Brain Research, 556 (1991) 329-332 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124780V

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BRES 24780

Collateral sprouting of serotonergic fibers in the cingulate cortex and the septum following cortical-hippocampal lesions Shuichi Ueda, Yutaka Sano and Mitsuhiro Kawata Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 30 April 1991)

Key words: Serotonin; Immunohistochemistry; Collateral sprouting; Cingulate cortex; Septum

The parietal cortex and dorsal hippocampus of adult rats were unilaterally ablated. One and 3 months after this operation, changes in serotonergic fiber distribution in the forebrain were studied immunohistochemically.At i month, increased numbers of serotonergic fibers were seen in the cingulate cortex and the medial and lateral septal nuclei of the lesioned side. This increase continued to 3 months in the ipsilateral cingulate cortex. In the present study, two different processes of serotonergic fiber collateral sprouting were noted. Plastic changes in serotonergic fibers in the ipsflateral septal nuclei and cingulate cortex may be attributable to the collateral sprouting underlying reactive synaptogenesis and the pruning effect, respectively.

Serotonergic fibers in the adult mammalian central nervous system have a remarkable regenerative capacity after chemical (selective) and traumatic (non-selective) axonal damage 2,3. After destruction of descending serotonergic fibers with 5,6-dihydroxytryptamine (5,6-DHT), serotonergic fibers reinnervate the brainstem and spinal cord. Using the formaldehyde-induced fluoresence (FIF) histochemistry, Wiklund and Bj6rklund 18 showed the regeneration of descending serotonergic fibers to the brainstem to be attributable to the collateral sprouting, presumably representing a pruning effect 15. However, little information is available concerning regeneration of the ascending serotonergic neuron system after selective and non-selective axonal damage. In the present study, we immunohistochemically studied the distribution of serotonergic fibers in the forebrain of the adult rat following partial ablation of the cerebral cortex and dorsal hippocampus, and demonstrated plastic changes in serotonergic fibers. Thirteen male Wistar rats weighing 180-200 g were used, 10 for experiments and 3 for controls. They were placed in a stereotaxic instrument under sodium pentobarbital anesthesia. After removal of the parietal bone, brain regions including the parietal and cingulate cortices and dorsal hippocampus (Fig. 1) were aspirated using a Pasteur capillary pipette connected to a suction pump. The animals were killed after 1 (n = 3) and 3 (n = 7) months. Under sodium pentobarbital anesthesia, all

animals were perfused with 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). Following postfixation in the same mixture, the brain was washed in 0.1 M phosphate buffer containing 20% sucrose for 48 h at 4 °C. The brain was frozen with CO 2 gas and 20 ~ m frontal and sagittal sections were cut using a cryostat. These sections were stored in 0.1 M phosphate-buffered saline containing 0.3% Triton X-100 at 4 °C, then stained with Cresyl violet or via the modified peroxidase-antiperoxidase method with serotonin antiserum (diluted 1:10,000) 17. Preparation of the antibody and specificity of the immunocytochemical staining have been reported elsewhere 17. In the septal nuclei of control rats, serotonergic fibers were most dense in the lateral part of the lateral septal nucleus, and the remainder of the nucleus containing only a moderate number of these fibers. The medial septal nucleus was relatively rich in serotonergic fibers. Fibers in the cingulate cortex of controls were arranged a laminar pattern. Dense plexuses of serotonergic fibers were observed in layers I, III and VI, while layers II, IV and V contained relatively few fibers. The cingulate bundle of serotonergic fibers I was observed in the supracallosal part of the cingulate cortex. These results in control rats agree with previous immunohistochemical studiesS-10,16. One month after operation, increased densities of serotonergic fibers were seen in the ipsilateral septal

Correspondence: S. Ueda, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan.

330 nuclei (medial and lateral), particularly, in the periventricular part of the lateral septal nucleus. These cases also showed a significant increase in fibers in the medial septal nucleus as compared with the contralateral side of the nucleus (Fig. 2). A slight increase in the remaining ipsilateral cingulate cortex was seen. Remarkably, many fibers showing an anomalous pattern of projection were seen in the cingulate cortex of this side (Fig. 3). These fibers originated from the cingulate bundle of serotonergic fibers, and extended towards the surface layer of the cortex. They were observed within the remaining cingulate cortex less than 1 mm rostral to the lesion, but were never seen in the proximal stump of lesioned serotonergic axons. On the other hand, these spiral fibers were not evident in the control or contralateral cingulate cortices. The dense accumulation of serotonergic fibers in the ipsilateral lateral septal nucleus was still seen 3 months after operation; however, density was similar to that at one month (Fig. 4). In comparison to the contralateral side all layers of the remaining ipsilateral cingulate cortex were densely innervated (Fig. 5a,b). The density of fibers in the ipsilateral cingulate bundle was much higher than in the contralateral side (Fig. 5a,b). These findings were evident in the cingulate cortex as far as 2 mm rostral from the lesion area. Based on electron microscopic observations, Raisman 13, and Raisman and Field 14 first reported the collateral sprouting of medial forebrain bundle (MFB) axons in response to fimbria-hippocampal lesions. These sprouting axons formed new synapses on the neurons of the medial and lateral septal nuclei. Using the FIF histochemical technique, Moore et al. 11 demonstrated that numbers of catecholaminergic fibers within the septal area ascending from the MFB increased following

Fig. 2. Frontal section of the medial septal nucleus 1 month after operation. The ipsilateral medial septal nucleus (to the fight of the interrupted black line designating the midline) shows an increase in serotonergic fibers in comparison to the contralateral side. x 150.

aspiration of the dorsal hippocampus with the overlying cortex, and suggested that some of the sprouting fibers from the MFB described by Raisman and Field 14 were catecholaminergic fibers. This collateral sprouting response had a fast onset (within 4-5 days) but a short duration (waned within a month). This type of collateral sprouting has been referred to as reactive synaptogenesis 4. In the present study, an increased number of serotonergic fibers was found in the medial and lateral septal nuclei following aspiration of dorsal hippocampus with the overlying cortex, but the process waned within a month of operation. Areas showing increased seroton-

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Fig. 1. Schematic representation of the position and extent of the lesion (black) in a dorsal view (a) and in the coronal plane (b).

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Fig. 3. Sagittal section of the cingulate cortex 1 month after operation. Spirally distorted serotonergic fibers (arrowheads) emerge from the cingulate bundle (CB) of serotonergic fibers. x210.

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Fig. 4. Frontal section of the lateral septal nucleus 3 months after operation. A greater density of serotonergic fibers is seen in the ipsilateral side (b) than in the contralateral side (a). x280. ergic fibers were agreed with the results of Moore et al. 11, It is well known that ascending serotonergic fibers project to the septal area via the MFB 1'12. The present result suggests that collateral sprouting of serotonergic fibers in the septal area occurs in response to ablation of the dorsal hippocampus with the overlying cortex as reactive synaptogenesis. Using the FIF histochemical method, Wiklund and Bj6rklund TM reported the degeneration and regeneration of the bulbospinal serotonergic system after 5,6-DHTinduced chemical axotomy. Several months after this treatment, hyperinnervation of serotonergic fibers was observed in the inferior olivary complex, while in the spinal cord there was a hypoinnervation of these fibers. They suggested that the drive of axonal growth was derived from a program inherent in the serotonergic neurons and that, according to this program, the regen-

eration of serotonergic fibers was established with a set number of terminal arborizations, although organization of axonal tree was altered. These effects are considered to be consistent with the concept of the pruning effect15. Frankfult and Azmitia 5 immunohistochemieally and biochemically studied the regeneration of serotonergic fibers in the hypothalamus following injection of 5,7-DHT into the dorsolateral hypothalamus, and reported hyperinnervation of serotonergic fibers in the hypothalamus as a result of the pruning effect. The present study also demonstrated hyperinnervation of serotonergic fibers in the ipsilateral cingulate cortex following unilateral ablation of dorsal hippocampus with the overlying cortex (parietal and cingulate cortices). One month after operation, distorted serotonergic fibers were seen to emerge from the cingulate bundle of serotonergic fibers, indicating collateral sprouting fibers. The number of these fibers

Fig. 5. Frontal section of the cingulate cortex 3 months after operation. All layers of the ipsilateral cingulate cortex (b), especially layers I, III, VI, and the cingulate bundle (,), contain a large number of serotonergic fibers in comparison to the contralateral side (a). x120.

332 gradually increased, expanding into the remaining cingulate cortex. Three months after operation, the ipsilateral cingulate cortex showed serotonergic hyperinnervation, possibly representing a compensation for ablated terminals in the distal cingulate cortex. Hyperinnervation observed in the cingulate cortex may therefore be attributed to the pruning effect. Serotonergic, noradrenergic and cholinergic fibers show homotypic (same transmitter) compensatory collat-

sprouting in the hippocampus following ablation of the dorsal hippocampus with the overlying cortex6*7. These previous studies and the present results indicate the different types of collateral serotonergic sprouting in different areas of the forebrain following non-specific traumatic lesions.

1 Azmitia, E.C. and Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and medial raphe nuclei in the rat, /. Comp. Neural., 179 (1978) 641-668. 2 Bjarkhmd, A. and Stenevi, U., Regeneration of monoaminergic and choline&z neurons in the mammalian central nervous system, Physiof. Rev., 59 (1979) 62-100. 3 Bj(irkhmd, A., Wikhmd, L. and Descarries, L., Regeneration and plasticity of central serotoninergic neurons: a review, J. Physiol., 77 (1981) 247-255. 4 Cotman, C.W. and Lynch, G.S., Reactive synaptogenesis in the adult nervous system: the effect of partial deafferentation on new synapse formation. In S. Barondes (Ed.), Neuronal Recognition, Plenum, New York, 1976, pp. 69-108. 5 Frankfurt, M. and Azmitia, E., Regeneration of serotonergic fibers in the rat hypothalamus following unilateral 5,7-dihydroxytryptamine injection, Brain Research, 298 (1984) 273-282. 6 Gage, F.H., Bjarklund, A. and Stenevi, U., Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Research, 268 (1983) 27-37. 7 Gage, F.H., Bjbrklund, A., Stenevi, U. and Dunnett, S.B., Functional correlates of compensatory collateral sprouting by [email protected] and cholinergic afferents in the hippocampal formation, Brain Research, 268 (1983) 39-47. 8 Gall, C. and Moore, R.Y., Distribution of enkephalin, substance-P, tyrosine hydroxylase and 5hydroxytryptamine immunoreactivity in the septal region of the rat, J. Comp. Neural., 225 (1984) 212-227. 9 K&ler, C., Chan-Palay, V. and Steinbusch, H., The distribution and origin of serotonin-containing fibers in the septal area: a

combined immunohistochemical and fluorescent retrograde tracing study in the rat, J. Camp. Neural., 209 (1982) 91-111. Lidov, H.G.W., Grzanna, R. and Molliver, M.E., The serotonin innervation of the cerebral cortex in the rat - an immunohistochemical analysis, Neuroscience, 5 (1980) 207-227. Moore, R.Y., Bjerklund, A. and Stenevi, U., Plastic changes in the adrenergic innervation of the rat septal area in response to denervation, Bruin Research, 33 (1971) 13-35. Moore, R.Y., Halaris, A.E. and Jones, B.E., Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neural., 180 (1978) 417-438. Raisman, G., Neuronal plasticity in the septal nuclei of the adult rat, Bruin Reseurch, 14 (1%9) 25-48. Raisman, G. and Field, P.M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Bruin Research, 50 (1973) 241-264. Schneider, G.E., Early lesions of the superior colliculus: factors effecting the formation of abnormal retina1 projections, Brain Behav. Evol., 8 (1973) 73-W. Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat. Cell bodies and terminals, Neuroscience, 6 (1981) 557-618. Takeuchi, Y., Kimura, H. and Sano, Y., Immunohistochemical demonstration of serotonin neurons in the brainstem of the rat and cat, Cell Tissue Res., 224 (1982) 247-267. Wiklund, L. and Bjiirklund, A., Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations, Bruin Research, 191 (1980) 129-160.

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This work was supported by a grant from the ministry of Education, Science and Culture, Japan.

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Collateral sprouting of serotonergic fibers in the cingulate cortex and the septum following cortical-hippocampal lesions.

The parietal cortex and dorsal hippocampus of adult rats were unilaterally ablated. One and 3 months after this operation, changes in serotonergic fib...
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