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Reinnervation after destruction of the dopaminergic system in the rat nucleus accumbens: a quantitative immunohistochemical analysis
N[UNIIZIltig[ ELSEVIER
NeuroscieneeLetters207(1996)21-24
I[II[HS
Reinnervation after destruction of the dopaminergic system in the rat nucleus accumbens: a quantitative immunohistochemical analysis Pieter
E. VOS a'*, Harry W.M.
S t e i n b u s c h b, J a n M . V a n R e e a
aDepartment of Pharmacology, Rudolf Magnus Institute for Neurosciences, Faculty of Medicine, University of Utrecht, Utrecht, The Netherlands bDepartment of Psychiatry and Neuropsychology, University of Maastricht, Maastricht, The Netherlands Received 22 December 1995; revised version received 1 February 1996; accepted 14 February 1996
Abstract
The recurrence of dopamine-immunoreactive (DAi) fibers and the effect of the adrenocorticotrophic hormone (ACTH)-(4-9) analog ORG 2766 on this process were investigated 1, 4, 12 and 24 weeks after a unilateral 6-hydroxydopamine (6-OHDA) lesion in the nucleus accumbens (NAc). DAi fibers were almost completely absent 1 week after the lesion. A gradual increase in DAi fibers throughout the NAc was observed, with subnormal values at 24 weeks. Treatment with ORG 2766 during the first week after the lesion resulted in more DAi fibers 4 weeks after the lesion as compared to placebo treatment, but not 12 and 24 weeks after the lesion. After 6-OHDA lesioning reinnervation of the NAc takes place and this process is transiently facilitated by ORG 2766.
Keywords: Regeneration; Ventral striatum; Nucleus accumbens; Dopamine; Immunoreactivity; Plasticity; ORG 2766; Adrenocorticotrophic hormone-(4--9) analog
The central nervous system of mammals is able to recover from lesion-induced disturbances in a functional way. Whether the recovery is a mere compensation (i.e. changes in turnover of neurotransmitters, receptor upregulation) or the result of a reorganization (i.e. neural circuitry changes, reinnervation) is still a matter of debate. Under certain conditions monoaminergic fibers display axonal regrowth after lesioning [2,7]. In the ventral striatum, i.e. the nucleus accumbens (NAc), no reinnervation with dopaminergic fibers was observed 9 months after a bilateral 6-hydroxydopamine (6-OHDA) lesion [5]. However, dense reinnervation of the NAc with dopaminergic fibers was found after transplantation of fetal dopaminergic neurons in this area after 6-OHDA lesioning. Novelty-induced locomotor activity is reduced if dopaminergic terminals in the NAc are lesioned bilaterally with 6-OHDA [3,10]. This reduction in locomotor activity recovered spontaneously in 3-4 weeks. Treatment * Corresponding author. Present address: Department of Neurology, University Hospital Utrecht, Hp C03.236, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Tel: +31 30 2507975; fax: +31 30 2542100.
with the adrenocorticotrophic hormone (ACTH)-(4-9) analog ORG 2766 shortened the recovery period to 1 week. The effects were explained by denervation supersensitivity of postsynaptic receptor systems, since apomorphine-induced behavioral responses changed in association with the compensation, but not [3H]dopamine uptake and the levels of dopamine and its metabolites [11]. The aim of the present investigations was twofold. (A) To clarify whether reinnervation occurred after a 6-OHDA lesion of the NAc. The recurrence of dopamine-immunoreactive (DAi) fibers in the NAc was therefore investigated 1, 4, 12 and 24 weeks after a unilateral 6 - O H D A lesion. (B) To evaluate whether ORG 2766 treatment influenced the recurrence of DA i fibers. This peptide facilitates functional recovery after lesions of the peripheral and the central nervous system [1,11]. A unilateral sham or 6-OHDA lesion of the right NAc was made as has been described earlier [10]. From day 1 until day 6 after the operation animals were treated subcutaneously with ORG 2766 (10/zg/kg, once daily) or placebo (saline). Animals were anesthetized with Nembutal (0.1 ml/100 g bodyweight) 1, 4, 12 or 24 weeks after the operation. Preperfusion through the ascending aorta with Krebs-Ringer solution containing 118 mM NACI,4.85 mM
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304- 3940(96) 12498-6
22
P.E. Vos et al. / Neuroscience Letters 207 (1996) 21-24
Fig. 1. Brightfield photomicrographs showing DA-immunoreactivity in the NAc of rats 1 (A), 4 (B), 12 (C) and 24 (D) weeks after a unilateral 6OHDA lesion. Note the almost complete absence of DA-immunoreactivity in the NAc 1 week after the lesion (A). Strong dopamine immunoreaetivity is present in the NAc 12 and 24 weeks after the lesion (C,D). CA, commissura anterior.
KCI, 1.15 mM KH2PO4, 1.15 mM MgSO4.7H20, 25 mM NaHCO 3, including 10 mM ascorbic acid (pH 7.2), was followed by perfusion with 500 ml freshly prepared icecold 5% glutaraldehyde in 0.05 M cacodylate buffer and 10 mM ascorbic acid (pH 7.2). The brains were removed, and the NAc was cut into a coronal block and postfixed in the same fixative for 30 min. Coronal sections (50#m thick) were incubated free floating in the following sequence: (1) rinsing three times in Tris-buffered saline (TBS) containing 10 mM ascorbic acid; (2) incubation with rabbit dopamine-antiserum [8], diluted 1:2000 at 4°C for 48 h; (3) rinsing three times for 10 min in TBS, TBS and 0.1% Triton X-100 (TBS-T) and TBS; (4) incu-
bation with goat anti-rabbit IgG, dilution 1:60 at room temperature for 2 h; (5) rinsing three times for 10 min inTBS, TBS-T and TBS; (6) incubation with PAPcomplex, dilution 1:600 at room temperature for 1 h; (7) rinsing three times for 10 min in TBS-T, and twice in Tris-HC1 (pH 7.6); (8) precipitation reaction with 3,3 diaminobenzidine and 0.0066% H202 at room temperature under light microscopic control. Sections were mounted on gelatin-coated glass slides, dried overnight at 37°C, dehydrated and coverslipped in Entellan. With an Olympus Vanox Microscope, using an ocular (10 x 10) square-grid at 40× magnification, DAi fiber frequencies in the mediodorsal (MV) and medioventral (MV) NAc
P.E. Vos et al./ Neuroscience Letters 207 (1996) 21-24
were counted (experimenter blinded to drug treatment). With an IBAS (Kontron) image analyzing system optical densities of the ipsilateral rostral NAc and caudal NAc (coordinates: 8920/~m anterior to interaural line, 1.0 mm lateral of midline, 1.6 mm below the horizontal zero plane as described [6]) were also measured. In unoperated animals dense DA i varicose fibers were evenly distributed throughout the NAc. Sham-operated rats (placebo- or ORG 2766-treated) showed a similar profile in the NAc after 1, 4, 12 and 24 weeks (data not shown). One week after a unilateral 6-OHDA lesion, DAimmunoreactivity was markedly decreased on the lesioned side in placebo-treated animals as compared to the sham-operated side. The MD NAc was more affected then the MV NAc. Many degenerated DAi fibers with swollen varicosities were seen as dark droplets with unsharp boundaries (Fig. 1A). Four weeks after operation a slight increase in DA-immunoreactivity (as compared to 1 week) was observed (Fig. 1B). After 12 weeks a steady increase was observed and an almost complete reappearance of DA-immunoreactivity was established 24 weeks after operation (Fig. 1C,D). To obtain a semi-quantitative measurement of the lesion-induced decrease and subsequent restoration of dopaminergic fibers in the NAc, frequencies of DA i fibers were counted in two perpendicular directions. Timecourse data were obtained from MD and MV NAc. Table 1A summarizes frequencies of DAi fibers. Over the 24-
23
week period an increase in DA i fiber frequencies from five to 106 and 13 to 105 was found in MD and MV NAc, respectively, in placebo-treated, 6-OHDA-lesioned animals. Frequencies of DA i fibers were higher in the y-then in the x-direction but this difference reached significance only 12 weeks after the lesion. DAi fiber frequency was higher in MV NAc as compared to MD NAc in placebotreated animals 1, 4 and 12 but not 24 weeks after the lesion. In ORG 2766-treated, 6-OHDA-lesioned animals frequencies of DAi fibers ranged from two to 88 and from 15 to 92 in MD and MV NAc, respectively, over the 24week time-period, with y-direction frequencies significantly higher than x-direction frequencies, 4 weeks after the lesion in MD and MV NAc and 12 weeks after the lesion in MV NAc (Table 1A). A significantly higher frequency of DA i fibers was found in MD and MV NAc after 4 weeks in ORG 2766- as compared to placebotreated, lesioned animals. One week after the lesion, the IBAS optical density value of total NAc in placebo-treated, lesioned animals was 10% from the control side. This increased to 80% after 24 weeks. A similar increase in optical density was found in MD and MV NAc. A significantly higher optical density was measured in the MV as compared to the MD NAc 1 and 4 weeks after the lesion; a tendency towards higher density was found after 12 weeks. In ORG 2766treated, lesioned animals IBAS scores ranged from 7% to 76% during the 24-week time period; a significant differ-
Table 1 DAi fiber frequencies(A) and optical densities(B) in ipsilateral NAc of rats, 1, 4, 12, or 24 weeks after a unilateral 6-OHDA lesion in the right NAc treated with placebo (pl) or ORG 2766 (org) from day 1 to day 6 after the lesion 1 week
4 weeks
12 weeks
24 weeks
pl
org
pl
org
pl
org
pl
org
2.2±1.4 2.4±1.6 4.6_+2.9 6.6-+0.7 6.4_+1.1 13.0_+1.8
0.7_+0.6 1.0_+0.9 1.7±1.5 7.5_+0.7 7.0_+1.5 14.5±2.1
7.6_+0.8 5.6_+0.8 13.2_+1.5 23.4±1.6 20.4_+2.1 43.8_+3.0
15.0±3.5" 9.5±1.7 24.5±4.8* 35.5_+0.4* 25.0±2.5 60.5_+2.6*
45.3±4.8 32.2±2.2 77.5±6.7 59.0_+3.9 41.2_+2.7 100.1_+5.9
45.5±6.5 31.5_+4.0 74.5±9.7 51.7_+4.9 37.5_+1.0 89.2_+3.9
60.0_+7.5 45.7_+7.7 105.7_+15.0 60.2_+8.6 48.0_+7.3 105.3±15.8
46.7±2.6 36.5±3.0 88.3±5.1 51.3_+5.9 41.0_+4.6 92.3±9.8
10_+3 10_ I 18_+3 80_+3
7_+3 9__. I 22_+2 86±3
46_+5* 31 _+6* 56_+7 93± 1
67_+7 44_+6 60__.4 81 ± 7
Aa
MD
MV
Y X T Y X T
Bb T MD MV PO
23_+3 14_+2 39_+ 10 79_+7
62_+8 58_+ 12 59---9 84_+2
80_+4 78_+4 85_+ 12 90_+5
76_+ 10 67_+ 10 73_+ 10 91 _+4
Each value represents mean score _ SEM per group of rats (n = 4-6/group). MD, medio-dorsal NAc; MV, medio-ventral NAc; PO, NAe posterior section; T, total Nac. *Different from placebo treatment (P < 0.05). ay, frequency of DA i fibe~ in y-direction; X, frequency of DA i fibers in x-direction. bFields of the rostral NAc observed through the microscope (magnification 1,25 × 1,6 at screen 62 were visualized on a monitor. Borders, traced with a digitizing pad, allowed integration of total surface areas in pixel units. Optical densities higher than background were measured. To measure the MD and the MV (magnification 5 × 1; at screen 149), a fixed square was used with its lower right corner on the upper left corner of the CA and its upper right corner on the lower left corner of the commissura anterior, respectively. Data are expressed as percentage of the contralateral NAc optical density.
24
P.E. Vos et al. / Neuroscience Letters 207 (1996) 21-24
ence between MD and MV NAc was observed 1 and 4 but not 12 and 24 weeks after the lesion. At 4 weeks a significantly higher optical density was measured in MD and total NAc, in ORG 2766- as compared to placebotreated, lesioned animals. A similar effect was present in the MV NAc, but this difference did not reach statistical significance. No differences in optical density in caudal NAc were found between ORG 2766- and placebo-treated animals. A slight decrease was present 1 week after the lesion (values 80% and 86% of contralateral side in placeboand ORG 2766-treated animals, respectively) and a small non-significant increase (values 90% and 91%, respectively) was measured after 24 weeks. Normally, a dense dopaminergic plexus is present in the NAc with afferent fibers originating in the ventral tegmental area (VTA) and the dorsal part of the substantia nigra (SN). Destruction of the cell bodies in the VTA-SN cell continuum with 6-OHDA results in a complete abolition of these fibers [9]. Based on the observed changes in motor behavior after bilateral 6-OHDA lesioning of the rostral part of the NAc [10], 6-OHDA was injected into this area in the present study. Because dopaminergic fibers in the medial forebrain bundle enter the NAc from ventral [4], a division was made in a ventral and dorsal part of the medial NAc, in order to detect a possible difference in reinnervation pattern. The dense DA network was almost completely abolished 1 week after the lesion in the rostral part of the NAc and degenerating fibers with irregularly shaped swollen varicosities were observed in both parts of the NAc. The injections with 6-OHDA were aimed at the rostral NAc, but diffusion of the neurotoxin probably resulted in a conical lesion with the rostral MD DA plexus most affected. In the MD and the MV part of the NAc the number of dopaminergic fibers increased gradually 4 and 12 weeks after the lesion; a subnormal density was found after 24 weeks. In placebo-treated, lesioned animals fiber frequencies in MD and MV NAc showed a difference in favor of the latter 1, 4 and 12 weeks after the lesion, but not 24 weeks after the lesion. The reinnervation process of the MV NAc precedes that of the MD NAc. As the fiber pattern between the two accumbal compartments is not different, it may be that axonal regrowth of dopaminergic fibers originates in the ventral part of the NAc. Reappearence of DAi fibers in the NAc may be a consequence of outgrowth of damaged fibers back to the terminal area (regeneration) or collateral sprouting of intact axons of adjacent cells (compensation). Both are examples of homotypic sprouting [2]. Due to the single source dopaminergic input to the NAc, possibly originating entirely in the VTA-SN cell continuum, homotypic sprouting is the most likely explanation for the observed results.
Disappearance of DA i fibers in the NAc was not prevented by treatment with ORG 2766 during the first 6 days after the lesion. However, DAi fiber density was increased in ORG 2766-treated animals within the first 4 weeks as compared to placebo-treated animals. The data suggest that the orientation of dopaminergic fibers reinnervating the NAc in ORG 2766-treated animals is not different from controls. The final outcome, i.e. reinnervation of the NAc with DA i fibers up to subnormal level after 24 weeks, is the same in ORG 2766- and placebotreated animals. No definite conclusions can be drawn on whether the differences are the result of an acceleration of fiber growth or a shift in the onset of regrowth in ORG 2766-treated animals. Supported by the Dutch Prinses Beatrix Fonds, Grant no. 88-2884. [1] Bijlsma, W.A., Jennekens, F.G.I., Schotman, P. and Gispen, W.H., Effects of corticotrophin (ACTH) on recovery of sensorymotor function in the rat: structure-activity study, Eur. J. Pharmacol., 76 (1981) 73 79. [2] Bj0rklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, Physiol. Rev., 59(1) (1979) 62-100. [3] Di-Chiara, G., Porceddu, M.L., Vargui, L., Argiolas, A. anti Gessa, G.L. Evidence for dopamine receptors mediating sedation in mouse brain, Nature, 264 (1976) 564-566. [4] Gerfen, C.R., Herkenham, M. and Yhibault, J., The neostriatal mosaic, lI: patch and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci.. 7 (1987) 3915-3934. [5] Herman, J.P., Choulli, K., Abrous, N., Dulluc, J. and Le Moal, M., Effects of intra-accumbens dopaminergic grafts on behavioral deficits induced by 6-OHDA lesions of the nucleus accumbens or AIO dopaminergic neurons: a comparison, Behav. Brain. Res. 29 (1988) 73-83. [6] K0nig, F.R. and Klippel, R.A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963, p. 162. [7] Onn. S.. Berger. T.W., Stricker, E.M. anti Zigmond, M.J.. Elfccls of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: histochemical and neurochemical analysis. Brain. Res., 376 (1986) 8 19. [8] Steinbusch, H.W.M., Van Viler, S.P., Bol, J.G.J.M. and De Vente. .I., Development and application of antibodies to primary (DA) and secondary (cGMP) messengers: a technical report. In A. Calas (Ed.), Neurocytochemical Methods, Springer Verlag, Berlin, 1991, pp, 13-41. [9] Voorn. P., Roest. G. and Grocnewegen, HJ. Increase ot enkephalin and decrease of substance P immunoreactivity in the striatum of the rat after midbrain 6-hydroxydopamine lesions. Brain. Res., 412 (1987) 391-396. [10] Wollerink, G., Van Zantcn, E, Kamsteeg, H., Radhakishun. FS. and Van Ree, J.M. Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats, I: behavioral and biochemical studies, Brain. Res., 507 (1990)92-100. [11] Wolterink, G , Van Zanten, E., Kamsteeg, H., Radhakishun, F.S, and Van Ree, J.M. Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. 11: facilitation by the ACTH-(4-9) analog ORG 2766, Brain. Res.. 507 (1990) 101-108.
NeuroscieneeLetters207(1996)21-24
I[II[HS
Reinnervation after destruction of the dopaminergic system in the rat nucleus accumbens: a quantitative immunohistochemical analysis Pieter
E. VOS a'*, Harry W.M.
S t e i n b u s c h b, J a n M . V a n R e e a
aDepartment of Pharmacology, Rudolf Magnus Institute for Neurosciences, Faculty of Medicine, University of Utrecht, Utrecht, The Netherlands bDepartment of Psychiatry and Neuropsychology, University of Maastricht, Maastricht, The Netherlands Received 22 December 1995; revised version received 1 February 1996; accepted 14 February 1996
Abstract
The recurrence of dopamine-immunoreactive (DAi) fibers and the effect of the adrenocorticotrophic hormone (ACTH)-(4-9) analog ORG 2766 on this process were investigated 1, 4, 12 and 24 weeks after a unilateral 6-hydroxydopamine (6-OHDA) lesion in the nucleus accumbens (NAc). DAi fibers were almost completely absent 1 week after the lesion. A gradual increase in DAi fibers throughout the NAc was observed, with subnormal values at 24 weeks. Treatment with ORG 2766 during the first week after the lesion resulted in more DAi fibers 4 weeks after the lesion as compared to placebo treatment, but not 12 and 24 weeks after the lesion. After 6-OHDA lesioning reinnervation of the NAc takes place and this process is transiently facilitated by ORG 2766.
Keywords: Regeneration; Ventral striatum; Nucleus accumbens; Dopamine; Immunoreactivity; Plasticity; ORG 2766; Adrenocorticotrophic hormone-(4--9) analog
The central nervous system of mammals is able to recover from lesion-induced disturbances in a functional way. Whether the recovery is a mere compensation (i.e. changes in turnover of neurotransmitters, receptor upregulation) or the result of a reorganization (i.e. neural circuitry changes, reinnervation) is still a matter of debate. Under certain conditions monoaminergic fibers display axonal regrowth after lesioning [2,7]. In the ventral striatum, i.e. the nucleus accumbens (NAc), no reinnervation with dopaminergic fibers was observed 9 months after a bilateral 6-hydroxydopamine (6-OHDA) lesion [5]. However, dense reinnervation of the NAc with dopaminergic fibers was found after transplantation of fetal dopaminergic neurons in this area after 6-OHDA lesioning. Novelty-induced locomotor activity is reduced if dopaminergic terminals in the NAc are lesioned bilaterally with 6-OHDA [3,10]. This reduction in locomotor activity recovered spontaneously in 3-4 weeks. Treatment * Corresponding author. Present address: Department of Neurology, University Hospital Utrecht, Hp C03.236, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Tel: +31 30 2507975; fax: +31 30 2542100.
with the adrenocorticotrophic hormone (ACTH)-(4-9) analog ORG 2766 shortened the recovery period to 1 week. The effects were explained by denervation supersensitivity of postsynaptic receptor systems, since apomorphine-induced behavioral responses changed in association with the compensation, but not [3H]dopamine uptake and the levels of dopamine and its metabolites [11]. The aim of the present investigations was twofold. (A) To clarify whether reinnervation occurred after a 6-OHDA lesion of the NAc. The recurrence of dopamine-immunoreactive (DAi) fibers in the NAc was therefore investigated 1, 4, 12 and 24 weeks after a unilateral 6 - O H D A lesion. (B) To evaluate whether ORG 2766 treatment influenced the recurrence of DA i fibers. This peptide facilitates functional recovery after lesions of the peripheral and the central nervous system [1,11]. A unilateral sham or 6-OHDA lesion of the right NAc was made as has been described earlier [10]. From day 1 until day 6 after the operation animals were treated subcutaneously with ORG 2766 (10/zg/kg, once daily) or placebo (saline). Animals were anesthetized with Nembutal (0.1 ml/100 g bodyweight) 1, 4, 12 or 24 weeks after the operation. Preperfusion through the ascending aorta with Krebs-Ringer solution containing 118 mM NACI,4.85 mM
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304- 3940(96) 12498-6
22
P.E. Vos et al. / Neuroscience Letters 207 (1996) 21-24
Fig. 1. Brightfield photomicrographs showing DA-immunoreactivity in the NAc of rats 1 (A), 4 (B), 12 (C) and 24 (D) weeks after a unilateral 6OHDA lesion. Note the almost complete absence of DA-immunoreactivity in the NAc 1 week after the lesion (A). Strong dopamine immunoreaetivity is present in the NAc 12 and 24 weeks after the lesion (C,D). CA, commissura anterior.
KCI, 1.15 mM KH2PO4, 1.15 mM MgSO4.7H20, 25 mM NaHCO 3, including 10 mM ascorbic acid (pH 7.2), was followed by perfusion with 500 ml freshly prepared icecold 5% glutaraldehyde in 0.05 M cacodylate buffer and 10 mM ascorbic acid (pH 7.2). The brains were removed, and the NAc was cut into a coronal block and postfixed in the same fixative for 30 min. Coronal sections (50#m thick) were incubated free floating in the following sequence: (1) rinsing three times in Tris-buffered saline (TBS) containing 10 mM ascorbic acid; (2) incubation with rabbit dopamine-antiserum [8], diluted 1:2000 at 4°C for 48 h; (3) rinsing three times for 10 min in TBS, TBS and 0.1% Triton X-100 (TBS-T) and TBS; (4) incu-
bation with goat anti-rabbit IgG, dilution 1:60 at room temperature for 2 h; (5) rinsing three times for 10 min inTBS, TBS-T and TBS; (6) incubation with PAPcomplex, dilution 1:600 at room temperature for 1 h; (7) rinsing three times for 10 min in TBS-T, and twice in Tris-HC1 (pH 7.6); (8) precipitation reaction with 3,3 diaminobenzidine and 0.0066% H202 at room temperature under light microscopic control. Sections were mounted on gelatin-coated glass slides, dried overnight at 37°C, dehydrated and coverslipped in Entellan. With an Olympus Vanox Microscope, using an ocular (10 x 10) square-grid at 40× magnification, DAi fiber frequencies in the mediodorsal (MV) and medioventral (MV) NAc
P.E. Vos et al./ Neuroscience Letters 207 (1996) 21-24
were counted (experimenter blinded to drug treatment). With an IBAS (Kontron) image analyzing system optical densities of the ipsilateral rostral NAc and caudal NAc (coordinates: 8920/~m anterior to interaural line, 1.0 mm lateral of midline, 1.6 mm below the horizontal zero plane as described [6]) were also measured. In unoperated animals dense DA i varicose fibers were evenly distributed throughout the NAc. Sham-operated rats (placebo- or ORG 2766-treated) showed a similar profile in the NAc after 1, 4, 12 and 24 weeks (data not shown). One week after a unilateral 6-OHDA lesion, DAimmunoreactivity was markedly decreased on the lesioned side in placebo-treated animals as compared to the sham-operated side. The MD NAc was more affected then the MV NAc. Many degenerated DAi fibers with swollen varicosities were seen as dark droplets with unsharp boundaries (Fig. 1A). Four weeks after operation a slight increase in DA-immunoreactivity (as compared to 1 week) was observed (Fig. 1B). After 12 weeks a steady increase was observed and an almost complete reappearance of DA-immunoreactivity was established 24 weeks after operation (Fig. 1C,D). To obtain a semi-quantitative measurement of the lesion-induced decrease and subsequent restoration of dopaminergic fibers in the NAc, frequencies of DA i fibers were counted in two perpendicular directions. Timecourse data were obtained from MD and MV NAc. Table 1A summarizes frequencies of DAi fibers. Over the 24-
23
week period an increase in DA i fiber frequencies from five to 106 and 13 to 105 was found in MD and MV NAc, respectively, in placebo-treated, 6-OHDA-lesioned animals. Frequencies of DA i fibers were higher in the y-then in the x-direction but this difference reached significance only 12 weeks after the lesion. DAi fiber frequency was higher in MV NAc as compared to MD NAc in placebotreated animals 1, 4 and 12 but not 24 weeks after the lesion. In ORG 2766-treated, 6-OHDA-lesioned animals frequencies of DAi fibers ranged from two to 88 and from 15 to 92 in MD and MV NAc, respectively, over the 24week time-period, with y-direction frequencies significantly higher than x-direction frequencies, 4 weeks after the lesion in MD and MV NAc and 12 weeks after the lesion in MV NAc (Table 1A). A significantly higher frequency of DA i fibers was found in MD and MV NAc after 4 weeks in ORG 2766- as compared to placebotreated, lesioned animals. One week after the lesion, the IBAS optical density value of total NAc in placebo-treated, lesioned animals was 10% from the control side. This increased to 80% after 24 weeks. A similar increase in optical density was found in MD and MV NAc. A significantly higher optical density was measured in the MV as compared to the MD NAc 1 and 4 weeks after the lesion; a tendency towards higher density was found after 12 weeks. In ORG 2766treated, lesioned animals IBAS scores ranged from 7% to 76% during the 24-week time period; a significant differ-
Table 1 DAi fiber frequencies(A) and optical densities(B) in ipsilateral NAc of rats, 1, 4, 12, or 24 weeks after a unilateral 6-OHDA lesion in the right NAc treated with placebo (pl) or ORG 2766 (org) from day 1 to day 6 after the lesion 1 week
4 weeks
12 weeks
24 weeks
pl
org
pl
org
pl
org
pl
org
2.2±1.4 2.4±1.6 4.6_+2.9 6.6-+0.7 6.4_+1.1 13.0_+1.8
0.7_+0.6 1.0_+0.9 1.7±1.5 7.5_+0.7 7.0_+1.5 14.5±2.1
7.6_+0.8 5.6_+0.8 13.2_+1.5 23.4±1.6 20.4_+2.1 43.8_+3.0
15.0±3.5" 9.5±1.7 24.5±4.8* 35.5_+0.4* 25.0±2.5 60.5_+2.6*
45.3±4.8 32.2±2.2 77.5±6.7 59.0_+3.9 41.2_+2.7 100.1_+5.9
45.5±6.5 31.5_+4.0 74.5±9.7 51.7_+4.9 37.5_+1.0 89.2_+3.9
60.0_+7.5 45.7_+7.7 105.7_+15.0 60.2_+8.6 48.0_+7.3 105.3±15.8
46.7±2.6 36.5±3.0 88.3±5.1 51.3_+5.9 41.0_+4.6 92.3±9.8
10_+3 10_ I 18_+3 80_+3
7_+3 9__. I 22_+2 86±3
46_+5* 31 _+6* 56_+7 93± 1
67_+7 44_+6 60__.4 81 ± 7
Aa
MD
MV
Y X T Y X T
Bb T MD MV PO
23_+3 14_+2 39_+ 10 79_+7
62_+8 58_+ 12 59---9 84_+2
80_+4 78_+4 85_+ 12 90_+5
76_+ 10 67_+ 10 73_+ 10 91 _+4
Each value represents mean score _ SEM per group of rats (n = 4-6/group). MD, medio-dorsal NAc; MV, medio-ventral NAc; PO, NAe posterior section; T, total Nac. *Different from placebo treatment (P < 0.05). ay, frequency of DA i fibe~ in y-direction; X, frequency of DA i fibers in x-direction. bFields of the rostral NAc observed through the microscope (magnification 1,25 × 1,6 at screen 62 were visualized on a monitor. Borders, traced with a digitizing pad, allowed integration of total surface areas in pixel units. Optical densities higher than background were measured. To measure the MD and the MV (magnification 5 × 1; at screen 149), a fixed square was used with its lower right corner on the upper left corner of the CA and its upper right corner on the lower left corner of the commissura anterior, respectively. Data are expressed as percentage of the contralateral NAc optical density.
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P.E. Vos et al. / Neuroscience Letters 207 (1996) 21-24
ence between MD and MV NAc was observed 1 and 4 but not 12 and 24 weeks after the lesion. At 4 weeks a significantly higher optical density was measured in MD and total NAc, in ORG 2766- as compared to placebotreated, lesioned animals. A similar effect was present in the MV NAc, but this difference did not reach statistical significance. No differences in optical density in caudal NAc were found between ORG 2766- and placebo-treated animals. A slight decrease was present 1 week after the lesion (values 80% and 86% of contralateral side in placeboand ORG 2766-treated animals, respectively) and a small non-significant increase (values 90% and 91%, respectively) was measured after 24 weeks. Normally, a dense dopaminergic plexus is present in the NAc with afferent fibers originating in the ventral tegmental area (VTA) and the dorsal part of the substantia nigra (SN). Destruction of the cell bodies in the VTA-SN cell continuum with 6-OHDA results in a complete abolition of these fibers [9]. Based on the observed changes in motor behavior after bilateral 6-OHDA lesioning of the rostral part of the NAc [10], 6-OHDA was injected into this area in the present study. Because dopaminergic fibers in the medial forebrain bundle enter the NAc from ventral [4], a division was made in a ventral and dorsal part of the medial NAc, in order to detect a possible difference in reinnervation pattern. The dense DA network was almost completely abolished 1 week after the lesion in the rostral part of the NAc and degenerating fibers with irregularly shaped swollen varicosities were observed in both parts of the NAc. The injections with 6-OHDA were aimed at the rostral NAc, but diffusion of the neurotoxin probably resulted in a conical lesion with the rostral MD DA plexus most affected. In the MD and the MV part of the NAc the number of dopaminergic fibers increased gradually 4 and 12 weeks after the lesion; a subnormal density was found after 24 weeks. In placebo-treated, lesioned animals fiber frequencies in MD and MV NAc showed a difference in favor of the latter 1, 4 and 12 weeks after the lesion, but not 24 weeks after the lesion. The reinnervation process of the MV NAc precedes that of the MD NAc. As the fiber pattern between the two accumbal compartments is not different, it may be that axonal regrowth of dopaminergic fibers originates in the ventral part of the NAc. Reappearence of DAi fibers in the NAc may be a consequence of outgrowth of damaged fibers back to the terminal area (regeneration) or collateral sprouting of intact axons of adjacent cells (compensation). Both are examples of homotypic sprouting [2]. Due to the single source dopaminergic input to the NAc, possibly originating entirely in the VTA-SN cell continuum, homotypic sprouting is the most likely explanation for the observed results.
Disappearance of DA i fibers in the NAc was not prevented by treatment with ORG 2766 during the first 6 days after the lesion. However, DAi fiber density was increased in ORG 2766-treated animals within the first 4 weeks as compared to placebo-treated animals. The data suggest that the orientation of dopaminergic fibers reinnervating the NAc in ORG 2766-treated animals is not different from controls. The final outcome, i.e. reinnervation of the NAc with DA i fibers up to subnormal level after 24 weeks, is the same in ORG 2766- and placebotreated animals. No definite conclusions can be drawn on whether the differences are the result of an acceleration of fiber growth or a shift in the onset of regrowth in ORG 2766-treated animals. Supported by the Dutch Prinses Beatrix Fonds, Grant no. 88-2884. [1] Bijlsma, W.A., Jennekens, F.G.I., Schotman, P. and Gispen, W.H., Effects of corticotrophin (ACTH) on recovery of sensorymotor function in the rat: structure-activity study, Eur. J. Pharmacol., 76 (1981) 73 79. [2] Bj0rklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, Physiol. Rev., 59(1) (1979) 62-100. [3] Di-Chiara, G., Porceddu, M.L., Vargui, L., Argiolas, A. anti Gessa, G.L. Evidence for dopamine receptors mediating sedation in mouse brain, Nature, 264 (1976) 564-566. [4] Gerfen, C.R., Herkenham, M. and Yhibault, J., The neostriatal mosaic, lI: patch and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci.. 7 (1987) 3915-3934. [5] Herman, J.P., Choulli, K., Abrous, N., Dulluc, J. and Le Moal, M., Effects of intra-accumbens dopaminergic grafts on behavioral deficits induced by 6-OHDA lesions of the nucleus accumbens or AIO dopaminergic neurons: a comparison, Behav. Brain. Res. 29 (1988) 73-83. [6] K0nig, F.R. and Klippel, R.A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963, p. 162. [7] Onn. S.. Berger. T.W., Stricker, E.M. anti Zigmond, M.J.. Elfccls of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: histochemical and neurochemical analysis. Brain. Res., 376 (1986) 8 19. [8] Steinbusch, H.W.M., Van Viler, S.P., Bol, J.G.J.M. and De Vente. .I., Development and application of antibodies to primary (DA) and secondary (cGMP) messengers: a technical report. In A. Calas (Ed.), Neurocytochemical Methods, Springer Verlag, Berlin, 1991, pp, 13-41. [9] Voorn. P., Roest. G. and Grocnewegen, HJ. Increase ot enkephalin and decrease of substance P immunoreactivity in the striatum of the rat after midbrain 6-hydroxydopamine lesions. Brain. Res., 412 (1987) 391-396. [10] Wollerink, G., Van Zantcn, E, Kamsteeg, H., Radhakishun. FS. and Van Ree, J.M. Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats, I: behavioral and biochemical studies, Brain. Res., 507 (1990)92-100. [11] Wolterink, G , Van Zanten, E., Kamsteeg, H., Radhakishun, F.S, and Van Ree, J.M. Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. 11: facilitation by the ACTH-(4-9) analog ORG 2766, Brain. Res.. 507 (1990) 101-108.