Brain Research, 119 (1977) 269-290

269

© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

SEROTONERGIC AFFERENTS TO THE DORSAL RAPHE NUCLEUS: EVIDENCE FROM HRP AND SYNAPTOSOMAL UPTAKE STUDIES

SARAH S. MOSKO, DEAN HAUBRICH and BARRY L. JACOBS Department of Psychology, Princeton University and (D.H.) E. R. Squibb and Sons, Princeton, N.J. 08540 (U.S.A.)

(Accepted May 5th, 1976)

SUMMARY Afferent connections of the serotonin (5-HT)-containing dorsal raphe nucleus were investigated in the rat utilizing the horseradish peroxidase (HRP)retrograde cell labeling technique. Small quantities (0.1-0.5 #1) of HRP solutions were infused into the dorsal raphe, and the brains were examined 19-72 h later for retrograde transport of the enzyme. Intrinsic connections within the dorsal raphe nucleus were revealed by this mapping technique, as was an input to the dorsal raphe from another serotonergic cell group, the median raphe nucleus. Little evidence was found for projections from other, more remote, brain sites. A serotonergic innervation of the dorsal raphe was also demonstrated by the presence of high affinity uptake of [ZH]5-HT (Kin = 0.17/zM) into synaptosomal suspensions of the dorsal raphe nucleus. Synaptosomal uptake of [aH]5-HT was blocked by selective destruction of serotonergic axon terminals induced by the intraventricular injection of 200 #g of 5,7-dihydroxytryptamine following desipramine HC1 pretreatment, but not by destruction of catecholaminergic axon terminals induced by intraventricularly injected 6-hydroxydopamine (2 x 250/~g). The uptake of [SH]5-HT by synaptosomes of the dorsal raphe was comparable to that of striatal and hypothalamic synaptosomes, and markedly greater than that of synaptosomes from the cerebellum or nearby dorsal central gray or midbrain reticular formation, indicating the presence of a relatively dense serotonergic innervation. These data together indicate that neurons in the dorsal raphe nucleus receive a prominent serotonergic input that is derived, at least in part, from other neurons within the dorsal nucleus and from a neighboring raphe nucleus.

INTRODUCTION The application of fluorescence histochemistry has provided a relatively corn-

270 plete picture of the localization of the cell bodies and axon terminals of the serotonin (5-HT)-containing neurons of the mammalian central nervous system. 5-HT-Containing cell bodies are found primarily within the raphe complex of the brain stem25,3v, 58,60, while their projections distribute to all levels of the brain and spinal cord 7,8,26,30, 31,42,64,70. In contrast, only limited information is available concerning sources of afferents to the raphe serotonin system. The only published study addressed specifically to this issue xs utilized stains for degenerating axons following brain lesions and described afferents primarily to the caudal raphe nuclei deriving from the cerebral cortex, cerebellum and spinal cord. However, because this work predated the development of fluorescence histochemistry, it is not clear how well those raphe neurons correspond to 5-HT neurons. In addition, this study provided little information regarding inputs to the rostral (midbrain) raphe nuclei which are the major source of forebrain 5-HTS,70. Other degeneration mapping studies have provided evidence for projections to nucleus raphe centralis superior from the septum 5~, lateral preoptic region56, lateral hypothalamus34, 5G, habenula 56 and prefrontal cortex 49, and to nucleus raphe magnus from the head of the caudate nucleus 72. Once again, however, it is not clear that the raphe neurons described in these studies actually contain 5-HT. Fluorescence histochemistry has revealed catecholamine-containing varicosities in contact with several fluorescent 5-HT cell groups of the bulbar, pontine and mesencephalic raphe nuclei 30. Evidence suggests that at least some of these derive from the norepinephrine-containing neurons of the locus coeruleus 51,61. Our lack of detailed knowledge of the afferents to the raphe-serotonin system is attributable to the fact that no satisfactory method for the identification of the inputs to a particular brain region has been available until recently. The horseradish peroxidase retrograde cell labeling technique has been successfully applied to the tracing of afferent connections in the CNS. The enzyme horseradish peroxidase (HRP) is taken up by nerve terminals and transported in a retrograde direction45,46,48, and its accumulation within cell bodies distal to the site of its injection provides direct evidence for an input from these distal cells to neurons at the injection site. In the present study, the HRP technique was employed to investigate the afferent connections of the dorsal raphe nucleus in the rat. This nucleus was chosen for study because it contains the largest, most homogeneous clusters of 5-HT-containing neurons and probably gives rise to the largest number of ascending 5-HT fibers ~,z5,52,59. This study provided evidence for a major input of the dorsal raphe nucleus deriving from other neurons within the nucleus, and a lesser input deriving from the nearby median raphe nucleus. A related study examined whether these inputs might represent significant serotonergic inputs to dorsal raphe neurons. The relative density of serotonin uptake sites within the dorsal raphe was estimated through an analysis of a high affinity uptake process for serotonin in synaptosomes derived from this region. Kuhar and his colleagues have demonstrated that at low media concentrations of 5-HT, synaptosomes from forebrain regions that receive serotonergic inputs actively accumulate 5-HT (see Kuhar 41 for a review). Synaptosomes from richly innervated regions, such as the hypothalamus and striatum, exhibit much greater 5-HT uptake than do synap-

271 tosomes from the cerebellum, a region relatively sparse in serotonergic afferents 42. That high affinity uptake is preferentially localized in sites where 5-HT terminals are present is demonstrated by dramatic decreases in regional and whole forebrain uptake activity following selective lesions of the midbrain raphe nuclei 42,44. Parallel decreases in 5-HT concentration, tryptophan hydroxylase activity and histochemical fluorescence of serotonergic axons and terminals also result from these lesions 42-44. Moreover, selective destruction of serotonin terminals by intracisternal injection of the neurotoxins 5,6- or 5,7-dihydroxytryptamine reduces high affinity uptake of 5-HT into synaptosomes prepared from different brain regions 32,33. METHODS

Horseradish peroxidase study Thirty-five adult, male Sprague-Dawley rats (250-300 g), anesthetized with sodium pentobarbital (50 mg/kg, i.p.), received injections of horseradish peroxidase (type II, Sigma) in the region of the dorsal raphe nucleus. With the bite bar 5.0 mm above the interaural line, HRP solutions (10 or 33 ~) were injected in volumes of 0.1-0.5/tl at the following coordinates: AP = --4). 1 to--0.2, ML = 0.0, DV = --0.8 to -1.0. Horseradish peroxidase, prepared daily in physiological saline just prior to use, was delivered via glass pipettes (20-35/~m in tip diameter) attached to a Gilmont microburet. Rats were sacrificed 19-72 h later by intracardiac perfusion with 0.9 saline followed by 10 % formal-saline. Brains were generally fixed overnight in formalsaline at 4 °C. Control sections 35/zm thick were cut on a freezing microtome and placed into cold 30 % sucrose. The sections were stained either immediately or the next day (4 °C storage) according to the following procedure modified from Lynch et al.54. Sections were rinsed briefly at 4 °C, first in two washes of distilled water, then in 15% ethanol. The sections were then incubated in a freshly prepared solution containing equal volumes of 0.2 % benzidine dihydroxychloride in 30 ~ ethanol and 0.06 % hydrogen peroxide for approximately 1 min at room temperature. For controls, alternate sections were incubated in the absence of benzidine or HeOz. After rinsing briefly in 35 % ethanol at 4 °C, the sections were transferred to a 6 ~ solution of sodium nitroprusside (nitroferricyanide) in 50 % ethanol for 15 min at 4 °C. Sections were washed briefly in cold 25 % ethanol, mounted onto albumin-coated slides from a distilled water rinse, air dried overnight and counter-stained with safranin. Every third section through the entire brain was examined under light- and dark-field illumination. Careful reconstruction of the injected area, on diagrammatic coronal section taken from KiSnig and Klippe140, permitted precise correlation of the localization and size of the injection with the resulting retrograde cell labeling.

Uptake of [3H]5-HT by dorsal raphe homogenates Routine assay conditions Male Sprague-Dawley rats (225-300 g) were decapitated and their brains re-

272

Fig. 1. Illustrated dissection of the dorsal raphe nucleus. Left: a schematic diagram (modified from Fuxe and Jonsson ~') of a midsagittal section showing the location of the serotonin-containing cell groups B1-B9 (notation of Dahlstr6m and Fuxe25). Dashed lines 'A' and 'B' indicate the position of coronal cuts made to obtain a slice of midbrain tissue containing the dorsal raphe nucleus (BT). The dorsal raphe was dissected from this slice as diagrammed in the photograph on the right. Right: a section stained with cresyl violet taken at level 'A'. The dorsal raphe was dissected as illustrated from the ventral central gray region.

moved within 1 min. The dorsal raphe nucleus was dissected out by first removing the cortex and hippocampus to expose the dorsal surface of the midbrain. A thin slice of midbrain tissue, extending from the posterior quarter of the inferior colliculus through the caudal one-third of the superior colliculus, was then obtained by making two cuts in the coronal plane. A small piece of tissue (3-4 mg), that contained almost exclusively the dorsal raphe nucleus, was dissected from the ventral portion of the central gray region (see Fig. 1). Care was taken to avoid inclusion of tissue immediately adjacent to the aqueduct to exclude ventricular serotonergic nerve endings 2a,~3 from the sample. Uptake of serotonin by synaptosomes was determined according to the method of Coyle and Snyder 24, with modifications. The tissue was homogenized by hand in 20 volumes of 0.32 M sucrose with 15 strokes of a loosely fitted glass pestle homogenizer. It was necessary to pool raphe tissues in order to obtain a workable volume of homogenate. Centrifugation of the homogenate at 1000 × g yielded a supernatant fraction (synaptosomal suspension 44) containing synaptosomes, as well as mitochondria, myelin and microsomes73; the remaining pellet consisting of sedimented myelin, nuclei and cell fragments 73 was discarded. Incubation was carried out in test tubes containing media warmed to 37 °C in a water bath. Each test tube contained 1.9 ml of freshly prepared Krebs-Ringer-phosphate solution (DeLuca and CohenZS), to which glucose (0.01 M) and ascorbic acid (0.001 M) were added, and 10 #1 of a solution containing radioactive substrate ([3H]5-HT, 2.72 Ci/mmole). The Krebs-Ringer-phosphate solution had been saturated with 1 0 0 ~ O~ for 30 min just prior to use. Nialamide, a monoamine oxidase inhibitor, was also present at a final concentration of 12.5/~M to prevent breakdown of the substrate.

273 Aliquots (I00/A) of the synaptosomal suspension were added to each incubation tube to start the incubation period, bringing the final incubation volume to 2.01 ml. Incubation was carried out at 37 °C with gentle shaking. Parallel samples were kept on ice during the incubation period to determine the amount of uptake due to passive diffusion and non-specific binding to the tissue. These zero degree blank values were subtracted from the total uptake observed at 37 °C. Ice-cold 5-HT creatinine sulfate (1 ml of a freshly prepared 2 mM solution) was added to each tube to terminate the uptake of radiolabeled 5-HT, and the tubes were immediately placed on ice. The samples were filtered by suction through dampened filter paper discs (Whatman glass fiber, Grade GF/A, 1/~m pore size) on a Millipore filtration unit and rinsed twice with 5 ml of 0.9 ~ saline. Radioactivity was eluted from the filter papers by shaking in 1 ml of 1 N formic acid. Radioactivity was then measured at 37 ~ efficiency using a Beckman liquid scintillation spectrometer (LS-355) and a fluorophor consisting of 0.5 ~ (w/v) 2,5-diphenyloxazole (PPO) and 10~ (w/v) naphthalene in dioxane. Under routine assay conditions, the concentration of [3H]5-HT in the incubation medium was 5 × 10-s M, and incubations were carried out for 5 rain. All determinations were performed in triplicate or quadruplicate, unless otherwise indicated.

Determination of kinetic constants of [3H]5-HT accumulation Kinetic constants (Kms and VmaxS) of the synaptosomal accumulation of [3H]5-HT were determined from Lineweaver-Burk plots 50 of data obtained from experiments in which the concentration of 5-HT ranged from 0.02 to 5.0/~M. Radiolabeled 5-HT was diluted by addition of unlabeled 5-HT creatinine sulfate, and the amount of 5-HT taken up was calculated from the specific activity of the added amine. Kinetic constants were determined by least-squares regression analysis using the ~curfit program supplied by the Tymshare Corporation of Englewood Cliffs, N.J.

Properties of the high affinity synaptosomal uptake of [3H]5-HT The high affinity uptake of [3H]5-HT by synaptosomes and tissue slices from other brain regions has been shown to be a saturable process that is both time and temperature dependent15,44,62,65,74. This process is also energy and sodium dependent since it is inhibited by either the presence of metabolic poisons15,44,62,65 or by the omission of sodium ions from the incubation medium65. In addition, synaptosomal uptake can be blocked by procedures known to disrupt the limiting membrane of the synaptosome, such as treatment with the non-ionic detergent Triton X-10029,73, or repeated freezing and thawing44. The presence of each of these properties was investigated in synaptosomal suspensions of the dorsal raphe. The time course of the accumulation of [3H]5-HT was examined by varying the incubation time from 30 see to 1 h. Temperature dependency was investigated by comparing the amount of [3H]5-HT accumulation occurring at 0 °C to that at 37 °C during 5 rain of incubation. In this case alone, blank values were obtained from the amount of [ZH]5-HT adhering to filter paper discs for samples incubated without tissue at 0 or 37 °C.

274 In other experiments, the effect of metabolic inhibitors (either ouabain or 2,4dinitrophenol at final concentrations of 10--4 M and 10-3 M, respectively) was determined. For these experiments, synaptosomal suspensions were preincubated for 5 min at 37 °C in the presence of the metabolic poison and [3H]5-HT was added to begin the uptake process. The amount of [3H]5-HT accumulated under routine assay conditions was essentially the same whether the uptake process was begun by the addition of substrates following a 5 min preincubation, or whether the preincubation was omitted and the uptake process begun by the addition of the homogenate. Equimolar replacement of sodium chloride by lithium chloride in the Krebs-Ringer-phosphate solution permitted the investigation of the effect of the removal of Na + ions on uptake activity. To examine the effects of procedures known to disrupt synaptosomes, either Triton X-100 was added to the incubation medium (final concentration 0.1 '~,~)or the synaptosomal suspension was subjected to repeated freezing and thawing (4 times) using a mixture of solid CO2 and acetone.

Effects of chlorimipramine, 5,7-dihydroxytryptamine and 6-hydroxydopamine Certain tertiary amine-containing tricyclic compounds are known to inhibit selectively the accumulation of 5-HT by serotonergic neurons 19,20,2z. Chlorimipramine (CMI) is the most potent of these compounds19,2°, 65. The ability of CMI to inhibit [3H]5-HT uptake into synaptosomes prepared from the dorsal raphe was examined. Synaptosomal suspensions were preincubated with CMI (0.2-5 × 10-8 M final concentration) in the absence of [3H]5-HT for 5 min, the substrate was then added to begin the uptake process. The IC50 of CMI was determined from a log probit plot of per cent inhibition at four CMI concentrations. A least-squares regression analysis (Tymshare Corp.) provided the linear regression through the data points. 5,7-Dihydroxytryptamine (5,7-DHT), when injected intraventricularly, enters serotonergic neurons via the membrane pump and induces extensive axonal degeneration 9,11. When uptake of 5,7-DHT into catecholamine neurons is prevented by pretreatment of rats with desipramine20, zl, the neurotoxic actions of 5,7-DHT are selective for serotonergic neurons 12,3z. If the high affinity uptake of 5-HT by homogenates of the dorsal raphe is selective for serotonergic synaptosomes, then treatment of rats with 5,7-DHT should reduce the uptake. Therefore, rats anesthetized with Nembutal received injections of 5,7-DHT creatinine sulfate (200/~g free base in 20 #1 sterile saline containing 1 mg/ml of ascorbic acid) through 28-gauge cannulae lowered stereotaxically into the lateral ventricle 8 days prior to sacrifice. Control rats received vehicle injections. Solutions were infused at a rate of 2.5/~l/min by a slow infusion pump. All animals were pretreated 60 min earlier with 25 mg/kg i.p. desipramine. Synaptosomal uptake activity, under routine assay conditions, was determined in dorsal raphe tissue from both 5,7-DHT-treated and control animals. In addition, 5-HT, dopamine and norepinephrine concentrations were measured in the forebrain regions (diencephalon plus telencephalon) of the same animals. Forebrains were frozen overnight on solid CO~ and all assays were performed the next day. The tissues were homogenized (Polytron, Brinkman, PCU-2)in 8.0 ml ice-cold 0.4 N perchloric

275 acid containing 0.1 70 ascorbic acid and 0.1 70 EDTA and were then centrifuged for 20 min at 20,000 × g. Aliquots (4.0 ml) of the supernatant fluid were withdrawn for the assay of catecholamines. Serotonin was measured in the remaining supernatant fluid according to a modification as of the method of Bogdanski et al. 17. Catecholamines were estimated by the assay described by Snyder and Taylor 6s. In contrast to the effects of 5,7-DHT, another neurotoxin, 6-hydroxydopamine (6-OHDA), induces marked degeneration of catecholamine neurons while essentially no damage is incurred by serotonergic neurons a5,~9,71. To further demonstrate the selectivity in [aH]5-HT uptake into synaptosomes from the dorsal raphe, [aH]5-HT accumulation by synaptosomal suspensions from rats pretreated with 6-OHDA was compared to accumulation by suspensions from vehicle-injected control rats. Cannulae were implanted unilaterally in the lateral ventricle, and the rats were allowed to recover for several days. Two infusions of 6-OHDA (each 250/~g free base in 20/~1 saline containing 1 mg/ml ascorbic acid) were given 24 h apart. Pargyline (50 mg/kg, i.p.), a monoamine oxidase inhibitor, was administered 30 min prior to the initial infusion. Rats were sacrificed 16 days after the second infusion for determination of the uptake of [aH]5-HT by the dorsal raphe. The forebrains were frozen on solid COg and 5-HT, norepinephrine and dopamine were measured the following day.

Drugs The following drugs were used: 5,7-dihydroxytryptamine creatinine sulfate (Regis); 5-hydroxytryptamine creatinine sulfate (Sigma); 6-hydroxydopamine HBr (Regis); nialamide (Pfizer); [aH]serotonin binoxylate (New England Nuclear); pargyline HC1 ( a gift from Abbott Laboratories); and desipramine (a gift from Lakeside Laboratories). RESULTS

Horseradish peroxidase study In 22 out of 35 brains, the HRP injection was completely restricted to the dorsal raphe nucleus. In the remaining 13 brains, the injections encroached on other areas of the central gray and immediately ventral or lateral midbrain structures. Complete, detailed reconstructions of the extent of the injection and analysis of retrograde HRP transport were performed only in those cases where the injection did not involve neighboring structures. As previously described 54, a blue HRP reaction product was obtained which appeared bright pink under dark-field illumination. No such reaction product was observed when alternate sections were developed in the absence of benzidine or H202. Neurons at the injection site showed a very dense, even, non-stippled accumulation of H R P in their cell bodies. In contrast, cells identified as labeled by retrograde transport always had a stippled appearance with individual HRP-positive granules clearly visible. Usually the entire soma and proximal dendritic processes were stippled and free of any background blue coloration. The neuronal nature of labeled cells was

276

Fig. 2. Left panel: dark-field illumination photomicrograph of a dorsal raphe cell filled with HRPpositive granules following an HRP injection restricted to the ventromedial portion of the dorsal raphe nucleus as indicated in the central panel. The arrow to the left indicates that the cell was photographed from the dorsolateral region of the nucleus. A few unlabeled cells counter-stained with safranin are discernible in the background. Right panel: HRP-positive cell from the dorsal region or" the median raphe nucleus following a similarly localized injection of HRP. Center panel : diagrammatic representation of a coronal section (K6nig and Klippel4°) illustrating a medium sized HRP injection and the resulting distribution of HRP-positive cells. The shaded area shows the extent of the injection and the dots indicate the approximate location and density of labeled ceils on a 35/~m thick section near the center of the injection. As diagrammed, only cells in the dorsal aspect of the median raphe nucleus revealed retrograde labeling. DR, dorsal raphe; MR, median raphe; PCS, superior cerebellar peduncle. Scale 10 tLm.

evidenced by a large pale nucleus, dense Nissl staining and the occasionally visible a c c u m u l a t i o n o f H R P along the entire length o f the a x o n . Endothelial cells frequently stained H R P - p o s i t i v e as well, b u t could be readily identified by larger, irregularly s h a p e d staining masses a n d by their p r o x i m i t y to b l o o d vessels. H R P - p o s i t i v e cells were identified in 17 o f the 22 b r a i n s in which H R P injections were restricted to the d o r s a l r a p h e region. W h e t h e r o r n o t visible r e t r o g r a d e t r a n s p o r t o f H R P occurred did n o t seem to d e p e n d on either the c o n c e n t r a t i o n o f the H R P solution injected or the survival time. R a t h e r , visible r e t r o g r a d e accumulation o f H R P failed to occur when very discrete injections, involving only a small fraction ( < 10~o a p p r o x i m a t e l y ) o f the d o r s a l r a p h e nucleus, were made. This was p a r t i c u l a r l y true o f injections restricted to a lateral or d o r s a l region o f the nucleus. G r e a t e r labeling o f cells occurred when the b u l k o f the injection i n v a d e d the m o r e medial a n d ventral aspects o f the nucleus and involved at least an e s t i m a t e d 20 ~ o f the entire nucleus. The m o s t consistent finding was t h a t H R P - p o s i t i v e cells were f o u n d within the d o r s a l r a p h e nucleus itself (Fig. 2). I n these cases, H R P injections always involved, a n d were often restricted to, the medial a n d / o r ventral zone o f the nucleus, a n d m a n y o f the cells d o r s a l a n d d o r s o l a t e r a l to the injection showed m a r k e d a c c u m u l a t i o n o f

277 HRP-positive granules. Cells in the dorsolateral portion of the nucleus were labeled more frequently than were the more midline cells. HRP-stained cells were generally small to medium sized, somewhat oval shaped and revealed prominent, centrally placed nuclei. That the labeling of these cells was attributable to retrograde transport of HRP rather than simple diffusion from the injection site was evidenced by the fact that cells completely devoid of label were invariably found scattered among clearly labeled ones. This selectivity of accumulation of HRP granules was also emphasized by the high granule density that characterized labeled cells. Ceils showing only a slight degree of stippling were seldom encountered. In addition, a zone essentially free of label was sometimes observed between the injection site and cells filled with HRP-positive granules. In those cases where the injection completely missed or involved only peripheral aspects of the dorsal raphe, visible retrograde labeling of dorsal raphe neurons did not take place. HRP-positive cells were also observed in the dorsalmost region of the median raphe nucleus (Fig. 2). Labeling was always restricted to just a small number of cells on any one section and was visible only when the injection involved a sizeable percentage of the dorsal raphe ( > 30% approximately). Cells in the ventral two-thirds of the median raphe were never visibly labeled. Labeled cells always had a stippled appearance and were clearly removed from the site of injection. Although the density of label was high enough to clearly differentiate labeled cells from background, accumulation of HRP was not, in general, as great as in cells in the dorsal raphe. No other brain regions outside these two raphe nuclei were clearly and consistently labeled. Careful examination of cells in the area of the more caudal 5-HTcontaining cell groups failed to provide any evidence for retrograde transport of the enzyme. Even structures immediately adjacent to the dorsal raphe, e.g. the trochlear or ventral tegmental nuclei, showed negative results. Occasionally a few cells in neighboring areas of the midbrain reticular formation or within nucleus linearis caudalis showed HRP-positive staining. However, these cells were always diffusely scattered, revealed very low granule density, and were observed in only a few brains. Therefore, it was not certain that the appearance of HRP granules in these two brain regions reflected true retrograde transport of the enzyme. Anterograde as well as retrograde transport of HRP away from an injection site has recently been reportedS4, 6~. In the present study, HRP-positive axons were often seen leaving the injection site. These axons proceeded ventrally from the dorsal raphe, turning rostrally before reaching the interpeduncular nucleus. Except for what appeared to be axonal endings within the nucleus linearis caudalis, these axons could not be traced to their sites of termination.

Uptake of [SH]5-HT by dorsal raphe homogenates Kinetics of [aH]5-HT accumulation In each of two separate experiments, plots of the reciprocals of the velocity of [aH]5-HT uptake as a function of the concentration of 5-HT were resolvable into two straight lines corresponding to two kinetically distinct uptake processes. The plot

278 28

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could not be resolved into a single straight line. The results of one such experiment are shown in Figs. 3 and 4, in which the Km and Vmax for the high affinity uptake process, as determined from uptake values at low concentrations of 5-HT, were 0.17 #M and 1.18 × 10-11 mol/5 mg tissue/5 rain, respectively. The Km and Vmax of the low affinity uptake process, as determined from uptake values at relatively higher concentrations of 5-HT, were 2.48 #M and 3.47 × 10-11 mol/5 mg tissue/5 min. In another experiment, the Km values for the high and low affinity processes were estimated to be 0.10/~M and 1.96 #M, respectively. These Km values compare favorably with values obtained in other laboratories for accumulation of 5-HT into slices or synaptosomes prepared either from whole brain or brain regions15,41,65,74. To minimize involvement of the low affinity uptake process in subsequent experiments designed to selectively study the high affinity process, homogenates were incubated with [SH]5-HT at a concentration lower than the Km for the high affinity process (i.e., 5 × 10-a M). With [3H]5-HT present at this concentration under routine assay conditions (5 mg tissue, 5 min incubation), the amount of [aH]5-HT taken up by synaptosomal suspensions of the dorsal raphe ranged from 21.4 × 10-13 to 31.8 × 10-13 moles, corresponding to approximately 14,900 disint./min. Blank values determined by incubation of tissue at 0 °C were about 1750 disint./min.

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Properties of [aH]5-HT accumulation The uptake of [aH]5-HT was dependent on both time and tissue concentration. At 37 °C, the amount of [aH]5-HT taken up increased rapidly as a function o f the incubation period for up to 30 min incubation (Fig. 5). After 30 min incubation, no further increase in uptake was observed for up to 1 h (data not shown). Doubling the concentration of tissue doubled the amount of uptake during 5 min of incubation, demonstrating a dependence on tissue concentration. As shown in Table I, [aH]5-HT accumulation was markedly reduced by a variety of manipulations known to inhibit synaptosomai transport processes. Replacement of Na + ions in the incubation medium with Li+ ions reduced uptake to 52 % of controls. Incubation of samples in ice inhibited the accumulation of [aH]5-HT by 95.8 %. The amount of radioactivity adhering to filter papers when tissue samples were incubated in ice was only slightly above that which adhered when samples containing radioactive substrate, but not tissue, were filtered. The metabolic inhibitors ouabain (10-4 M) and dinitrophenol (10-a M) decreased uptake of [SH]5-HT by 80.8% and 61.2%, respectively. Thus, [aH]5-HT uptake is an energy-dependent

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MINUTES Fig. 5. Time course of the accumulation of [aH]5-HT by synaptosomes of the dorsal raphe. Synaptosomal suspensions (5 mg of tissue) were incubated in the presence of 5 × 10 a M [aH]5-HT for periods ranging from 30 sec to 30 rain.

process blocked by both low temperature and metabolic poisons. That synaptosomes are actually responsible for the accumulation of [aH]5-HT by synaptosomal suspensions is suggested by the significant reductions in uptake activity that resulted from manipulations known to disrupt synaptosomes. Incubation with Triton X-100 (0.1 ~o) or repeated freezing and thawing of the homogenate prior to incubation lowered uptake activity to 1.0 ~o and 31.7 ~ of normal, respectively. Under routine assay conditions, the accumulation of [3H]5-HT by synaptosomal TABLE i

Effect of various treatments on uptake of [SHJ5-HT by synaptosomes of the dorsal raphe Treatment

[ 3H]5-HT uptake, % Inhibition*

No Na + Low temperature (0 °C) Ouabain (10 -4 M) Dinitrophenol (10 -3 M) Triton X-100 (0.1%) Freeze and thaw (4 times)

48.1 95.8 80.8 61.2 99.0 68,3

* The percentage inhibition for each treatment was calculated from control samples analyzed in the same experiment. Uptake by controls ranged from 21.41 i 0.29 × 10 la to 23.61 4- 0.16 × 10 -13 tool/5 mg tissue/5 rain. For each of the 6 treatments, inhibition of uptake was highly significant (P < 0.001, t-test).

281 TABLE II Accumulation of f3HJ5-HT by synaptosomesfrom various brain regions* Region

f3H]5-HT accumulation (pmol/5 mg tissue/5 min)

Dorsal raphe Hypothalamus Striatum Dorsal central gray Midbrain reticular formation Cerebellum

2.34 + 0.02 2.26 -4-0.03 2.03 ± 0.09** 1.27 ± 0.05' ** 1.31 ~ 0.01*** 0.40 -4-0.01***

* The accumulation of [3H]5-HT (5 × 10-8 M) was determined using the 1000 × g supernatant fraction prepared in 0.32 M sucrose. ** Different from dorsal raphe, P < 0.02, t-test. *** Different from dorsal raphe, P < 0.001, t-test. suspensions of dorsal raphe was compared to [aH]5-HT accumulation by synaptosomal suspensions of hypothalamic, striatal and cerebellar tissue. Cerebellar uptake activity however, was measured in whole homogenates since it has been shown that 5-HT nerve endings in this region sediment during the 1000 × g centrifugation4L The hypothalamus and striatum are known to receive a rich serotonergic innervation, and synaptosomes prepared from these regions exhibit substantial high affinity uptake of 5-HT 42. Uptake of 5-HT into synaptosomes from the cerebellum, a region with only a sparse serotonergic innervation, is comparatively low 42. In addition, uptake of [aH]5-HT by synaptosomal suspensions of two relatively discrete midbrain regions proximal to the dorsal raphe, the dorsal central gray and a portion of the midbrain reticular formation, was compared to uptake by dorsal raphe suspensions. The results of these comparisons are given in Table II. Uptake of [aH]5-HT by whole homogenates of cerebellar tissue was quite low; the amount of radioactivity accumulated at 37 °C was less than 3 times that occurring at 0 °C. In contrast, synaptosomal suspensions of the dorsal raphe, hypothalamus and striatum all exhibited uptake of [aH]5H T at 37 °C greater than 8 times that of samples incubated at 0 °C. In this and several other experiments, the uptake of [3H]5-HT by the dorsal raphe always exceeded that of both hypothalamic and striatal tissues. Uptake of [aH]5-HT in all 3 of these regions was nearly twice as great as that of synaptosomal suspensions of either the nearby dorsal central gray or the midbrain reticular formation. In the experiment described in Table II, uptake of [3H]5-HT by these midbrain regions was 45.6 ~o and 44.2 ~ less, respectively, than that obtained for the dorsal raphe. Effects o f CMI, 5,7-DHT and 6-OHDA The tricyclic compound CMI, in concentrations ranging from 1 to 5 × 10-8 M, significantly inhibited [aH]5-HT uptake by synaptosomal suspensions of the dorsal raphe nucleus in a concentration-dependent fashion (Fig. 6). However, the uptake of [3H]5-HT was not altered when CMI was present in the incubation medium at 0.2 × 10-a M or 0.5 × 10-s M (P > 0.05, t-test). The ICso of CMI-induced inhibition of

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Log(CMl) Fig. 6. Graphical determination of the IC~0of CMI-induced inhibition of [3H]5-HTaccumulation by synaptosomes of the dorsal raphe. CMI in concentrations ranging from 0.5 to 5.0 × 10-8 M was preincubated for 5 min with synaptosomes. Uptake was then measured in the presence of 5 x 10 8 M [3H]5-HT. Control uptake activity was 22.69 4:2.29 × 10-13 moles/5 mg tissue/5 min. The data are expressed as per cent inhibition of control uptake activity. The linear regression through the data points was determined by least-squares regression analysis and the IC50 was calculated from the linear equation. [aH]5-HT accumulation was estimated graphically to be 2.0 × 10-s M. Comparable ICs0s have been reported for 5-HT uptake in hypothalamic slices 65. In an experiment independent of the one depicted in Fig. 6, uptake of 5-HT by synaptosomal suspensions of dorsal raphe and hypothalamus was determined under routine assay conditions in the presence of 2 × 10-8 M CMI. Comparable reductions in uptake activity near the 50 % level resulted in the two regions. Uptake activity was lowered to 44.9 ~o of normal in the dorsal raphe and to 49.8 % in the hypothalarnus. Eight days after the intraventricular administration of 200/~g of 5,7-DHT in animals pretreated with DMI, the concentration of 5-HT in the forebrain region was reduced to 29.4 ~ 4.8 % of levels found in sham-operated controls (Fig. 7). Since serotonergic cell bodies are restricted to midbrain and hindbrain regions ~5, this decrease in 5-HT content reflects the destruction of serotonin-containing axons and axon terminals. Forebrain levels of norepinephrine and dopamine, in contrast, were not significantly altered, suggesting that D M I pretreatment was effective in protecting catecholaminergic neurons from the neurotoxic action of 5,7-DHT. The forebrain

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Fig. 7. Effects of 5,7-DHT and 6-OHDA on the uptake of [aH]5-HT by synaptosomes of the dorsal raphe and on forebrain levels of serotonin, norepinephrine and dopamine. Rats were sacrificed 8 days after 5,7-DHT administration (200/~g) and 16 days following 6-OHDA (2 x 250/~g). All results are expressed as percentages of mean values measured in sham operated controls. Difference from control values: * P < 0.05, *** P < 0.001 ; t-test (N = 6--7rats per group). content of norepinephrine was 99.6 4- 5.3% that of sham operated controls, and although dopamine levels were apparently reduced to 79.1 ~: 16.7% of controls, this decrease did not reach statistical significance. In parallel with the decrease in the concentration of 5-HT in the forebrains, administration of 5,7-DHT also caused a marked reduction in the uptake of [aH]5-HT by synaptosomal suspensions of the dorsal raphe. Under routine assay conditions, accumulation of [3H]5-HT in preparations from rats treated with 5,7-DHT was only 27.8 ~: 2.3 % of that observed in sham preparations. In contrast to the selectivity of the effect of 5,7-DHT on serotonergic neurons, treatment with 6 - O H D A (2 × 250/~g) had profound effects on catecholamine neurons with comparatively little effect on serotonin-containing neurons (Fig. 7). Sixteen days after 6 - O H D A administration, the concentrations of norepinephrine and dopamine in the forebrain were reduced to 1.60 -4- 0.68 % and 10.36 ± 3.83 %, respectively, of concentrations measured in sham operated controls. In contrast to the changes in catecholamines, the concentration of 5-HT in the forebrain was not significantly

284 reduced by treatment with 6-OHDA, and the uptake of [3H]5-HT was reduced only slightly (14.1 ~o, P < 0.05) in the dorsal raphe. DISCUSSION These data provide evidence for intrinsic connections between neurons of the dorsal raphe, as well as for an input to this nucleus from a subpopulation of median raphe neurons. The latter projection was also, very recently, demonstrated in the cat by autoradiographic tracing of the efferents of the nucleus centralis superior (corresponding to the median raphe of the rat) 16. Other observations from past literature can be cited in support of serotonergic inputs to the dorsal raphe. (1) The presence of yellow fluorescent varicosities over dendrites and cell bodies of midbrain raphe neurons4; (2) selective uptake of [3H]5-HT into axon terminals within the caudal midbrain central gray region2; and (3) the sensitivity of midbrain raphe units to iontophoretically applied 5-HT 6. Although alternative interpretations of these three lines of support are possible, when taken in conjunction with the results of the present experiments, they favor the interpretation that the dorsal raphe receives a serotonergic innervation.

Horseradish peroxidase study Since cell labeling by the process of retrograde axonal transport permits identification of the presynaptic but not postsynaptic neuron, the identity of dorsal raphe elements which receive the serotonergic inputs cannot be ascertained directly. Serotonin synapses may be present on raphe neurons and/or neighboring interneurons. However, the presence of large, homogeneous clusters of 5-HT-containing neurons, which comprise the bulk of the dorsal raphe nucleus, implies that HRP was taken up by axon terminals in synaptic contact with serotonin-containing raphe neurons. Aghajanian and Haigler 5 have described three such closely packed clusters within the dorsal raphe, a dorsomedial one and two ventral ones that meet on the midline. By far the most conspicuous retrograde labeling of dorsal raphe neurons took place when HRP injections were restricted to the ventromedial aspect of the nucleus. Therefore, it is reasonable to assume that, at least in these cases, cells presynaptic to actual serotonin-containing raphe neurons were being labeled. As described above, the direct sensitivity of raphe neurons to 5-HT 6 and the existence of yellow fluorescent varicosities over midbrain raphe neurons 4 favor this interpretation as well. The accumulation of HRP-positive granules within cells of the dorsal and median raphe is most likely attributable to the retrograde transport of the enzyme. Only those cells with a stippled appearance of deposits were considered labeled by retrograde transport. According to Nauta et al. 57, only cell bodies labeled by retrograde transport reveal such a stippled appearance since the enzyme, when accumulated by retrograde transport, is confined to cell organelles. In addition, the fact that moderately to heavily labeled cells were always scattered among label-free cells in both the dorsal and median raphe suggests that diffusion alone was not responsible. Because

285 of the proximity of labeled cells to the actual site of HRP injection, it is also worthwhile to consider whether uptake into axons of passage, rather than into axon terminals, might have occurred. However, the application of the HRP tracing technique to other brain regions has shown that uptake and transport by axons of passage rarely, if ever, occurs 47,54,57. The fact that, especially for HRP-positive cells of the dorsal raphe, the number of labeled ceils and the density of accumulated granules were high makes it unlikely that axonal uptake alone can account for these results. In addition, an instance of uptake and retrograde transport of HRP by dendrites has been reported recently 67. This raises the possibility that the accumulation of HRP by dorsal and median raphe perikarya resulted from retrograde transport from the dendrites, rather than the axon terminals, of these neurons. We feel this is unlikely since no more than a single labeled process was ever observed leaving a labeled cell soma and traveling to the site of HRP injection. Multiple process labeling would be expected to occur, in at least some instances, if dendritic transport were involved. A detailed topographical analysis of the connections between cells of the dorsal raphe, and from median raphe to dorsal raphe cells, was not feasible. This was due primarily to the fact that small, discretely localized injections typically failed to result in appreciable retrograde labeling. Furthermore, visible transport of HRP was generally limited to those cases where the injection involved a considerable portion of the medial and/or ventral aspect of the dorsal raphe. Therefore, although it was clear that cells of the dorsolateral and dorsomedial regions innervate the more medial and ventral regions of the nucleus, it was less certain whether other connections (e.g., reciprocal ones) exist within the nucleus. Likewise, projections from the median raphe nucleus to the medial zone of the dorsal raphe were demonstrated far more convincingly than were projections to other parts of the nucleus. No cells outside the midbrain raphe were reliably labeled following dorsal raphe injections. However, because of inherent limitations of the technique, negative findings cannot be taken as evidence against the existence of afferents from more distal brain sites. It appears that not all axon terminals visibly take up and transport the protein 54,57. Although the factors which determine the degree of retrograde labeling are not well understood, there is some indication that a cell's capacity to accumulate HRP in a retrograde manner is related to the number and size of its terminalsSL Other possible impediments to successful retrograde labeling of afferents include (1) the diluting effect of retrograde transport from axon collaterals distributed to areas outside the injection site and (2) interposed barriers, e.g. glial sheaths, preventing exposure of axonal endings to the HRP 57. Therefore, other inputs to the dorsal raphe may have escaped detection. This does not seem unlikely since neurons outside the raphe which purportedly supply afferents to the dorsal raphe, e.g. the locus coeruleus 51, were consistently label-free.

Uptake of [3H]5-HT by dorsal raphe homogenates The results of the uptake studies, in conjunction with the results of the HRP transport study, provide evidence for a serotonergic innervation of the dorsal raphe

286 nucleus. This conclusion is based on the resolution of a high affinity transport process for 5-HT in synaptosomal suspensions of dorsal raphe tissue that possesses properties which characterize uptake sites for 5-HT known to be associated with serotonergic nerve endings. These include time, temperature, sodium and energy dependence. Treatment of synaptosomal suspensions with Triton X-100 or repeated freezing and thawing markedly reduced the accumulation of 5-HT, suggesting that synaptosomes are indeed responsible for the uptake. The specificity of 5-HT accumulation was demonstrated through various types of pharmacological intervention. Chlorimipramine, a selective competitive inhibitor of 5-HT uptake el,65, and selective destruction of 5-HT axons with the neurotoxin 5,7-DHT both markedly reduced the accumulation of 5-HT by the dorsal raphe. On the other hand, widespread destruction of catecholamine axons by 6-OHDA produced only a slight decrement in the accumulation of 5-HT by synaptosomes. It has recently been demonstrated that dendritic terminals of certain catecholaminergic neurons contain a3,3G,e6 and accumulate13 their transmitter. By analogy this might raise the possibility that uptake by raphe cell dendrites, not axon endings, was responsible for the accumulation of [3H]5-HT by homogenates of the dorsal raphe. Although this is an interesting issue, there is currently no indication that the dendrites of raphe or other neurons are specialized to actively take up their own transmitter. If 5-HT uptake by dendrites does occur in raphe cells, there is no evidence that it would exhibit the same kinetic properties as the high affinity uptake of 5-HT by the axon terminal. Furthermore, it is not clear that dendritic terminals pinch-off during sucrose homogenization, as do axon terminals, thus being sensitive to the same manipulations which disrupt the limiting membrane of the synaptosome. In a light and electron microscopic autoradiographic study of the distribution of radioactive particles following the intraventricular injection of [3H]5-HT, Aghajanian and Bloom ~ reported a preponderance of grain clusters in the caudal portion of the midbrain central gray, corresponding to the locus of the dorsal raphe nucleus. While 78 % of the grains were seen over nerve endings and axons, only a small percentage were scattered over dendrites, cell bodies and other structures. The failure of these authors to observe appreciable uptake of [3H]5-HT by dendrites in the region of the dorsal raphe is consistent with a synaptosomal rather than a dendritic site of [3H]5-HT uptake by homogenates Of the dorsal raphe. Finally, the present observation that 5,7-DHT (the neurotoxic actions of which are reportedly selective for the terminal and non-terminal portions of the serotonergic neuron 1°,a4,2'~) effectively blocks [3H]5-HT uptake by dorsal raphe homogenates favors a synaptosomal site of 5-HT uptake as well. However, we recognize that these data are insufficient to completely rule out the possibility that raphe neurons may communicate through dendrodendritic or dendrosomatic rather than axodendritic or axosomatic synapses. An investigation into the nature of synaptic elements within the dorsal raphe which stain positively for an antibody to tryptophan hydroxylase might resolve this issue. Because the uptake activity of dorsal raphe synaptosomes was at least comparable to that of synaptosomes from the striatum and hypothalamus, areas known to receive a relatively dense serotonergic innervation42, and was markedly greater than

287 uptake by synaptosomes prepared from the nearby midbrain reticular formation and dorsal central gray, the density of serotonergic synapses within the dorsal raphe must be relatively high. However, the sources of these 5-HT afferents were not investigated in the synaptosomal uptake study. The H R P transport study suggests that at least some derive from the median raphe and other dorsal raphe cells. Visible H R P transport to any of the more caudal 5-HT-containing cell groups was not found. It is also conceivable that recurrent collaterals of dorsal raphe neurons project back onto the parent neuron and account for some of the 5-HT accumulation by dorsal raphe homogenates. ImpOcations

As discussed in the following paper, connections between serotonergic neurons bear on an important issue concerning the regulation of the functional activity of these neurons. Based on the observation that increases in the synaptic availability of 5-HT decrease the discharge rate of midbrain raphe neurons, it has been postulated that impulse flow in 5-HT-containing neurons is subject to regulation via negative neuronal feedbackL A compensatory, neuronally mediated decrease in the discharge rate of raphe neurons is hypothesized to take place in response to increased activity at postsynaptic 5-HT receptors. The existence of raphe-raphe connections, however, raises the alternative possibility that manipulations which increase the concentration of 5-HT in the synapse depress the discharge of raphe neurons directly via serotonergic synapses on raphe neurons. ACKNOWLEDGEMENTS This research was supported by Grants M H 23433 and M H 13445 from the National Institute of Mental Health, and the Spencer Foundation. We would like to thank Pfizer, Inc. (PCPA) and Lakeside Laboratories (DMI) for their generous gift of drugs. We thank Dr. Gary S. Lynch for generously donating his time and facilities in making the H R P portion of this paper possible and Thomas J. Chippendale for his assistance in the uptake studies.

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