way to more precise quantification of other fluorophores as well, a benefit to be reaped in the future by the growing field of immunohistochemistry.

Cytofluorimetry

Microspectrofluorimetry

In contrast to the CAs, the development of quantitative cytofluorimetric measures of cellular serotonin (5-HT) was complicated by the rapid photodecomposition of the 5-HT fluorophore under exposure to ultraviolet light. While some early reports did demonstrate significant correlations between cytofluorimetric and standard measures of SHT, these methods were limited in sensitivity and susceptible to sampling bias [7, 20, 321. In our initial attempt to quantify cellular 5-HT, we circumvented the fading problem by using substage red light and a phase-contrast condenser to align the specimen, this precluding both photodecomposition and sampling bias [lo]. With the photometric system open, we then admitted exciting light to the specimen from above and recorded the initial peak intensity of fluorescence. Though this method worked satisfactorily without drug pretreatments to artificially increase 5-HT levels, the overlap in emission wavelengths prevented a clear distinction between 5-HT and CAs. Noting that the largest difference between the CA and 5-HT fluorophores was in their rates of photodecomposition (half-lives of 5+ min and 3.5 set, respectively [13]), we reasoned that a measure of the decrement in fluorescence intensity over a fixed period of time should be proportional to 5-HT concentration and relatively independent of background or CA fluorescence. This reasoning was confirmed by a series of in vitro and in vivo studies demonstrating that a

‘This work was supported by a grant from the National Science Foundation (BNS 04676). E.H.Y.L. was supported in part by the PEO Scholarship Foundation. 2Address reprint requests to Dr. Mark A. Geyer, T-004, Department of Psychiatry, UCSD, La Jolla, CA 92093.

Copyright 0 1982 ANKHO International

Inc.-0361-9230/82/070719-07$03.00/O

LEE AND GEYER

770

“fading measure” provides for sensitive and reliable quantification of changes in 5-HT at the cellular level even in the presence of CAs [13]. For example, after various doses of the monoamine oxidase inhibitor, pargyline, in vivo, a correlation of 0.927 was observed between a standard fluorimetric assay of 5-HT and the cytofluorimetric fading measures of 5-HT fluorescence in the cytoplasm of midbrain raphe neurons in rats [13]. Using this approach, we have shown a variety of environmental and drug-induced changes in the levels and distribution of S-HT within the dorsal and median raphe nuclei, some being regionally specific [10,14], others being specific to either the intracellular or extracellular measures [ 13,221 made possible by the anatomical precision of cytofluorimetry. For example, lysergic acid diethylamide, which inhibits firing of raphe cells [I], selectively increases intracellular S-HT [13], while the 5-HT reuptake inhibitor fluoxetine increases 5-HT extraneuronally without affecting intracellular levels [ 131. Thus, the classic Falck-Hillarp histochemical method coupled with recent technological and methodological advances now provides a sensitive tool with which psychopharmacologists can examine drug effects on both catecholaminergic and serotonergic neurons with an exquisite degree of anatomical specificity. The present paper illustrates the application of such cytofluorimetric methods to the study of potential interactions between the serotonergic and dopaminergic systems in rat brain. The experiments reported here reveal regionally specific changes in cellular 5-HT following treatment with drugs whose primary action is on brain dopaminergic systems. Apomorphine is believed to be a specific dopaminergic agonist in the central nervous system [2, 8, 91. However. biochemical studies have shown that apomorphine also accelerates the turnover of 5-HT [15] and elevates the concentration of both S-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in whole brain and in several separate brain regions in rat and mouse [17,29]. These findings were corroborated by Smialowska with histofluorescence studies in which apomorphine (20 mg/kg) significantly increased the subjective estimates of 5-HT fluorescence in the dorsal raphe nucleus (B7) [l&30]. The effect of apomorphine on the serotonergic system appears to be mediated by central dopaminergic neurons, since haloperidol, spiroperidol and pimozide antagonized apomorphine’s effect [ 171and a transection between the substantia nigra and dorsal raphe also abolished apomorphine’s effect on S-HT in B7 [ 181. The purpose of the present study was to use our quantitative fluorescence measures [13] to further investigate the interaction between central dopaminergic and serotonergic systems and to determine whether there is a differential effect of apomorphine on the dorsal and median (B8) raphe nuclei. The latter comparison was prompted by the difference in the degree of catecholaminergic innervation to these two serotonergic nuclei [23,25] and by our previous observation that both amphetamine and methylphenidate affect the intracellular fluorescence levels in dorsal but not median raphe neurons [IO]. METHOD

rats were housed in group cages of three rats each located in a temperature-regulated (2522°C) animal room on a 12112hr light/dark cycle. Purina Rat Chow and water were available ad lib. After 5 to 7 days in the animal room. the rats were transferred to the laboratory. All animals were sacrificed by decapitation after drug manipulation. fhgs

Apomorphine hydrochloride (Merck & Company, Rahway, NJ) and haloperidol (McNeil Laboratories) were used. Doses refer to the salt form. Drugs were generally dissolved in isotonic saline immediately before use and were injected intrapetitoneally in a volume of 2 ml/kg.

The microspectrofluorimeter is described in detail elsewhere [ 131. Briefly, a 1000 watt Xenon lamp and grating monochromater (Schoeffel) provides 410 mm excitation light that is admitted to the specimen under computer control through an electronic shutter. A Leitz microscope and MPV system with sub-stage phase-contrast red light and a Ploem epi-illuminator allow for alignment of the specimen in phase-contrast and the isolation of a 5 km circular area of the cytoplasm adjacent to the nucleus for fluorescence measurement. Alternatively, the measurement aperture is aligned in the regions between cell bodies. This extraperikaryal measure has been found to respond to some drugs independently of changes in the intracellular measures [ 131. A small grating monochromater is used to select the optimal emission wavelength (512 nm) for biogenic amines to be detected by photometer. After alignment of each cell, readings are taken automatically at one per second and stored on magnetic tape for subsequent analyses. Both model droplet analyses and irl \ivo studies with pargyline have shown that the fluorescence intensity remaining after 14 seconds of excitation is proportional to the concentration of CAs, while a fading measurethe difference in fluorescence intensities from the first to the fourteenth second of excitation-is the best predictor of 5-HT concentration and independent of CA levels. To allow reliable comparisons among a large number of tissue samples, the formaldehyde histochemical method has been modified so that all samples within an experiment are batch-processed together from sacrifice until sectioning.

Using red-light phase-contrast, the slides containing the regions of interest were selected by reference to predetermined landmarks. In these experiments, one rostrocaudal plane through the midbrain was selected for the B7 and B8 raphe measures. The B7 area is designated as the dense cell cluster immediately below the Aqueduct of Sylvius; and B8 is the median nucleus just below the decussation of the superior cerebellar peduncle [5]. For each region, at least six intracellular and six extraperikaryal readings were taken from different although adjacent slides. Individual raphe neurons from both sides were chosen evenly for microspectrofluorimetry. Four background readings were taken from non-fluorescent cell bodies in the lateral reticular formation.

Animals

Ti.\.surYrrparatio~z

The animals were 131 experimentally naive male Sprague-Dawley rats weighing from 125-150 g. Upon receipt from the supplier (Hilltop Laboratories, Scottdale, PA), the

At the appropriate time after injection, animals were sacrificed by decapitation and their brains were removed within 90 sec. After dissection, as described previously [131, brain

SEROTONIN

721

AND APOMORPHINE

samples were frozen in propane, freeze-dried over phosphorous pentoxide for four weeks at -60°C treated with gaseous formaldehyde, embedded in paraffin, and sectioned at eight pm on a rotary microtome. Sections were mounted on slides in Entellan (Merck). High Performance

Liquid

Chromatography

(HPLC).

The chromatographic system used in these studies was the Bioanalytical Systems LC-17 equipped with a “pBondapack” C-18 reverse-phase column (3.9 by 30 cm; Waters Ass.), an Altex pump and an LC-4 amperometric detector coupled to a TL-5 glassy carbon electrode (Bioanalytical Systems) and an Ag-AgCl reference electrode. Stainless steel tubing was used throughout. The solvent system consisted of 0.1 M sodium acetate, 0.06 M citric acid and 20% (v:v) methanol at pH 4.1, which was degassed before use. Serotonin and 5-HIAA were estimated according to the method of Mefford [26]. Briefly, this method is as follows. Tissues were weighed while still frozen and 300 pl of 0.1 M perchloric acid was added to each 1.5 ml polypropylene tube containing the tissue. Tissue was then thoroughly disrupted by sonication for 30 set in an ice bath. After sonication, the tissue was centrifuged at 6,000 rpm for 10 min using a refrigerated centrifuge and 20 pl of the clean supernatant was injected directly into the chromatographic system. A flow rate at 1.O ml/min was maintained and the column was operated at ambient temperature. The electrochemical detector was set on a current-to-voltage gain (sensitivity) of 5 nA/V and at a potential of +0.58 volt with the recorder set to give a full scale deflection with 10 mV. Serotonin and 5-HIAA were resolved at 6 and 11 min, respectively. Statistics

Separate analyses of variance (ANOVAs) were done for both intracellular, extraperikaryal and biochemical measures from each brain region examined. In microspectrofluorimetric experiments, the appropriate values from each animal were averaged after subtraction of the corresponding blank value so that each animal contributed only one value to each ANOVA. The Newman-Keuls and Dunnett’s tests [33] were used to make specific comparisons between groups when the overall ANOVA revealed a significant group effect. Experiment

I

To define the most sensitive region of B7 to the effects of apomorphine, a regional mapping study was conducted. We have depicted elsewhere [ 131 several arbitrary subdivisions of B7 and B8 in one rostrocaudal plane (A 350 of Konig and Klippel [21]) in a single rat. We have also described the relative densities of serotonergic cell bodies in these subdivisions 11I]. The dorsal and ventral portions of B8 are reasonably comparable in all variables measured in an untreated animal. Similarly, three subdivisions of B7, ventrolateral B7 (B7-3), ventromedial B7 (B7-4) and ventral B7 (B7-5), are relatively homogenous. The dorsomedial and dorsolateral portions of B7 (B7-1 and B7-2) contain the brightest 5-HT cells and are heavily innervated by CA terminals [I 31. Five saline and five 1.O mg/kg apomorphine animals were used in this experiment to see whether apomorphine has differential effects on 5-HT levels among these

subdivisions.

Experiment

2

Forty-eight animals were used in this experiment to exam-

ine the dose-dependency of the effects of apomorphine on 5-HT fluorescence in B7 and B8 neurons. In B7, the B7-4 subdivision was chosen in this and subsequent experiments since it was the region showing the most significant apomorphine effect from the mapping study. The doses used ranged from 0.025 mg/kg to 20 mg/kg, as listed in Table 2. In addition, 5-HT and 5-HIAA concentrations were also measured in the corpus striata of most of these animals. Experiment

3

Thirty-five animals were randomly assigned to 7 groups to examine the time-course of the effect of 0.1 mg/kg apomorphine on the dorsal raphe. The time intervals selected ranged from ten to 80 min following injection. Experiment

4

To test the hypothesis that dopaminergic systems mediate the effect of apomorphine on B7 cells, the specific dopaminergic antagonist, haloperidol, was used in the present experiment. Twenty-one animals were randomly divided into four groups. Group 1 (N=6) received a single injection of saline. Group 2 (N=5), Group 3 (N=5), and Group 4 (N=5) received single injections of 0.2 mg/kg, 0.4 mg/kg and 0.8 mg/kg haloperidol, respectively, one hr before sacrifice. Striatal 5-HT and 5-HIAA were also assayed in the 0.2 mg/kg and 0.8 mg/kg haloperidol groups. Experiment

5

Following the dose-response study, a dose of haloperidol which had no effect alone was administered together with apomorphine to determine whether haloperidol antagonizes apomorphine’s effect. An additional 17 animals were randomly assigned to three groups: Group 1 (N=6) received a single injection of saline; Group 2 (N=5) received an injection of 0.4 mg/kg haloperidol; Group 3 (N=6) received an injection of 0.4 mg/kg haloperidol 30 min before injection of 1.0 mg/kg apomorphine. All animals were sacrificed 60 min after haloperidol treatment. The results of this experiment were combined with the previous experiment. RESULTS

Experiment

I

Table 1 shows the means of the 5-HT fading measure in each of the subdivisions of B7 from tive saline and five 1 mg/kg apomorphine animals. In general, apomorphine slightly increased intracellular 5-HT in each subdivision except B7-2 but the effect was significant only in the B7-4 subdivision, F(l,9)=46.86, pcO.01. Readings in control animals showed the same pattern and order as in our previous study [13]. Experimcwt

2

The dose-dependent effects of apomorphine on the cytofluorimetric measures of 5-HT fluorescence are summarized in Table 2. Doses of apomorphine above 0.5 mg/kg all preferentially increased the intracellular fading measure of 5-HT in B7 except 6 mg/kg and 20 mg/kg apomorphine. The latter decreased the intracellular 5-HT level significantly, as shown in Table 2. The dose response curve of apomorphine approximated an inverted U, with 1 mg/kg showing the most significant effect. Extracellularly, there was a slight dose-dependent aug-

122

LEE AND GEYER TABLE

1

REGIONAL DIFFERENCES FOR VARIOUS SUBDIVISIONS OF DORSAL RAPHE IN SALINE AND APOMORPHINE ANIMALS Intracellular Subdivisions of Dorsal Raphe

N

Dorsomedial Dorsolateral Ventrolateral Ventromedial Ventral

5 5 5 5 5

Sal 3693 4187 2739 2685 2709

? ? t + i_

Apo (1 mgfkg) F(l,8) 84 193 103 62 77

3836 4153 2835 3388 2807

t 80 t 211 t- 143 % 82* + 85

p’

1.53 n.s. 0.01 n.s. 0.30 n.s. 46.86 0.01 0.74 n.s.

Data are expressed as group means t SEM for the fading measure of 5-HT after subtraction of the background readings, which ranged from 150-350. Numbers are in nanoamperes of photometric current. *p
TABLE 2 DOSE-DEPENDENT EFFECT OF APOMORPHINE ON 5-HT CONTENT IN RAPHE NEURONS Median Raphe

Dorsal Raphe Dose of Apo (m&g)

N

Intracellular

Extracellular

Intracellular

b 0.025 0.05 0.1 1.0 3.0 6.0 10.0 20.0

6 5 5 6 6 5 5 5 5

3397 -t 183 3480 2 127 3261 2 66 3818 r 138t 4069 2 206t 3669 + 114* 3592 -+ 48 3823 ? 3lt 3011 2 88t

1843 i 1936 i1812 i1912 i 1940 i 1995 -t 1907 2 20.58 i 2359 t

4738 4619 4828 4818 4871 3884 4610 5101 4009

Data are expressed

63 61 86 64 47 62* 26 146* 1451

+ 157 ? 127 t- 147 t 62 2 213 -t 154* +_ 71 2 206 ? 270*

Extracellular 2067 2 1994 -t 1910 + 2038 .+ 2090 2 2112 -t 1998 f 2219 t2004 &

106 67 90 41 83 74 36 91 14

as in Table 1.

*p<0.05; tp<&ol. Statistical significance was evaluated using analyses of variance (ANOVA’s) and multiple comparisons

among means

mentation of 5-HT fluorescence, with this increase being significant for the 3, 10, and 20 mg/kg doses. The marked effect of the highest dose was opposite to that seen intracellularly with this dose. Table 2 also shows that of the

eight doses of apomorphine studied, only 3 and 20 mg/kg apomorphine produced significant increases in intracellular 5-HT in B8. None of the doses had any significant effect on extracellular 5-HT fluorescence in B8. Doses of apomorphine above 1 mg/kg also increased the final fluorescence intensity, a measure of CAs outside the B7 cell bodies [13], with 3 and 20 mglkg showing significant effects, q(4,14)=4.11, p
These results are consistent with previous [ 15,301 and confirm our histochemical the dorsal raphe cell body area.

ports

Experiment

biochemical reobservations in

3

As shown in Table 4, 0.1 mglkg apomorphine elevated intracellular 5-HT levels in B7 significantly at all the time points studied except at 10 min, F(6,28)=9.75, p
4

The effects of 0.2, 0.4 and 0.8 mg/kg haloperidol on the cytofluorimetric measures of 5-HT fluorescence are summarized in Table 5. Although there was a dose-dependent

SEROTONIN

723

AND APOMORPHINE TABLE

3

EFFECTS OF APOMORPHINE AND HALOPERIDOL ON 5-HT AND 5-HIAA CONCENTRATIONS IN STRIATUM

Dose of Apo (mg/kg) 0.0 0.025 0.05 3.00 6.00 20.00 Dose of Hal (m&g) 0.2 0.8

N

S-HT (rig/g tissue)

6 5 5 5 5 5

692 704 651 880 938 903

5 5

694 f 555 r

% Control

+ 50 -i- 60 f 42 t 86 2 103* 2 65

36 70

5-HIAA (rig/g tissue)

% Control

100 102 94 127 136 130

961 f 880 + 875 f 1457 f 1083 f 1140 f

69 120 54 167t 72 48

100 92 91 152 113 119

100 80

875 ? 853 f

64 59

91 89

*p
TABLE 4 TIME COURSE OF APOMORPHINE’S

EFFECT ON 5-m

CONTENT

IN RAPHE NEURONS

Dorsal Raphe Time After Apo (min) 0

10 20 30 40 60 80

Median Raphe

N

Intracellular

Extracellular

5 5 4 6 5 4 5

2955 2 59 3093 k 68 3482 -+ 136t 3395 ? 65t 3447 + 70t 3584 k 38t 3294 + 63

1642 ‘1841 f 1783 k 1736 k 1854 2 19% k 1764 t

58 81 49 30 51 100* 79

Intracellular 3515 + 3426 k 3435 k 3575 2 3526 -c 3687 f 3408 ”

62 74 89 46 91 68 76

Extracellular 1735 k 1784 ? 1753 2 1712 k 1791 t 1862 2 1835 2

38 75 53 35 42 73 51

Data are expressed as in Table 1. *p
TABLE 5 EFFECTS OF HALOPERIDOL

ON S-HT CONTENT AND APOMORPHINE-INDUCED CONTENT IN RAPHE NEURONS

Dorsal Raphe Dose of Hal (mgkg) 0.0

0.2 0.4 0.8 Hal (0.4) + Apo (1.0)

N

Intracellular

6 5 5 5 6

3425 2 69 3415 f 84 3287 5 120 2997 k 54* 3234 2 56

OF S-HT

Median Raphe

Extracellular 1877 + 1713 f 1868 k 1647 2 1729 t

ELEVATION

71 64 70 72 37

Intracellular 4725 4507 4612 4647 4589

t * ” T f

91 130 95 119 98

Extracellular 2071 + 63 1912 2 46 2048 + 16 2OOOk46 2042 ‘- 33

Data are expressed as in Table 1. *p
LEE AND GEYER

724

decrease in the intracellular fading measure in B7, of the three doses tested, only 0.8 mg/kg haloperidol decreased this measure significantly, F(4,22)=5.57, p
As shown in Table 5, 0.4 mg/kg haloperidol had no effect alone but completely antagonized apomorphine’s effect on 5-HT in B7 both intracellularly and extraperikaryally. A two-way ANOVA using apomorphine treatment and haloperidol treatment as two between-subject factors also showed a significant interaction effect on intracellular and extraperikaryal 5-HT fading measures in B7, F( 1,16)= 19.65, p
The present results indicate that most doses of apomorphine significantly augment the intracellular 5-HT content in B7 without significantly affecting the cells in B8. With a very high dose (20 mg/kg) of apomorphine, intracellular 5-HT was reduced and an augmentation was observed in the extraperikaryal spaces, giving the visual impression of an overall increase in 5-HT fluorescence in B7 [30]. This latter effect may be attributable to an increased rate of 5-HT release and/or turnover [1.5]. The regional difference in apomorphine’s effect may be related to the differential amounts of catecholaminergic innervation to these two nuclei [23,25]. Previously, we reported a similar regional difference in the effects of amphetamine on 5-HT fluorescence in B7 versus B8 [lo]. although amphetamine’s effect was to decrease rather than increase intracellular 5-HT. Anatomical data demonstrate that B7 and B8 constitute the origins of two distinct serotonergic projections to the forebrain: a mesostriatal pathway originating in B7 and a mesolimbic pathway derived from B8 [3,11]. These two serotonergic systems appear to be functionally distinct as well, since lesions of B8, but not B7, reproduce many of the behavioral changes seen with wholebrain depletions of 5-HT [3,12]. The regional mapping study of apomorphine’s effect on 5-HT in B7 indicated that the interaction site between dopaminergic and serotonergic systems occurred in the ventromedial portion of B7. This result is consistent with studies using the horseradish peroxidase technique (HRP) in rats and cats, which show that the medial and medio-lateral portions of B7, particularly its rostra1 part, receives a dopaminergic projection directly from the substantia nigra [27,28]. We have recently obtained preliminary data using HRP which confirms this localization of the nigral input to B7 in rats (Lee and Geyer, unpublished observations). The time-course study suggested that the effects of apomorphine on 5-HT in B7 were not significant until 20 min

after administration, peaked at 60 min, and were still significant at 80 min. Typically, apomorphine’s pharmacological actions on the physiology of substantia nigra neurons, as well as most behaviors mediated through dopaminergic systems, are more rapid and short-lived. The relatively slow action of apomorphine on 5-HT in B7 may reflect an indirect effect, probably mediated via dopamine receptors. Our results also indicated that haloperidol at dose of 0.8 mg/kg significantly decreased intracellular 5-HT level in B7. The dose-dependent decrease of 5-HT concentration in the extraperikaryal space may indicate a decreased rate of 5-HT release and/or turnover. These results were consistent with the mean decrease in the levels of both 5-HT and 5-HIAA in striatum, although these effects were not statistically reliable with our group sizes. However, Grabowska [ 161 reported that 0.5 mg/kg haloperidol decreased 5-HT levels in rat striatum. In general, the biochemical results with both apomorphine and haloperidol agree with the histofluorescence measures. However, the opposite effects of the high dose of apomorphine on cellular 5-HT in B7 and 5-HT levels in homogenates of the corresponding terminal area, the corpus striatum, may reflect a more rapid rate of 5-HT catabolism or a marked redistribution of the amine in the cell body region. The fact that the effects of both apomorphine and haloperidol were specific to dorsal as opposed to median raphe neurons suggests that their effects were indirectly mediated by dopaminergic systems. Further support for the dopaminergic nature of apomorphine’s effect comes from the interaction between apomorphine and haloperidol. While 0.4 mg/kg haloperidol had no significant influence on B7 alone. it still antagonized the apomorphine-induced elevation of 5-HT in B7. This result corroborates the work of Grabowska ct 01. 1171, who demonstrated that dopamine antagonists block the apomorphine-induced increase in whole-brain 5-HT. Low doses of apomorphine have been suggested to act preferentially on the presynaptic dopaminergic autoreceptors intrinsic to the substantia nigra. inhibiting the neuronal firing and releasing the postsynaptic neurons from tonic inhibition [ 191. High doses of apomorphine, on the other hand. are believed to act mainly on the post-synaptic dopamine receptors. In our microspectrofluorimetric results, the maximal apomorphine effect was observed at 1 mg/kg. and a:, doses increased above I mg/kg apomorphine’s effect declined. However, the present results do not permit a specitication of whether postsynaptic or nigral dopamine receptors are responsible for the effects of apomorphine and haloperido1 on B7 neurons. While electrical stimulation of the substantia nigra is reported to decrease firing in B7 [3l]. and haloperidol appears to have no effect on firing rates in B7 1I I. the effects of either apomorphine or dopamine itself on B7 cells have not been established electrophysiologically. The indirectly acting dopamine agonist, amphetamine. increases firing [I] and reduces intracellular 5-HT levels in B7 [ 101, but this effect may be attributable to the release of norepinephtine. Perhaps the strongest indication that nigral autoreceptars are responsible for the effects of apomorphine on B7 neurons is the report that a transection between the substantia nigra and the midbrain raphe prevents the augmentation by apomorphine of midbrain 5-HT [18]. Additional experiments are required to more clearly define the nature of the interaction between central dopaminergic and serotonergic systems and to establish the precise dopaminergic mechanism responsible for the effects of apomorphine and haloperidol on dorsal raphe neurons.

SEROTONIN

725

AND APOMORPHINE

REFERENCES 1. Aghajanian, G. K. and R. Y. Wang. Physiology and pharmacology of central serotonergic neurons. In: Psychopharmacology: A Generation of Progress, edited by M. A. Lipton, A. DiMascio

and K. F. Killam. New York: Raven Press, 1978, pp. 171-183. 2. Anden, N. E., A. Rubeson, K. Fuxe and T. Hokfelt. Evidence for dopamine receptor stimulation by apomorphine. /. Pharm. Pharmac.

19: 627-629,

1967.

3. Azmitia, E. C. The serotonin-producing neurons of the midbrain median and dorsal raphe nuclei. In: Handbook of Psychopharmacology, edited by L. L. Iversen, S. D. Iversen and S. H. Snyder. New York:.Plenum Press, 1978, pp. 233-314. 4. Baconoulos. N. G.. R. K. Bhatnaaar. W. J. Schnute and L. S. Van &den,’ III. On the use of the fluorescence histochemical method to estimate catecholamine content in brain. Neuropharmacology

14: 291-299,

1975.

5. Dahlstrom, A. and K. Fuxe. Evidence for the existence of monoamine-containing neurons in the central nervous system. Acta physiol. stand. 62: Suppl. 232, l-55, 1974. 6. Einarsson, P., H. Hallman and G. Jonsson. Quantitative microfluorimetry of formaldehyde induced fluorescence of dopamine in the caudate nucleus. Med. Biol. 53: 15-24, 1975. 7. Enerback, L. and J. Jarlstedt. A cytofluorimetric and radiochemical analysis of the uptake and turnover of S-hydroxytryptamine in mast cells. J. Histochrm. Cvtochem. 23: 128-135, 1975. 8. Ernst, A. M. Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychopharmacologiu 10: 316323, 1967. 9. Ernst, A. M. and P. G. Smelik. Site of action of dopamine and apomorphine on compulsive gnawing behavior in rats. Experienria 22: 837-838, 1966. 10. Geyer, M. A., W. 3. Dawsey

and A. .I. Mandell. Differential effects of caffeine, d-amphetamine and methylphenidate on individual raphe cell fluorescence: A microspectrofluorimetric demonstration. Brain Res. 85: 135-139, 1975. II. Geyer, M. A., A. Purerto, W. J. Dawsey, S. Knapp, W. P. Bullard and A. J. Mandell. Histologic and enzymatic studies of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 241-256, 1976. 12. Geyer, M. A., A. Puerto, D. B. Menkes, D. S. Segal and A. J. Mandell. Behavioral studies following lesions of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 257270, 1976. 13. Geyer, M. A., W. J. Dawsey and A. J. Mandell. Fading: A new cytofluorimetric measure quantifying serotonin in the presence of catecholamines at the cellular level in brain. J. Pharmac. exp. Ther. 207: 650-667, 1978. 14. Geyer, M. A., C. Flicker and E. H. Y. Lee. Effects of tactile

startle on serotonin content of midbrain raphe neurons in rats. Behav. Brain Res. 4: 369-376, 15. Grabowska,

1982.

M. Influence of apomorphine on brain serotonin turnover rate. Pharmac. B&hem. Behav. 3: 58%591, 1975. 16. Grabowska, M. Butyrophenones and brain serotonin metabolism. Pol. J. Pharmac. Pharm. 28: 253-257. 1976. 17. Grabowska, M., L. Antkiewiez, J. Maj and J. Michaluk. Apomorphine and central serotonin neurons. Pal. J. Pharmac. Pharm.

25: 2%39,

1973.

18. Grabowska, M., R. Przewlocki and M. Smialowska. On the direct or indirect influence of apomorphine on central serotonin neurons. J. Pharm. Pharmac. 28: W65, 1976. 19. Groves, P. M., C. J. Wilson, S. J. Young and G. V. Rebec. Self-inhibition by dopaminergic neurons. Science 190: 522-529, 1975. 20. Jonsson, G., P. Einarsson, K. Fuxe and H. HalIman. Microspectrofluorimetric analysis of the formaldehyde induced fluorescence in midbrain raphe neurons. Med. Biol. 53: 25-39, 1975. 21. Konig, J. R. F. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas of the Forebrain

and Lower Parts of the Brain Stem.

Baltimore: Williams & Wilkins, 1963. 22. Lee, E. H. Y. and M. A. Geyer. Persistent effects of chronic administration of LSD on intracellular serotonin content in rat midbrain. Neuropharmacology 19: 1005-1007, 1980. 23. Lindvall, 0. and A. Bjorklund. The organization of the ascending catecholamine neuron systems in the rat brain. Acta physiol. stand.

412: l-48,

1974.

24. Lofstrom, A., G. Jonsson and K. Fuxe. Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. 1. Aspects on the distribution of dopamine and noradrenaline nerve terminals. J. Hisfochem. Cytochem. 24: 415-429, 1976. 25. Loizou, L. A. Projections of the nucleus locus coeruleus in the albino rat. Brain Res. 15: 563-566, 1969. 26. Mefford, I. N. Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain. J. Neurosci. Metho. 3: 207-224, 1981. 27. Pasquier, D. A., T. L. Kemper, W. B. Forbes and P. J. Morgane. Dorsal raphe, substantia nigra and locus coeruleus: Interconnections with each other and the neostriatum. Brain Res. Bull. 2: 323-339,

1977.

28. Sakai, K., D. Salvert, M. Touret and M. Jouvet. Afferent connections of the nucleus raphe dorsalis in the cat as visualized by the horseradish peroxidase technique. Brain Res. 137: 11-35, 1977. 29. Scheel-Kruger, J. and E. Hasselager. Studies of various amphetamines, apomorphine and clonidine on body temperature and brain 5-hydroxytryptamine metabolism in rats. Psychopharmacologia 36: 189-202, 1974. 30. Smialowska, M. The effect of apomorphine on serotonin neurons in dorsal raphe nucleus and catecholamine nerve terminals in paraventricular hypothalamic nucleus, histofluorescence studies. Pol. J. Pharmac. Pharm. 27: 419428, 1975. 31. Stern, W. C., A. Johnson, J. D. Bronzino and P. J. Morgane. Influence of electrical stimulation of the substantia nigra on spontaneous activity of raphe neurons in the anesthetized rat. Brain Res. Bull. 4: 561-565, 1979. 32. Tilders, F. J. H., J. S. Ploem and P. G. Smelik. Quantitative

microfluorimetric studies on formaldehyde-induced fluorescence of 5-hydroxytryptamine in the pineal gland of the rat. J. Hisrochem. Cytochem. 22: 967-975, 1974. 33. Winer, B. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1971.