Regeneration of central serotonin neurons after axonal degeneration induced by 5, 6-dihydroxytrptamine

214

Brain Research, 50 (1973) 214- 220 :~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Regeneration of central serotonin neurons after axonal degeneration induced by 5,6-dihydroxytryptamine

ANDERS BJORKLUND, ANDERS NOBIN AND ULF STENEVI

Department of Histology, University of Lund, S-223 62 Lund (Sweden) (Accepted October 19th, 1972)

Catecholamine and indolamine neurons in the adult rat brain have been found to possess remarkable growth capacity after axonal damage. By monoamine histochemistry this is observed after mechanical or electrolytic lesions as vigorous sprouting from the severed axons 7,13. The growth of the developing sprouts is partly seemingly random and undirected - - into the necrosis of the lesion and into the brain tissue surrounding the lesioned axons - - and partly directed, e.g. into and along the walls of intracranial blood vessels and within myelinated fiber bundles, such as cranial nerves or spinal nerve roots. A major part of these regenerated fibers persist for at least one year, and if true synaptic connections were formed in these abnormal locations they thus appear to be permanent. Following a substantial mechanical or electrolytic lesion, the growing monoamine axon sprouts do not appear to reach across the necrosis into the original axonal pathway and we have not observed a reestablishment of the normal connections of the lesioned axons, although this has been reported to occur for noradrenaline neurons after 6-hydroxydopamine-induced lesions in the spinal cord 16. In experiments with transplants of peripheral tissue to the brain 9 we have observed that regenerating central catecholamine fibers can grow to considerable distances and reestablish a persistent fiber pattern in the grafted tissue that at least to some extent has a normal morphological appearance. From these observations it seems that when the conditions are favorable the monoamine neurons in the CNS are capable of extensive, directed regeneration. Recently, it was found that intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT), a strongly reducing congener of 5-hydroxytryptamine (5-HT, serotonin), causes axonal degeneration of central indolamine neurons, as evidenced by a long-lasting reduction in brain and spinal cord serotonin levels~,4, accompanied by ultrastructural 1 and fluorescence microscopical 5,~5 signs of axonal degeneration, and by the disappearance of 5-HT-containing terminals and a loss of 5-HT uptake sites s. In a previous chemical study 3 there were signs of a recovery of the 5-HT levels during the third and fourth weeks after the 5,6-DHT injection, suggesting that regenerative phenomena might occur in the chemically lesioned 5-HT neurons. If regeneration actually takes place in the 5,6-DHT-treated animals they offer a very interesting

SHORTCOMMUNICATIONS

215

growth situation: regenerating nerve fibers in 5,6-DHT-treated rats will grow in brain tissue that is grossly morphologically intact as opposed to the growth stiuation after electrolytic or mechanical lesions with their inevitable necroses and scars. In the present paper we report findings in support of such a regeneration of chemically lesioned central 5-HT neurons. Adult female Sprague-Dawley rats (180-200 g body wt) received one injection of 75 pg 5,6-DHT (creatinine sulfate-H20-complexis, calculated as the free base). The injection volume was 20 pl and was given stereotaxically into the lateral ventricle. Groups of rats were killed after 10-17 days and 2-3 months. In all experiments the 5,6-DHT-injected animals were compared with controls of the same age. Some of the animals of each experimental group and untreated controls were used for fluorescence histochemistry of intraneuronal monoamines according to the Falck-Hillarp method (for technical details, see ref. 6). These rats were either treated with the MAO-inhibitor nialamide (Pfizer, 300 mg/kg i.p.) 3-5 h before killing, to facilitate the demonstration of 5-HT-containing neurons, or with the catecholamine depleting drug a-methyl-mtyrosine (AB H~issle, Gothenburg, Sweden, 2 × 350 mg/kg, i.p., 18-20 h and 3-5 h before sacrifice) plus nialamide (as above), to obtain a fluorescence picture largely free of catecholamine fluorescence. Specimens from controls and 5,6-DHT treated animals were processed in strict parallel. The fluorescence microscopy was restricted to diencephalon, medulla oblongata (including the most cranial portion of the spinal cord), and thoracic and lumbar segments of the spinal cord. Uptake of [3H]5-HT was measured in vitro on thin circular slices (3 mm in diameter and about 0.5-1.0 mm thick) prepared from cortex, hypothalamus, medulla oblongata, and thoracic segments of the spinal cord according to Hamberger 11 and Sachs and Jonsson 17. The slices were preincubated for 10 min in a Krebs-Ringer bicarbonate buffer (pH 7.4, saturated with 95 ~o 02 and 5 ~o CO2 ,and containing 1.8 g/l glucose) in a metabolic shaker at +37 °C. The incubation volume was 2 ml for 4-6 slices. After the preincubation, [3H]5-HT ([3H]5-HT creatinine sulfate, 6.8-11.7 Ci/mmole, gen. labelled, Radiochemical Centre, Amersham, England) was added to a final concentration of 0.5 × 10-7 M, and the incubation was continued for another 10 min. The incubation was terminated by the addition of 5 ml ice-cold buffer to each incubation flask and the slices were then transferred to fresh ice-cold buffer. To obtain values for [SH]5-HT uptake at 0 °C, parts of the slices from each region and each control or experimental animal were incubated as above, but the temperature was kept at 0 °C. Total tritium and unchanged [3H]5-HT were measured according to the procedure described by Bj6rklund et al. s. The recovery of 5-HT in this procedure averaged 45 ~o; the [aH]5-HT uptake values have been corrected for recovery. The 0 °C uptake was very similar in slices from control and 5,6-DHT-treated animals; it was 4-8 (unchanged [aH]5-HT) and 7-15 ~o (total tritium) of the uptake at 37 °C. Endogenous 5-HT levels in selected brain regions and in the spinal cord have been determined fluorometrically2. Ten days after 75 #g 5,6-DHT, the 5-HT concentrations were 13 ~o (4- 1.0; S.E.M. of 4 determinations) of the controls of the same age in the spinal cord, 81 ~o (4- 4.8) in the medulla oblongata plus pons preparation, and 57 ~ (4- 3.9) in the hypothalamus (for mode of dissection, see ref. 3). At 2-3 months

Fig. 1. a and b, Inferior olivary complex at the emergence of the radices oF the hypoglossal nerve (XII). a, Nialamide-treated control rat with a cluster of 5-HT-containing cell bodies at the ventral surface. Some faintly fluorescent terminals, not clearly visible in the photograph, are present in the dorsal accessory olivary nucleus (AO). b, Nialamide-treated rat, 3 months after one injection of 75 Hg 5,6-DHT. A dense plexus of yellow-fluorescent, probably 5-HT-containing fibers have now developed in the dorsal accessory olivary nucleus (AO) and in the lateral reticular nucleus (LR). c and d, The ventral horn of the spinal cord just caudal to the decussation of the pyramidal tract. The neuronal catecholamines had been depleted with a-methyl-rn-tyrosine, followed by nialamide, c, 10 days after 75 Hg 5,6-DHT. The 5-HT-containing terminals normally present in the ventral horn have almost completely disappeared, d, 3 months after 75 Pg 5,6-DHT, showing the re-appearance of a seemingly normal supply of yellow-fluorescent fibers in the ventral horn. No such re-appearance occurred in more distal portions of the cord. ~: 120.

SHORT COMMUNICATIONS

217

after injection the 5-HT level in the spinal cord was still very low, 12 ~o (4- 0.8; S.E.M. of 6 determinations) as compared to the controls of the same age. In the other regions, there was an increase in 5-HT to 108 ~o ( + 3.2) of control in medulla oblongata plus pons, and to 123 ~ ( i 14.8) in hypothalamus. These increases between 10 days and 2-3 months were statistically highly significant (P < 0.001). As reported elsewhere s there was an almost complete disappearance of histochemically detectable, yellow-fluorescent indolamine-containing terminals by 10-17 days after 5,6-DHT treatment in the suprachiasmatic nuclei, the subcommissural organ and the ventral horn of the spinal cord (Fig. lc), i.e. in those areas of the diencephalon and spinal cord where indolamine fibers can be reliably visualized in nialamide-treated animals. In these areas only single scattered yellow-fluorescent fibers remained. In contrast, other regions such as areas of the inferior olivary complex seemed to have a largely intact indolamine innervation pattern. In the fluorescence microscope, signs of axonal sprouting were observed at 10-17 days after 5,6-DHT injection, and appeared as delicate smooth or varicose strongly yellow-fluorescent fibers that developed at the swollen, distorted and strongly fluorescent stumps of the damaged indolamine axons in the medial forebrain bundle (MFB) of the caudal hypothalamus and rostral mesencephalon, and in the bulbospinal pathway in the caudal medulla oblongata just lateral to the pyramidal tract, and at the junction between medulla oblongata and spinal cord. The swollen fibers most probably represent the dilated, amine-filled ends of the lesioned axons (cf refs. 7 and 13), and the newly-appearing, delicate fibers could frequently be traced back to these structures. Three months after 5,6-DHT injection abundant systems of newly-appearing, yellow-fluorescent fibers were detected in diencephalon and medulla oblongata. These fibers had very fine varicosities, and their morphology and fluorescence characteristics were very similar to those of normal indolamine-containing terminals in the brain. In the diencephalon the newly-appearing fibers formed dense, irregular patterns within and around the MFB, i.e. ventrally and laterally in the mammillary region, and throughout the lateral hypothalamic area as far rostral as the retrochiasmatic region. The suprachiasmatic nuclei were still devoid of yellow-fluorescent fibers, but in the subcommissural organ yellow-fluorescent fibers had now re-appeared. Although their density was less than normal, these fibers had a normal appearance and distribution within the subcommissural organ. In the medulla oblongata of the 3-month animals the newly-appearing fibers occurred in abundance in areas where few or no indolamine-containing fibers were detected in the control animals as well as in areas normally innervated by such fibers (cf ref 10). The fibers had a remarkably high fluorescence intensity and penetrated densely the entire inferior olivary complex (Fig. la and b) and large areas of the reticular formation situated lateral to the inferior olivary complex. Bundles of smooth yellow-fluorescent fibers, with the appearance of non-terminal axon bundles, could be followed for long distances laterally and dorsally along the inner surface of the spinal tract of the trigeminal nerve. In the most cranial portion of the spinal cord the yellow-fluorescent newly-appearing fibers could be traced among the ventrally and medially situated myelinated fiber tracts and within the decussation of the

2l8

SHORT COMMUNICATIONS [ ] TOTAL UPTAKE •

% 150

HYPOTHALAMUS

5-HT UPTAKE

MED. OBL

CORTEX

SPINAL coRD

100.

50-

II CONTR. ~-OHT ~:DHI I0-17 3MONTHS DAY$

cO~,#R %S-OHT 6-t~rr 10-17 DAYS

3MONTHS

coNTR S~-0HT5,B'~ 10:17

DAYS

3 MONTH5

CONTRS,e:~T 10-17

~-DHT 3 MONTHS

DAYS

Fig. 2. Uptake of total tritium and unchanged pH]5-HT by brain slices and slices from thoracic segments of the spinal cord in vitro. For each region the values are expressed in percent of the total tritium uptake of the control slices, and are given as means 4- S.E.M. of 5-10determinations. The values represent active uptake, calculated as the uptake measured at ÷ 37 °C minus the uptake measured at 0 °C. Differences from control values, *** = P < 0.001; ** = 0.01 > P > 0.001; * = 0.05 > P > 0.01. Student's t-test. pyramidal tract. Interestingly, a dense supply of seemingly normal yellow-fluorescent varicose fibers had now re-appeared in the ventral horn of the grey matter in the most rostral part of the spinal cord (Fig. lc and d). They had a distribution within the grey matter that was similar to the normal innervation. At more caudal levels, in thoracic and lumbar segments, the ventral horn was still devoid of yellow-fluorescent fibers, except for a low number of strongly fluorescent fibers with an irregular and abnormal looking morphology. That at least part of the newly-appearing fibers in the long-term, 5,6-DHTtreated animals were indeed newly-formed is supported by the strong increase in 5-HT uptake sites demonstrated between 10-17 days and 3 months after 5,6-DHT injection (Fig. 2). At 10-17 days, there was a reduction in [3H]5-HT uptake by slices from all regions analyzed, and this reduction was highly significant (P < 0.001) in hypothalamus, medulla oblongata and spinal cord (solid bars in Fig. 2). A similar reduction was also seen in the accumulation of total tritium (corresponding to 5-HT plus metabolites; open bars in Fig. 2) in cortex, hypothalamus and spinal cord, while in the medulla oblongata the reduction in total tritium uptake was clearly less than the reduction in the uptake of unchanged [aH]5-HT. That this [aH]5-HT uptake is almost exclusively neuronal and largely confined to indolamine (probably 5-HT) neurons is supported by parallel experiments 8 with the 5-HT uptake inhibitor chlorimipramine and with slices from 6-hydroxydopamine-treated rats. In these experiments it was shown that the

SHORT COMMUNICATIONS

219

5,6-DHT-induced reduction in the [aH]5-HT uptake was due to a reduction in the chlorimipramine-sensitive part of the uptake, i.e. the part of the uptake that most probably is neuronal (cfi ref. 14). Further, 6-hydroxydopamine did not cause any significant reduction in the [3H]5-HT uptake, indicating that uptake into catecholamine neurons did not interfere at the low [aH]5-HT concentrations in the incubation medium (0.5 × 10-7 M) used in the present study (cfi refs. 12 and 19). Ten to 17 days after the 5,6-DHT injection the reduction in [3H]5-HT uptake sites was 83 ~ in the spinal cord slices, 65 ~o in slices from medulla oblongata, 53 ~ in slices from hypothalamus, and 35 ~o in slices from cerebral cortex. By 3 months, there was a marked increase in the [3H]5-HT uptake in medulla oblongata and hypothalamus, i.e. in those regions where the recovery of the endogenous 5-HT levels and the reappearance of indolamine fibers were observed. This increase in [3H]5-HT uptake sites was recorded both in the total tritium uptake (differences between 10-17-day and 3-month values: P < 0.001 in medulla oblongata and 0.01 > P > 0.001 in hypothalamus) and in the uptake of unchanged [3H]5-HT (0.01 > P > 0.001 in medulla oblongata and 0.05 > P > 0.01 in hypothalamus). In thoracic segments of the spinal cord and in cortex the [3H]5-HT uptake was similar at 10-17 days and 3 months after injection. In spinal cord this conforms well to the fluorescence microscopical and the chemical observations. The present results provide fluorescence microscopical and biochemical evidence for a significant regenerative growth of 5-HT neurons in rat hypothalamus and medulla oblongata after 5,6-DHT-induced axonal destruction. The new fibers occurred most abundantly in regions in the vicinity of the proximal parts of the damaged indolamine axon bundles within the MFB and the bulbo-spinal tracts. They appeared both as an excess fiber supply of areas where a normal indolamine innervation can be established histochemically (such as in the caudal and lateral parts of the dorsal accessory olivary nucleus; Fig. la and b), as an apparently abnormal supply to areas where few or no indolamine-containing fibers are seen in control animals (e.g. in other areas of the inferior olivary complex and in the reticular formation lateral to the inferior olivary complex), and as a re-appearance of a normal or almost normal pattern of indolaminecontaining fibers in areas that most probably had been denervated by the 5,6-DHT treatment (the subcommissural organ and the ventral horn of the most cranial portion of the spinal cord; Fig. lc and d). To us, the most likely interpretation of these findings is that, when the damaged 5-HT neurons are allowed to regenerate in a brain tissue with an intact general morphology, part of the growing fibers are directed to areas normally innervated by 5-HT fibers, making possible the reestablishment of connections disrupted by the lesion. Other fibers appear to grow into abnormal positions in a manner that resembles the growth observed after mechanical lesions of monoamine axon bundlesT,13. At this point we do not know the more exact extent and the final result of these regeneration processes within the CNS. The study was supported by grants from the Magnus Bergvall Foundation, the Ake Wiberg Foundation, and the Swedish Medical Research Council (Grant No. B73-04X-3874-01).

220

SHORT COMMUNICATIONS

W e t h a n k D r . H. G. S c h l o s s b e r g e r f o r synthesis and gilt o f 5 , 6 - d i h y d r o x y t r y p t a m i n e , a n d K e r s t i n F o g e l s t r 6 m for skillful t e c h n i c a l assistance.

1 BAUMGARTEN,H. G., BJORKLUND,A., HOLSTEIN, A. F., AND NOBIN, A., Chemical degeneration

of indolamine axons in rat brain by 5,6-dihydroxytryptamine, an ultrastructural study, Z. Zellforsch., 129 (1972) 256-271. 2 BAUMGARTEN,n . G., LACHENMAYER,L., BJORKLUND,A., NOBIN, A., AND ROSENGREN,E., Longterm recovery of serotonin concentrations in the rat CNS following 5,6-dihydroxytryptamine, To be published. 3 BAUMGARTEN, H. G., BJORKLUND, A., LACHENMAYER,L., NOBIN, A., AND STENEVI, U., Longlasting selective depletion of brain serotonin by 5,6-dihydroxytryptamine, Acta physiol, scand., Suppl. 373 (1971). 4 BAUMGARTEN,H. G., EVETTS,K. D., HOLMAN, R. B., IVERSEN,L. L., VOGT, M., AND WILSON,G., Effects of 5,6-dihydroxytryptamine on monoaminergic neurones in the central nervous system of the rat, J. Neurochem., 19 (1972) 1587-1597. 5 BAUMGARTEN,H. G., LACHENMAYER,L., AND SCHLOSSaERGER,H. G., Evidence for a degeneration of indolamine-containing nerve terminals in rat brain, induced by 5,6-dihydroxytryptamine, Z. Zellforsch., 125 (1972) 553-569. 6 BJORKLUND, A., FALCK, B., AND OWMAN, CH., Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In J. E. RALL AND I. J. KOPIN (Eds.), Methods in Investigative and Diagnostic Endocrinology, Vol. 1, The Thyroid and Biogenic Amines, North Holland, Amsterdam, 1972, pp. 318-363. 7 BJORKLUND,m., KATZMAN,R., STENEVI,U., AND WEST, K. A., Development and growth of axonal sprouts from noradrenaline and 5-hydroxytryptamine neurones in the rat spinal cord, Brain Research, 31 (1971) 21-33. 8 BJ()RKLUND, A., NOBIN, m., AND STENEVI, U., Effects of 5,6-dihydroxytryptamine on nerve terminal serotonin and serotonin uptake in the rat brain, Brain Research, (1973) In press. 9 BJ6RKLUND, A., AND STENEVI, U., Growth of central catecholamine neurones into smooth muscle grafts in the rat mesencephalon, Brain Research, 31 (1971) 1-20. 10 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol, scand., 64, Suppl. 247 (1965) 37-85. 11 HAMBERGER,B., Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta physiol, scand., Suppl. 295 (1967). 12 IVERSEN, L. L., Neuronal uptake processes for amines and amino acids. In E. COSTA AND E. GIACOBINI(Eds.), Biochemistry of Simple Neuronal Models, Vol. 2, Advances in Biochemical Psyehopharmacology, Raven Press, New York, 1970, pp. 109-132. 13 KATZMAN,R., BJ6RKLUND,A., OWMAN,CH., STENEVI,U., AND WEST, K. m., Evidence for regenerative axon sprouting of central catecholamine neurons in the mesencephalon following electrolytic lesions, Brain Research, 25 (1971) 579-596. 14 LIDBRINK,P., JONSSON,G., AND FUXE, K., The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenatine and 5-hydroxytryptamine neurons, Neuropharmacol., 10 (1971) 521-536. 15 NOBIN, A., BAUMGARTEN,H. G., BJORKLUND, A., LACHENMAYER,L., AND STENEVI, U., Axonal degeneration and regeneration of the bulbospinal indolamine neuron systems after 5,6-dihydroxytryptamine treatment, Brain Research, (1973) in press. 16 NYGREN, L.-G., OLSON, L., AND SEIGER,ilk., Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections, Histochemie, 28 (1971) 1-15. 17 SACHS, CH., AND JONSSON, G., Degeneration of central and peripheral noradrenaline neurons produced by 6-hydroxy-DOPA, J. Neurochem., 19 (1972) 1561-1575. 18 SCHLOSSBERGER,H. G., AND KUCH, H., Synth6se des 5,6-dihydroxytryptamins, Chem. Ber., 93 (1960) 1318-1323. 19 SHASKAN,E. G., AND SNYDER, S. H., Kinetics of serotonin uptake into slices from different regions of rat brain, J. Pharmacol. exp. Ther., 175 (1970)404-418.