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Effects of parachloroamphetamine upon the serotonergic innervation of the rat hippocampus
Brain Research, 577 (1992) 253-260 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
253
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Effects of parachloroamphetamine upon the serotonergic innervation of the rat hippocampus John H. Haring, Lewis Meyerson and Terri L. Hoffman Department of Anatomy and Neurobiology, St. Louis University Medical Center, St. Louis, MO 63104 (USA) (Accepted 19 November 1991) Key words: Serotonin; Para-chloroamphetamine; Hippocampal formation; Synaptosomal uptake; Immunocytochemistry; WGA-HRP; PHA-L; Plasticity
The ability of hippocampal serotonergic (5-HT) axons to proliferate in response to damage by para-chloroamphetamine (PCA) was examined in this study. Synaptosomal uptake of 5-HT in the hippocampal formation was decreased to 40% of control 3 days after systemic administration of PCA. Six weeks after PCA, uptake values were 44% of control. Retrograde tracing combined with 5-HT immunocytochemistry showed a significant reduction (18% of control) in the number of 5-HT raphe neurons projecting to the hippocampus 3 days after PCA. The number of 5-HT neurons projecting to the hippocampal formation increased to 69% of control by 6 weeks. The dorsal raphe nucleus was not retrogradely labeled after PCA; the increase in labeled neurons was observed in the median raphe nucleus. PHA-L, injections of the median raphe nucleus demonstrated a reduction of raphe axons in the hippocampal formation after PCA. In rats treated with PCA, raphe axons labeled with PHA-L also appeared to have fewer boutons than raphe axons labeled in control cases. The density of PHA-L containing axons in the hippocampal formation of rats injected 3 days and 6 weeks after PCA was less than control but there was no difference between the experimental groups. Based upon the results from synaptosomal uptake and anterograde tracing experiments, we feel that compensatory proliferation of 5-HT axons does not occur within 6 weeks of PCA-induced damage to the 5-HT plexus of the hippocampal formation. The data derived from the retrograde tracing experiment are thought to reflect reduced uptake and transport of WGA-HRP as an acute effect of PCA. INTRODUCTION The serotonergic (5-HT) projections within the central nervous system are capable of responding to injury by c o m p e n s a t o r y proliferation of u n d a m a g e d axons 1-5'8'24' 25. Studies demonstrating the plasticity of 5-HT projections have used the 5-HT-selective neurotoxin 5,7-dihyd r o x y t r y p t a m i n e ( 5 , 7 - D H T ) to p r o d u c e partial lesions of 5-HT inputs. These studies have shown that within 6 weeks of 5 , 7 - D H T lesions, evidence of 5-HT axon proliferation can be o b t a i n e d by analysis of a n t e r o g r a d e tracing, 5 - H T u p t a k e , 5-HT content, r e t r o g r a d e tracing and 5-HT i m m u n o c y t o c h e m i s t r y 1'4'5'8'24'25. Systemic administration of p a r a - c h l o r o a m p h e t a m i n e ( P C A ) has been widely used to d e p l e t e forebrain 5-HT in a variety of neurobiological studies (e.g. refs. 6,12,16, 20,21). P C A is a halogen-substituted a m p h e t a m i n e that is toxic to ascending 5 - H T projections but appears to have little or no effect on the neurons of the dorsal ( D R N ) and m e d i a n ( M R N ) raphe nuclei o r the B9 cell group 6'12'13'14. L o n g - t e r m decreases in t r y p t o p h a n hydroxylase activity have also b e e n r e p o r t e d 18'19. Until very recently, there have b e e n no reports of 5-HT axon
plasticity after P C A treatment. Evidence suggesting that 5-HT axons regenerate several months after P C A has now been r e p o r t e d 16. Because the m e t h o d s frequently used to detect changes in the 5-HT axon plexus rely on the presence of 5-HT (e.g. immunocytochemistry and H P L C ) , the proliferation of 5-HT axons could be missed or seriously u n d e r e s t i m a t e d due to decreased 5 - H T levels caused by the suppression of t r y p t o p h a n hydroxylase. In the present study, we have asked whether raphe axons proliferate in P C A - t r e a t e d animals within the 6-week p e r i o d d o c u m e n t e d in cases of partial 5-HT depletion by 5,7-DHT. M e t h o d s used in this study include 5-HT-specific and 5-HT-non-specific techniques in an a t t e m p t to ascertain whether 5-HT axon plasticity has been undetected due to t r y p t o p h a n hydroxlyase inhibition by P C A . MATERIALS AND METHODS Subjects for these experiments were adult, male SpragueDawley rats (250-300 g, Harlan). Rats in the experimental groups received two subcutaneous injections of PCA 24 h apart (10 mg/ kg). These rats were treated with sodium pentobarbital (30 mg/kg, i.p.) 10 min prior to PCA injection to prevent seizures resulting from the PCA-induced release of monoamines21. Control rats received subcutaneous injections of normal saline. PCA-treated rats
Correspondence: J.H. Hating, Department of Anatomy and Neurobiology, St. Louis University Medical Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. Fax (1) (314) 772-1307.
254 were studied 3 days and 6 weeks after injection.
Synaptosomal uptake of 5-HT Rats (3 from each experimental group and 3 controls) were euthanized by sodium pentobarbital overdose; their brains were quickly removed and the hippocampi dissected on a chilled glass plate. Each hippocampus was homogenized and a crude synaptosomal pellet obtained as described by Whitaker-Azmitia et al. 22. Incubations for uptake were performed in triplicate in 1.5 ml microcentrifuge tubes with a total reaction medium volume of 200 #1. The reaction medium consisted of artificial CSF containing 0.40 mM pargyline, 1 mM ascorbic acid and 50 nM [3H]5-HT (ARC, 26.7 Ci/mmol). Non-specific uptake was determined by the addition of 1/~M fluoxetine. The temperature of the incubation tubes was raised to 37°C and the reaction begun by the addition of 20 ~1 of the synaptosomal suspension. The reaction was terminated after 20 min by the addition of 1 ml of ice-cold artificial CSF to each tube. Samples were filtered through Gelman glass filters and washed with 0.05 M phosphate-buffered saline using a Millipore cell harvester. Protein levels in the synaptosomal suspension were determined using the BCA protein analysis reagent (Pierce). Uptakes were expressed as CPM/mg protein.
Retrograde tracing with 5-HT immunocytochemistry Rats (5 in each experimental group and 3 control) were anesthetized with sodium pentobarbital (65 mg/kg) and placed in a stereotaxic frame (Kopf). Injections of 2% WGA-HRP (Sigma) were made in the dorsal area dentata using the following stereotaxic coordinates: AP = -3.3, ML = -1.5, DV = -3.7; bregma zero; incisor bar -3.3. A total tracer volume of 50 nl was delivered over 5 min using a glass micropipette (tip diameter 30 ~m) attached to a 1-pl Hamilton syringe. After 48 h, rats were overdosed with sodium pentobarbital and perfused transcardially with 0.1 M phosphatebuffered saline followed by 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Sections through the hippocampal formation and brainstem were reacted for HRP histochemistry by the TMB method of Mesulam Ls and the reaction product was stabilized by the DAB-cobalt technique ~7. Brainstem sections were then incubated in 5-HT antiserum (1:2000, Eugene Tech) and processed by the avidin-biotin-peroxidase method 9, (Vector) using 3-amino-9-ethylcarbazole as the chromagen to produce a red reaction product. Raphe neurons containing WGA-HRP alone or in combination with 5-HT immunoreactivity were counted in every other section through the brainstem. The distribution of labeled cells was plotted on standardized diagrams at three levels through the dorsal (DRN) and median (MRN) raphe nuclei.
Anterograde tracing with PHA-L The status of the MRN projection to the hippocampal formation was studied by anterograde transport of PHA-L 7 in 13 rats (5 in each experimental group and 3 controls). Deposition of PHA-L in the MRN was accomplished by iontophoresis (5 mA, 7 s on -7 s off for 20 min) using the following coordinates: AP = 12.3, ML = 0.0, DV = -9.5; bregma zero; incisor bar -3.3; micromanipulator tilted 30° posterior. After 5 days, rats were perfused as described above and sections through the hippocampal formation and brainstem were processed for PHA-L immunocytochemistry. PHA-L antiserum (Vector) was diluted 1:2000 and DAB was the chromagen for the avidin-biotin-peroxidase technique.
RESULTS
The mean hippocampal 5-HT uptake value obtained from control synaptosomal preparations was 12,199.25 + 2464.23 CPM/mg protein. 5-HT uptake was reduced to 40% of control (4922.81 _+ 1178.96 CPM/mg protein) in rats studied 3 days after PCA treatment. Six weeks after PCA, 5-HT uptakes were 44% of control (5332.5 + 779.72 CPM/mg protein). Uptake values at both 3 days and 6 weeks were significantly (P < 0.05, Mann-Whitney U-test) less than control but were not statistically different from each other. The W G A - H R P injection sites included the area dentata and parts of the overlying CA1 region (Figs. 1 and 2) and extended from the septal pole to the hippocampal flexure (Fig. 2). Neurons in both the DRN and MRN were retrogradely labeled from the hippocampal WGAH R P injections. Retrogradely labeled 5-HT neurons were distinguished by black, granular reaction product from the transported W G A - H R P and red reaction product from the carbazole reaction from the 5-HT immuncytochemical processing (Fig. 1). With few exceptions, DRN neurons labeled from the hippocampal formation of control cases were also 5-HT immunopositive (Fig. 2). In the MRN, a significant number (26%) of neurons projecting to the hippocampal formation were not 5-HT-immunoreactive (Fig. 2). After PCA treatment, no retrograde labeling was seen in the DRN and labeling of MRN 5-HT neurons was reduced. Three days after PCA the total number of 5-HT neurons having projections to, the hippocampal formation was 18% of control (Fig. 3). The ratio of 5-HT to non-5-HT MRN neurons projecting to the hippocampal formation was not significantly different from control cases. Six weeks after PCA, DRN labeling was still absent but the number of labeled 5-HT MRN cells had risen to 69% of control (Fig. 3). Non5-HT projection neurons accounted for 35% of the total number of MRN neurons labeled with W G A - H R P in these cases. Iontophoresis of PHA-L resulted in small injection sites in the MRN (Fig. 4) that contained extracellular reaction product as well as labeled neurons. The size of the effective injection site was determined by the number and distribution of labeled neurons in each case. Control and experimental cases were matched for anal-
Fig. 1. Brightfield photomicrographs illustrating a WGA-HRP injection site (A) and retrogradely labeled, 5-HT-immunopositive raphe neurons (B). WGA-HRP injections of the dorsal hippocampal formation included the area dentata and parts of the overlying subiculum and CA1 (A). There was no spread of reaction product into neocortex and anterograde labeling of mossy fibers was present (A, arrows). Retrogradely labeled, 5-HT-immunopositive neurons of the dorsal and median raphe nuclei were identified by the presence of black granular reaction product (B, small arrows) within neurons containing the red reaction product generated by the use of carbazole in the 5-HT immunocytochemical procedure. Bars: A = 200 ~m: B = 20/~m.
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Fig. 2. In this diagram, A, B and C illustrate the regions of the dorsal hippocampal formation included in a typical WAG-HRP injection. D, E and F show the distribution of raphe neurons retrogradely labeled from the injection depicted in A-C. In this control case, the distribution of labeled neurons and the percentage of 5-HT to non-5-HT cells is consistent with previous reports of retrograde raphe labeling after hippocampal injections ]°. Each symbol represents one labeled neuron; dots indicate cells containing WGA-HRP and 5-HT immunoreactivity, circles indicate cells containing WGA-HRP alone, bc, brachium conjunctivum; cc, corpus callosum; CTX, cerebral cortex; DRN, dorsal raphe nucleus; fx, fimbria-fornix; HF hippocampal formation; IV, trochlear nucleus; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MRN, median raphe nucleus; PAG, periaquaductal grey; RT, retieulotegmental nucleus; VT, ventral tegmental nucleus; xbc, decussation of the brachium conjunctivum. Bars = 1 mm
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Fig. 3. This diagram illustrates the distribution of retrogradely labeled neurons in the dorsal and median raphe nuclei in rats receiving hippocampal imeetions of WGA-HRP 3 days (A-C) and 6 weeks (D-F) after systemic administration of PCA. DRN projections to the hippocampal formation are eliminated by PCA treatment and no recovery is seen by 6 weeks. Fewer MRN neurons are labeled from the hippocampal formation after PCA, but an increase in the numbers of retrogradely labeled MRN neurons is observed between 3 days post-PCA and 6 weeks. Symbols, abbreviations and calibration bars in this figure are identical to those defined in Fig. 2
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Fig. 4. Brightfieid photomicrographs showing the axonal labeling in the area dentata of the hippocampal formation that results from PHA-L injections in the median raphe nucleus of control rats. A: PHA-L injection site (arrow) in the ventrolateral part of the median raphe nucleus. B: this panel illustrates the distribution of median raphe axons in the area dentata. Axons are seen in the molecular layer (small arrows) as well as in the hilar re#on (large arrows). Note the presence of prominent boutons that identify these axons as arising from the median raphe nucleus (11). C and D: higher power views of PHA-L labeled axons (arrows) in the molecular layer (C) and dentate granule cell layer and hilar region (D). DGC, dentate granule cell layer; DRN, dorsal raphe nucleus; H, hflar region; Mol, molecular layer; MRN, median raphe nucleus. Bars: A = 200/lm; B = 100 #m; C and D = 50/tm.
ysis on the basis of similar numbers of PHA-L-containing neurons in the MRN. P H A - L injections of the MRN in control animals resulted in the labeling of numerous axons throughout the hippocampal formation. Within the area dentata, raphe axons were seen in the molecular layer and granule cell layer and were particularly prominent in the hilar region (Fig. 4). Regardless of their location, MRN axons exhibited a common morphology (Fig. 4) that featured prominent boutons 11. Following PCA treatment, the number of axons labeled from MRN injections was diminished and, P H A - L labeled axons appeared to have fewer boutons, (Fig. 5).
MRN projections to the molecular layer and granule cell layer appeared to be more severely affected by PCA than was the hilar region (Fig. 5). No difference in the density or distribution of P H A - L labeled MRN axons was apparent between the 3-day and 6-week PCA groups. DISCUSSION
The present study has confirmed and extended previous observations of the effects of PCA administration on the 5-HT innervation of the hippocampal formation. The
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Fig. 5. Brightfield photomicrographs illustrating the axon labeling resulting from a P H A - L injection in the median raphe nucleus made 6 weeks after P C A treatment. A: P H A - L injection site (arrow) in the ventrolateral M R N . B and D: fewer PHA-L-labeled axons are seen in the dorsal area dentata 6 weeks after P C A than are present in control rats (arrows). Note that the majority of labeled axons are located in the hilar region adjacent to the granule cell layer. C: this photomicrograph demonstrates P H A - L labeled axons with prominent boutons related to the dentate granule cell layer (arrows). Abbreviations are identical to those defined in Fig. 4. Bars: A = 100 # m ; B and D = 50 #m;C = 25#m.
approximately 40% decrease in 5-HT uptake in crude synaptosomal preparations observed 3 days after PCA is comparable to values reported after PCA administration paradigms similar to our own 2°. We have shown that this reduction in 5-HT uptake persists for at least 6 weeks. This observation suggests that 5-HT axons are not proliferating during this time in contrast to the increase in 5-HT uptake reported 6 weeks after partial MRN lesions using 5,7 D H T 1'4'5'8'24'25. Anterograde labeling of MRN axons with PHA-L supports the interpretation of the 5-HT uptake data. Fewer hippocampal axons were labeled from MRN injections in PCA-treated rats and no difference was noted
between the 3-day and 6-week experimental groups. The reduction in MRN axons projecting to the hippocampal formation indicates that the change in 5-HT uptake is the result, at least in part, of the loss of raphe axons and not due to an effect of PCA on the 5-HT transporter alone. These data also show that MRN axons are susceptible to damage by PCA. The observations obtained by combining retrograde tracing with 5-HT immunoeytochemistry demonstrate the loss of DRN projections to the hippocampal formation after PCA administration. The number of MRN neurons labeled is also reduced but the majority of these cells contained 5-HT. Thus one can infer that the majority of
259 PHA-L-labeled axons projecting to the hippcampal formation of PCA-treated are from 5-HT MRN neurons. A severe reduction in MRN neurons labeled from the hippocampal formation 3 days after PCA is followed by an increase in labeling to almost 70% of control by 6 weeks. This change might indicate the regeneration or sprouting of 5-HT axons in the hippocampal formation 23'24 were it not for the results of 5-HT uptake and anterograde tracing experiments. Because 5-HT-specific and 5-HT-non-specific axon marking techniques fail to show any change in the axon plexus of PCA-treated rats, the change in retrograde labeling must reflect an acute effect of PCA on the uptake and transport of WGAHRE The retrograde labeling data also support the contention that some 5-HT MRN neurons are affected by PCA. Although no change was noted in the ratio of 5-HT to non-5-HT MRN neurons labeled from the hippocampal formation 3 days after PCA, an increase of about 10% was seen in the population of labeled non-5-HT neurons from rats studied 6 weeks after PCA. PCA is known to inhibit tryptophan hydroxylase in the projection areas of the DRN and MRN 13'14'1s'19. The increase in the number of 5-HT-immunonegative MRN cells with projections to the hippocampal formation suggests that tryptophan hydroxylase activity may be suppressed in some raphe neurons after PCA administration. Preliminary counts of 5-HT-immunopositive neurons in the DRN and MRN of PCA-treated rats indicate that the number
of DRN 5-HT neurons is decreased by about 20% while the MRN contains about 10% fewer 5-HT neurons 6 weeks after PCA (Haring, unpublished observations). No evidence of raphe neuron degeneration has yet been obtained, therefore these changes are thought to indicate inhibition of 5-HT synthesis among certain 5-HT neurons of the DRN and MRN. In summary, compensatory proliferation of hippocampal 5-HT axons does not occur within 6 weeks of 5-HT denervation by systemic administration of PCA. This is in contrast to the reported sprouting and regeneration of 5-HT axons that follows 6 weeks after partial denervations of CNS structures by 5 , 7 - D H T 1'5'6'8'24'25. The basis for this difference is unclear. That complex effects of PCA upon cell function and metabolism may limit the plastic response of raphe neurons is suggested by the observation that 5-HT axon proliferation may occur months after PCA treatment 16. The time at which this proliferation of 5-HT axons has been observed coincides with times at which the restoration of tryptophan hydroxylase activity has been reported 18'19. Thus it is possible that the recovery of tryptophan hydroxylase activity signals a wider recovery of 5-HT neuronal function from the effects of PCA.
REFERENCES
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1 Azmitia, E.C., Buchan, A.M. and Williams, J.H., Structural and functional restoration by collateral sprouting of hippocampal 5-HT axons, Nature, 274 (1978) 374-376. 2 Bj6rklund, A. and Stenevi, U., Regeneration of monaminergic and cholinergic neurons in the mammalian central nervous system, Physiol. Rev., 59 (1979) 62-100. 3 Bj6rklund, A., Wiklund, L. and Descarries, L., Regeneration and plasticity of central serotonergic neurons: a review, J. Physiol. (Paris), 77 (1981) 247-255. 4 Frankfurt, M. and Azmitia, E.C., Regeneration of serotonergic fibers in the rat hypothalamus following unilateral 5,7-dihydroxytryptamine injection, Brain Res., 298 (1984) 273-282. 5 Frankfurt, M. and Beaudet, A., Ultrastructural organization of regenerated serotonin axons in the dorsomedial hypothalamus of the adult rat, J. Neurocytol., 16 (1987) 799-809. 6 Fritschy, J.M., Lyons, W.E., Molliver, M.E. and Grzanna, R., Neurotoxic effects of p-chloroamphetamine on the serotonergic innervation of the trigeminal motor nucleus, Brain Res., 473 (1988) 261-270. 7 Gerfen, C.R. and Sawchenko, EE., An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris-leukoagglutinin (PHA-L), Brain Res., 343 (1984) 219-238. 8 Hating, J.H., Reorganization of the area dentata serotoninergic plexus after lesions of the median raphe nucleus, J. Comp. Neurol., 306 (1991) 576-584.
Acknowledgements. The technical assistance of L. Beaudet is gratefully acknowledged. This study was supported by USPHS Grants NS25752 and DE07734.
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superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. Molliver, M.E. and Axt, K.J., Specific regional patterns of serotonergic innervation in ventral areas of forebrain in the rat; selective denervation and reinnervation after administration p-chloroamphetamine (PCA), Soc. Neuroci. Abstr., 16 (1990) 1032. Rye, D.B., Saper, C.B. and Wainer, B.H., Stabilization of the tetramethyl benzidine (TMB) reaction product: application for retrograde and anterograde tracing and combination with immunohistochemistry, J. Histochem. Cytochem., 32 (1984) 11451153. Sanders-Bush, E., Bushing, J. and Sulser, F., Long-term effects of p-chloroamphetamine on tryptophan hydroxylase activity and on the levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in brain, Eur. J. Pharmacol., 20 (1972) 385-388. Sanders-Bush, E. and Steranka, L.R., Immediate and longterm effects of p-chloroamphetamine on brain amines. In J.H. Jacoby and L.D. Lytle (Eds.), Serotonin Neurotoxins, Annals of the New York Academy of Sciences, Vol. 305, The New York Academy of Sciences, New York, 1978, pp. 209-221. Srebro, B., Azmitia, E.C. and Winson, J., Effects of 5-HT de-
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253
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Effects of parachloroamphetamine upon the serotonergic innervation of the rat hippocampus John H. Haring, Lewis Meyerson and Terri L. Hoffman Department of Anatomy and Neurobiology, St. Louis University Medical Center, St. Louis, MO 63104 (USA) (Accepted 19 November 1991) Key words: Serotonin; Para-chloroamphetamine; Hippocampal formation; Synaptosomal uptake; Immunocytochemistry; WGA-HRP; PHA-L; Plasticity
The ability of hippocampal serotonergic (5-HT) axons to proliferate in response to damage by para-chloroamphetamine (PCA) was examined in this study. Synaptosomal uptake of 5-HT in the hippocampal formation was decreased to 40% of control 3 days after systemic administration of PCA. Six weeks after PCA, uptake values were 44% of control. Retrograde tracing combined with 5-HT immunocytochemistry showed a significant reduction (18% of control) in the number of 5-HT raphe neurons projecting to the hippocampus 3 days after PCA. The number of 5-HT neurons projecting to the hippocampal formation increased to 69% of control by 6 weeks. The dorsal raphe nucleus was not retrogradely labeled after PCA; the increase in labeled neurons was observed in the median raphe nucleus. PHA-L, injections of the median raphe nucleus demonstrated a reduction of raphe axons in the hippocampal formation after PCA. In rats treated with PCA, raphe axons labeled with PHA-L also appeared to have fewer boutons than raphe axons labeled in control cases. The density of PHA-L containing axons in the hippocampal formation of rats injected 3 days and 6 weeks after PCA was less than control but there was no difference between the experimental groups. Based upon the results from synaptosomal uptake and anterograde tracing experiments, we feel that compensatory proliferation of 5-HT axons does not occur within 6 weeks of PCA-induced damage to the 5-HT plexus of the hippocampal formation. The data derived from the retrograde tracing experiment are thought to reflect reduced uptake and transport of WGA-HRP as an acute effect of PCA. INTRODUCTION The serotonergic (5-HT) projections within the central nervous system are capable of responding to injury by c o m p e n s a t o r y proliferation of u n d a m a g e d axons 1-5'8'24' 25. Studies demonstrating the plasticity of 5-HT projections have used the 5-HT-selective neurotoxin 5,7-dihyd r o x y t r y p t a m i n e ( 5 , 7 - D H T ) to p r o d u c e partial lesions of 5-HT inputs. These studies have shown that within 6 weeks of 5 , 7 - D H T lesions, evidence of 5-HT axon proliferation can be o b t a i n e d by analysis of a n t e r o g r a d e tracing, 5 - H T u p t a k e , 5-HT content, r e t r o g r a d e tracing and 5-HT i m m u n o c y t o c h e m i s t r y 1'4'5'8'24'25. Systemic administration of p a r a - c h l o r o a m p h e t a m i n e ( P C A ) has been widely used to d e p l e t e forebrain 5-HT in a variety of neurobiological studies (e.g. refs. 6,12,16, 20,21). P C A is a halogen-substituted a m p h e t a m i n e that is toxic to ascending 5 - H T projections but appears to have little or no effect on the neurons of the dorsal ( D R N ) and m e d i a n ( M R N ) raphe nuclei o r the B9 cell group 6'12'13'14. L o n g - t e r m decreases in t r y p t o p h a n hydroxylase activity have also b e e n r e p o r t e d 18'19. Until very recently, there have b e e n no reports of 5-HT axon
plasticity after P C A treatment. Evidence suggesting that 5-HT axons regenerate several months after P C A has now been r e p o r t e d 16. Because the m e t h o d s frequently used to detect changes in the 5-HT axon plexus rely on the presence of 5-HT (e.g. immunocytochemistry and H P L C ) , the proliferation of 5-HT axons could be missed or seriously u n d e r e s t i m a t e d due to decreased 5 - H T levels caused by the suppression of t r y p t o p h a n hydroxylase. In the present study, we have asked whether raphe axons proliferate in P C A - t r e a t e d animals within the 6-week p e r i o d d o c u m e n t e d in cases of partial 5-HT depletion by 5,7-DHT. M e t h o d s used in this study include 5-HT-specific and 5-HT-non-specific techniques in an a t t e m p t to ascertain whether 5-HT axon plasticity has been undetected due to t r y p t o p h a n hydroxlyase inhibition by P C A . MATERIALS AND METHODS Subjects for these experiments were adult, male SpragueDawley rats (250-300 g, Harlan). Rats in the experimental groups received two subcutaneous injections of PCA 24 h apart (10 mg/ kg). These rats were treated with sodium pentobarbital (30 mg/kg, i.p.) 10 min prior to PCA injection to prevent seizures resulting from the PCA-induced release of monoamines21. Control rats received subcutaneous injections of normal saline. PCA-treated rats
Correspondence: J.H. Hating, Department of Anatomy and Neurobiology, St. Louis University Medical Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. Fax (1) (314) 772-1307.
254 were studied 3 days and 6 weeks after injection.
Synaptosomal uptake of 5-HT Rats (3 from each experimental group and 3 controls) were euthanized by sodium pentobarbital overdose; their brains were quickly removed and the hippocampi dissected on a chilled glass plate. Each hippocampus was homogenized and a crude synaptosomal pellet obtained as described by Whitaker-Azmitia et al. 22. Incubations for uptake were performed in triplicate in 1.5 ml microcentrifuge tubes with a total reaction medium volume of 200 #1. The reaction medium consisted of artificial CSF containing 0.40 mM pargyline, 1 mM ascorbic acid and 50 nM [3H]5-HT (ARC, 26.7 Ci/mmol). Non-specific uptake was determined by the addition of 1/~M fluoxetine. The temperature of the incubation tubes was raised to 37°C and the reaction begun by the addition of 20 ~1 of the synaptosomal suspension. The reaction was terminated after 20 min by the addition of 1 ml of ice-cold artificial CSF to each tube. Samples were filtered through Gelman glass filters and washed with 0.05 M phosphate-buffered saline using a Millipore cell harvester. Protein levels in the synaptosomal suspension were determined using the BCA protein analysis reagent (Pierce). Uptakes were expressed as CPM/mg protein.
Retrograde tracing with 5-HT immunocytochemistry Rats (5 in each experimental group and 3 control) were anesthetized with sodium pentobarbital (65 mg/kg) and placed in a stereotaxic frame (Kopf). Injections of 2% WGA-HRP (Sigma) were made in the dorsal area dentata using the following stereotaxic coordinates: AP = -3.3, ML = -1.5, DV = -3.7; bregma zero; incisor bar -3.3. A total tracer volume of 50 nl was delivered over 5 min using a glass micropipette (tip diameter 30 ~m) attached to a 1-pl Hamilton syringe. After 48 h, rats were overdosed with sodium pentobarbital and perfused transcardially with 0.1 M phosphatebuffered saline followed by 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Sections through the hippocampal formation and brainstem were reacted for HRP histochemistry by the TMB method of Mesulam Ls and the reaction product was stabilized by the DAB-cobalt technique ~7. Brainstem sections were then incubated in 5-HT antiserum (1:2000, Eugene Tech) and processed by the avidin-biotin-peroxidase method 9, (Vector) using 3-amino-9-ethylcarbazole as the chromagen to produce a red reaction product. Raphe neurons containing WGA-HRP alone or in combination with 5-HT immunoreactivity were counted in every other section through the brainstem. The distribution of labeled cells was plotted on standardized diagrams at three levels through the dorsal (DRN) and median (MRN) raphe nuclei.
Anterograde tracing with PHA-L The status of the MRN projection to the hippocampal formation was studied by anterograde transport of PHA-L 7 in 13 rats (5 in each experimental group and 3 controls). Deposition of PHA-L in the MRN was accomplished by iontophoresis (5 mA, 7 s on -7 s off for 20 min) using the following coordinates: AP = 12.3, ML = 0.0, DV = -9.5; bregma zero; incisor bar -3.3; micromanipulator tilted 30° posterior. After 5 days, rats were perfused as described above and sections through the hippocampal formation and brainstem were processed for PHA-L immunocytochemistry. PHA-L antiserum (Vector) was diluted 1:2000 and DAB was the chromagen for the avidin-biotin-peroxidase technique.
RESULTS
The mean hippocampal 5-HT uptake value obtained from control synaptosomal preparations was 12,199.25 + 2464.23 CPM/mg protein. 5-HT uptake was reduced to 40% of control (4922.81 _+ 1178.96 CPM/mg protein) in rats studied 3 days after PCA treatment. Six weeks after PCA, 5-HT uptakes were 44% of control (5332.5 + 779.72 CPM/mg protein). Uptake values at both 3 days and 6 weeks were significantly (P < 0.05, Mann-Whitney U-test) less than control but were not statistically different from each other. The W G A - H R P injection sites included the area dentata and parts of the overlying CA1 region (Figs. 1 and 2) and extended from the septal pole to the hippocampal flexure (Fig. 2). Neurons in both the DRN and MRN were retrogradely labeled from the hippocampal WGAH R P injections. Retrogradely labeled 5-HT neurons were distinguished by black, granular reaction product from the transported W G A - H R P and red reaction product from the carbazole reaction from the 5-HT immuncytochemical processing (Fig. 1). With few exceptions, DRN neurons labeled from the hippocampal formation of control cases were also 5-HT immunopositive (Fig. 2). In the MRN, a significant number (26%) of neurons projecting to the hippocampal formation were not 5-HT-immunoreactive (Fig. 2). After PCA treatment, no retrograde labeling was seen in the DRN and labeling of MRN 5-HT neurons was reduced. Three days after PCA the total number of 5-HT neurons having projections to, the hippocampal formation was 18% of control (Fig. 3). The ratio of 5-HT to non-5-HT MRN neurons projecting to the hippocampal formation was not significantly different from control cases. Six weeks after PCA, DRN labeling was still absent but the number of labeled 5-HT MRN cells had risen to 69% of control (Fig. 3). Non5-HT projection neurons accounted for 35% of the total number of MRN neurons labeled with W G A - H R P in these cases. Iontophoresis of PHA-L resulted in small injection sites in the MRN (Fig. 4) that contained extracellular reaction product as well as labeled neurons. The size of the effective injection site was determined by the number and distribution of labeled neurons in each case. Control and experimental cases were matched for anal-
Fig. 1. Brightfield photomicrographs illustrating a WGA-HRP injection site (A) and retrogradely labeled, 5-HT-immunopositive raphe neurons (B). WGA-HRP injections of the dorsal hippocampal formation included the area dentata and parts of the overlying subiculum and CA1 (A). There was no spread of reaction product into neocortex and anterograde labeling of mossy fibers was present (A, arrows). Retrogradely labeled, 5-HT-immunopositive neurons of the dorsal and median raphe nuclei were identified by the presence of black granular reaction product (B, small arrows) within neurons containing the red reaction product generated by the use of carbazole in the 5-HT immunocytochemical procedure. Bars: A = 200 ~m: B = 20/~m.
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Fig. 2. In this diagram, A, B and C illustrate the regions of the dorsal hippocampal formation included in a typical WAG-HRP injection. D, E and F show the distribution of raphe neurons retrogradely labeled from the injection depicted in A-C. In this control case, the distribution of labeled neurons and the percentage of 5-HT to non-5-HT cells is consistent with previous reports of retrograde raphe labeling after hippocampal injections ]°. Each symbol represents one labeled neuron; dots indicate cells containing WGA-HRP and 5-HT immunoreactivity, circles indicate cells containing WGA-HRP alone, bc, brachium conjunctivum; cc, corpus callosum; CTX, cerebral cortex; DRN, dorsal raphe nucleus; fx, fimbria-fornix; HF hippocampal formation; IV, trochlear nucleus; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MRN, median raphe nucleus; PAG, periaquaductal grey; RT, retieulotegmental nucleus; VT, ventral tegmental nucleus; xbc, decussation of the brachium conjunctivum. Bars = 1 mm
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Fig. 3. This diagram illustrates the distribution of retrogradely labeled neurons in the dorsal and median raphe nuclei in rats receiving hippocampal imeetions of WGA-HRP 3 days (A-C) and 6 weeks (D-F) after systemic administration of PCA. DRN projections to the hippocampal formation are eliminated by PCA treatment and no recovery is seen by 6 weeks. Fewer MRN neurons are labeled from the hippocampal formation after PCA, but an increase in the numbers of retrogradely labeled MRN neurons is observed between 3 days post-PCA and 6 weeks. Symbols, abbreviations and calibration bars in this figure are identical to those defined in Fig. 2
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Fig. 4. Brightfieid photomicrographs showing the axonal labeling in the area dentata of the hippocampal formation that results from PHA-L injections in the median raphe nucleus of control rats. A: PHA-L injection site (arrow) in the ventrolateral part of the median raphe nucleus. B: this panel illustrates the distribution of median raphe axons in the area dentata. Axons are seen in the molecular layer (small arrows) as well as in the hilar re#on (large arrows). Note the presence of prominent boutons that identify these axons as arising from the median raphe nucleus (11). C and D: higher power views of PHA-L labeled axons (arrows) in the molecular layer (C) and dentate granule cell layer and hilar region (D). DGC, dentate granule cell layer; DRN, dorsal raphe nucleus; H, hflar region; Mol, molecular layer; MRN, median raphe nucleus. Bars: A = 200/lm; B = 100 #m; C and D = 50/tm.
ysis on the basis of similar numbers of PHA-L-containing neurons in the MRN. P H A - L injections of the MRN in control animals resulted in the labeling of numerous axons throughout the hippocampal formation. Within the area dentata, raphe axons were seen in the molecular layer and granule cell layer and were particularly prominent in the hilar region (Fig. 4). Regardless of their location, MRN axons exhibited a common morphology (Fig. 4) that featured prominent boutons 11. Following PCA treatment, the number of axons labeled from MRN injections was diminished and, P H A - L labeled axons appeared to have fewer boutons, (Fig. 5).
MRN projections to the molecular layer and granule cell layer appeared to be more severely affected by PCA than was the hilar region (Fig. 5). No difference in the density or distribution of P H A - L labeled MRN axons was apparent between the 3-day and 6-week PCA groups. DISCUSSION
The present study has confirmed and extended previous observations of the effects of PCA administration on the 5-HT innervation of the hippocampal formation. The
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Fig. 5. Brightfield photomicrographs illustrating the axon labeling resulting from a P H A - L injection in the median raphe nucleus made 6 weeks after P C A treatment. A: P H A - L injection site (arrow) in the ventrolateral M R N . B and D: fewer PHA-L-labeled axons are seen in the dorsal area dentata 6 weeks after P C A than are present in control rats (arrows). Note that the majority of labeled axons are located in the hilar region adjacent to the granule cell layer. C: this photomicrograph demonstrates P H A - L labeled axons with prominent boutons related to the dentate granule cell layer (arrows). Abbreviations are identical to those defined in Fig. 4. Bars: A = 100 # m ; B and D = 50 #m;C = 25#m.
approximately 40% decrease in 5-HT uptake in crude synaptosomal preparations observed 3 days after PCA is comparable to values reported after PCA administration paradigms similar to our own 2°. We have shown that this reduction in 5-HT uptake persists for at least 6 weeks. This observation suggests that 5-HT axons are not proliferating during this time in contrast to the increase in 5-HT uptake reported 6 weeks after partial MRN lesions using 5,7 D H T 1'4'5'8'24'25. Anterograde labeling of MRN axons with PHA-L supports the interpretation of the 5-HT uptake data. Fewer hippocampal axons were labeled from MRN injections in PCA-treated rats and no difference was noted
between the 3-day and 6-week experimental groups. The reduction in MRN axons projecting to the hippocampal formation indicates that the change in 5-HT uptake is the result, at least in part, of the loss of raphe axons and not due to an effect of PCA on the 5-HT transporter alone. These data also show that MRN axons are susceptible to damage by PCA. The observations obtained by combining retrograde tracing with 5-HT immunoeytochemistry demonstrate the loss of DRN projections to the hippocampal formation after PCA administration. The number of MRN neurons labeled is also reduced but the majority of these cells contained 5-HT. Thus one can infer that the majority of
259 PHA-L-labeled axons projecting to the hippcampal formation of PCA-treated are from 5-HT MRN neurons. A severe reduction in MRN neurons labeled from the hippocampal formation 3 days after PCA is followed by an increase in labeling to almost 70% of control by 6 weeks. This change might indicate the regeneration or sprouting of 5-HT axons in the hippocampal formation 23'24 were it not for the results of 5-HT uptake and anterograde tracing experiments. Because 5-HT-specific and 5-HT-non-specific axon marking techniques fail to show any change in the axon plexus of PCA-treated rats, the change in retrograde labeling must reflect an acute effect of PCA on the uptake and transport of WGAHRE The retrograde labeling data also support the contention that some 5-HT MRN neurons are affected by PCA. Although no change was noted in the ratio of 5-HT to non-5-HT MRN neurons labeled from the hippocampal formation 3 days after PCA, an increase of about 10% was seen in the population of labeled non-5-HT neurons from rats studied 6 weeks after PCA. PCA is known to inhibit tryptophan hydroxylase in the projection areas of the DRN and MRN 13'14'1s'19. The increase in the number of 5-HT-immunonegative MRN cells with projections to the hippocampal formation suggests that tryptophan hydroxylase activity may be suppressed in some raphe neurons after PCA administration. Preliminary counts of 5-HT-immunopositive neurons in the DRN and MRN of PCA-treated rats indicate that the number
of DRN 5-HT neurons is decreased by about 20% while the MRN contains about 10% fewer 5-HT neurons 6 weeks after PCA (Haring, unpublished observations). No evidence of raphe neuron degeneration has yet been obtained, therefore these changes are thought to indicate inhibition of 5-HT synthesis among certain 5-HT neurons of the DRN and MRN. In summary, compensatory proliferation of hippocampal 5-HT axons does not occur within 6 weeks of 5-HT denervation by systemic administration of PCA. This is in contrast to the reported sprouting and regeneration of 5-HT axons that follows 6 weeks after partial denervations of CNS structures by 5 , 7 - D H T 1'5'6'8'24'25. The basis for this difference is unclear. That complex effects of PCA upon cell function and metabolism may limit the plastic response of raphe neurons is suggested by the observation that 5-HT axon proliferation may occur months after PCA treatment 16. The time at which this proliferation of 5-HT axons has been observed coincides with times at which the restoration of tryptophan hydroxylase activity has been reported 18'19. Thus it is possible that the recovery of tryptophan hydroxylase activity signals a wider recovery of 5-HT neuronal function from the effects of PCA.
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Acknowledgements. The technical assistance of L. Beaudet is gratefully acknowledged. This study was supported by USPHS Grants NS25752 and DE07734.
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