Neuroscience Letters, 116 (1990) 51-57
51
Elsevier Scientific Publishers Ireland Ltd.
NSL 07046
Quantitative autoradiographic changes in 5-[3H]HT labeled 5-HT1 serotonin receptors in discrete regions of brain in the rat model of persistent dyskinesias induced by iminodipropionitrile (IDPN) Serge Przedborski, Mia Wright, Stanley F a h n and Jean L u d Cadet Neurological Institute, College of Physicians and Surgeons, Columbia University, New York, N Y 10032 (U.S.A.) (Received 2 March 1990; Revised version received 15 April 1990; Accepted 19 April 1990)
Key words." Iminodipropionitrile; Dyskinesias; Serotonin receptor; Autoradiographic; Frontal cortex; Striatum; Hippocampus; Septum; Superior Colliculus Chronic injections of iminodipropionitrile (IDPN) to rat cause a persistent motor hyperactivity, lateral and vertical sustained twisting movement of the neck, random circling and increased startle response. These abnormalities are similar to those observed after the acute administration of serotonin (5-HT) agonists in rodents. Significant changes in 5-HT concentration and in 5-HT2 receptor density in several motorrelated brain regions have been observed in IDPN-treated rats. The present quantitative autoradiographic study was undertaken to assess the possibility that IDPN may also affect 5-HTt receptors in rat brain. IDPN caused significant increases of 5-[3H]HT binding in the oriens and pyramidal layers of the CA3 field of hippocampus. In contrast, there were significant decreases of 5-[3H]HT binding in the frontal and cingulate cortices, the olfactory tubercle, the ventromedial aspect of the caudate-putamen, the nucleus accumbens, the superior colliculus, and the lateral septal nuclei. These results provide further evidence for the involvement of the 5-HT system in the development of the IDPN-induced dyskinetic syndrome.
Chronic treatment with the neurotoxin iminodipropionitrile (IDPN) induced in rodents and in nonhuman primate a complex and persistent behavioral syndrome characterized by motor hyperactivity, sustained twisting lateral (laterocollis) and vertical (retrocollis) movements of the neck and increased startle response (see ref. 3 for review). These behavioral abnormalities are analogous to those induced by acute injections of serotonin (5-HT) agonists [12]. Because of the similarities between the IDPNand the 5-HT-induced syndromes, we have undertaken a series of studies in order to investigate the possible involvement of the 5-HT system in the behavior abnormal-
Correspondence.. S. Przedborski, Neurological Institute, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
52 ities observed in IDPN-treated rats [3]. Significant increases in 5-HT concentrations were found in the nucleus accumbens and in the caudate-putamen nucleus in IDPNtreated rats [5]. Homogenate [7] and autoradiographic [6] receptor binding experiments have revealed significant changes in 5-HT receptor subtype 2 (5-HT2) density in various brain regions in similarly treated animals. Although it was initially thought that the activation of the 5-HT2 receptors mediated most of the 5-HT-induced behavioral abnormalities [12, 19], recent evidence has implicated 5-HTj receptors in the manifestation of these behavioral changes [16, 22]. For example, the administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT), a potent 5-HTI agonist, elicited most of the 5-HT-induced manifestations such as motor hyperactivity, head weaving and forepaw treading [13, 22]. Similarly, in the IDPN model recent data also support the possible involvement of the 5-HTI receptors in some of the behavioral abnormalities [12]. Moreover, the administration of 8-OHDPAT to rat has been reported to inhibit the IDPN-induced dystonic movements of the neck and backward locomotion [9]. The following quantitative autoradiographic study was thus carried out in order to determine whether chronic treatment with IDPN would cause specific topographical changes in 5-[3H]HT-labeled 5-HT1 receptors in rat brain. Adult male Sprague-Dawley rats weighing 220-250 g at the beginning of the experiment were maintained on a 12-h light,lark cycle and were provided with food and water ad libitum. Rats were divided into two groups; one group received daily i.p. injections of IDPN (100 mg/kg) until the behavioral syndrome developed (5-7 days) and a second group received daily i.p. injections of saline vehicle (1 ml/kg). All rats treated with IDPN developed the abnormalities which persisted without any need for further injections. Four weeks after the last injection, 5 IDPN-treated and 5 control animals were killed by decapitation. This time period was chosen according to our previous behavior and biochemical studies [3]. The brains were rapidly removed, frozen on dry ice and stored at -70°C. Sections (12/~m) were cut at -20°C with a cryostat, thaw-mounted onto chrome alum/gelatin-subbed glass slides and stored at - 70'~C until use. The sections were labeled in vitro for receptor binding assay autoradiography according to the method described by Pazos and Palacios [17]. Sections were preincubated (25°C; 30 min) in 50 mM Tris-HCl (pH 7.6) containing 4 mM CaCI2 and 0.01% ascorbic acid before being incubated (25°C; 60 min) in the same buffer with 2 nM 5-[3H]HT (30 Ci/mmol, New England Nuclear). Non-specific binding was determined by incubating adjacent sections in the presence of I/~M unlabeled 5-HT and represented 5-10% of total binding. After incubation, the sections were washed in fresh ice-cold buffer for 5 min, dipped in ice-cold distilled water and dried rapidly with cold air. Dried sections were placed in an X-ray cassette with plastic standards ([3H]-micro-scales, Amersham) and were apposed to LKB 3H-Ultrafilm for 8 weeks. After the films were developed, the sections were stained with cresyl violet for histological reference. The developed autoradiographic films were quantified using a Loats PC-based computerized image analysis system (Amersham). The film optical densities were converted to fmol of 5-[3H]HT bound per mg tissue using a standard curve generated by the [3HI-micro-scales. Statistical differences between the
53
TABLE I E F F E C T O F C H R O N I C A D M I N I S T R A T I O N OF I D P N O N T H E B I N D I N G O F 2 n M 5-[3H]HT TO 5-HTI R E C E P T O R S IN T H E R A T B R A I N Brain regions
Cerebral cortex Prefrontal Frontal, layersI-III layers IV-VI Parietal Temporal Cingulate Entorhinal Olfactory tubercle Caudate-putamen Dorsolateral Ventromedial Nucleus Accumbens Globus Pallidus Claustrum Hippocampus Dentate C y r u s CA4 (hilus) CAI CA2 CA3, oriens layer pyramidal layer molecular layer Subiculum Interpeduncular nucleus Substantia nigra Pars compacta Pars reticulata Pars lateralis Ventral tegmental area Centralgrey Superior colliculus Septal nucleus Dorsal lateral Ventral lateral
Specific binding (fmol/mg tissue + S.E.M.)
Control
IDPN-treated
P-values
225.66__+ 9.88 154.50__+ 7.40 233.55+ 7.13 149.50+ 7.90 190.75+ 8.31 284.13 + 8.66 286.87 ___i 1.36 389.15 ___24.31
228.75___ 8.16 154.00+ 6.54 212.50+ 4.76 149.83+ 7.23 192.13__+ 10.97 246.00___ 9.88 294.00 + 12.82 277.50 + 20.45
n.s. n.s. 0.045 n.s. n.s. 0.0085 n.s. 0.0012
189.60+ 6.46 204.25+ 4.21 311.00__+ 15.50 499.25 ___13.69 307.83 -+ 10.68
176.66+ 4.07 178.00_+ 3.79 269.00+ 13.09 472.50___ 10.23 299.50___ 8.25
n.s. 0.019 0.045 n.s. n.s.
576.13 + 15.07 399.62 -+ 23.48 376.50+ 8.36 415.40___ 11.45 403.16-+27.59 201.25 __+12.79 586.50___ 23.93 494.38 + I 1.78 415.00+ 9.50
535.25 __+19.51 362.50-+ 21.64 396.00+__ 19.58 406.13___ 11.58 571.10+25.88 281.86 __+13.46 592.83 + 26.31 467.25-+ 14.67 424.00+10.71
n.s. n.s. n.s. n.s. 0.0013 0.0036 n.s. n.s. n.s.
189.00 ___I 1.62 594.75 ___12.73 276.35+ 15.93 179.50+ 5.88 320.66+ 9.26 339.63 _+ 10.12
201.83 ___13.31 588.63 + 11.34 237.25 + 18.52 161.13+ 8.24 301.60+ 6.06 276.13 + 11.78
n.s. n.s. n.s. n.s. n.s. 0.012
580.17 + 16.05 475.80__+ 21.69
483.33 ___21.45 336.33 + 11.96
0.003 0.002
The values represent the means + the standard error of the mean (SEM) of 5-[3H]HT bound for 5 control (saline-treated) and 5 IDPN-treated rats. Non-specific binding was defined by 1 g M unlabeled 5-HT and was subtracted from all readings.
54
55 two groups were determined with Student's t-test. The null hypothesis was rejected at the 0.05 level. The distribution of 5-[3H]HT binding was comparable to what has been previously reported [17] and is summarized.in Table I. Chronic injections of I D P N were associated with significant increases of 5-[3H]HT binding in the oriens (+41%) and in the pyramidal ( + 40 %) layers of the CA3 field of hippocampus (Table I). In contrast, as figure 1 shows, there were significant decreases in 5-[3H]HT binding in layers IVVI of the frontal cortex ( - 10%), the cingulate cortex ( - 14%), the olfactory tubercle ( - 29 %), the ventromedial part of the caudate-putamen nucleus ( - 13 %), the nucleus accumbens ( - 14%), the superior colliculus ( - 19%) and in the dorsal lateral ( - 17%) and ventral lateral ( - 29 %) septal nuclei (see Table I for specific values of binding). Since biochemical studies have indicated that 5-[3H]HT binds to 5-HT1 receptors [17, 18], the present quantitative autoradiographic study indicates that chronic administration of IDPN causes significant changes in 5-HT1 receptors in several discrete regions of the brain of treated rats. The decreases in cortical 5-[3H]HT binding in the brain of IDPN-treated rats are in contrast to the increases previously observed in both 5-HTIA [20] and 5-HT2 [6, 7] receptors in similarly treated animals. Since the majority of 5-[3H]HT-labeled sites in rat frontal cortex are of 5-HTIB subtype [17], it is likely that the decreases in cortical 5-[3H]HT binding reflect changes in 5-HT1B receptors which are thought to be located on presynaptic terminals [18]. This idea is supported by the finding of significant decreases in cortical 5-HT concentration after chronic administration of IDPN [15]. These observations raise the possibility that chronic treatment with IDPN may result in loss of cortical 5-HT nerve terminals. Immunohistochemical studies may help to test that assumption. Since both the binding of [125I]lysergic acid diethylamide-labeled 5-HT2 [6] and 5[3H]HT-labeled 5-HTI receptors are significantly decreased in the nucleus accumbens and the ventromedial part of the striatum in IDPN-treated rats, it is likely that 5-HTz and 5-HTI receptors are regulated in a similar fashion in these regions of rat brain. It is also possible that the changes observed in those receptors are related to compensatory down-regulation secondary to the sustained increase of 5-HT concentration found in the striatum and the nucleus accumbens of similarly treated animals [5]. From a behavioral perspective, it is likely that these changes play a role in the manifestation in the IDPN-induced syndrome since these brain regions have been implicated in motor phenomena such as increased locomotion [8] and circling behavior [21] which are prominent aspects of the IDPN-induced abnormalities. Although it
Fig. 1. Representativedark-field transform autoradiograms illustrating the effectof chronic administration of IDPN on 5-13H]HTbinding to 5-HT-1 receptors in rat brain. 5-[3H]HTbinding in hemicoronal sections of saline-treated rats (A-C) and IDPN-treated rats (D-F). Autoradiograms were generated as described in the text. Fr, frontal cortex; Cg, cingulatecortex; CPu, caudate-putamennucleus;Acb, nucleus accumbens; OTu, olfactory tubercle; LS, lateral septal nucleus; HIP, hippocampus; SuG, superficialgrey layer of the superior colliculus; CG, central grey; SN, substantia nigra; IP, interpeduncularnucleus. Bar = 140/tin.
56 remains to be determined how I D P N might have led to these abnormalities in the rat brain, the decreases in 5-[3H]HT binding in the superior colliculus might also be related to some aspects of the behavioral syndrome. This idea is supported by neuroanatomical studies which have identified projections from the substantia nigra pars reticulata to the superior collicutus [1, 1 1] which, in turn, sends efferents to cell bodies in the brainstem [10]. This interpretation is also supported by 2-deoxy-D-[l-lnC]glucose studies which have revealed abnormalities in glucose utilization in the superior colliculus of IDPN-treated animals [4] and in a genetic model of dystonia (rat mutant dystonic) [2]. I D P N caused increases in 5-[3H]HT binding in the oriens and pyramidal layers of the CA3 field of the hypocampus. These changes probably reflect increases in 5-HT-IB receptors since we reported previously decreases in 8-OH[3H]DPAT-Iabeled 5-HTIA receptors in these regions [20]. The overexcitability observed in I D P N treated animals [3] may be related to these changes since lesion of the septo-hippocampal axis is associated with a similar behavioral pattern [14]. In summary, the present study provides further evidence for the involvement of the 5-HT system in the IDPN-induced behavioral syndrome. Most of the changes observed in 5-HTI and 5-HT2 receptors can be explained by reciprocal alterations in receptor numbers in response to sustained increases in 5-HT levels in the same regions of the brain. The alternative explanation that some of those changes may be due to neuronal death is not plausible in view of the absence of any neuropathological studies which have been able to document cell death in these brain regions (reviewed in ref. 3). These studies also provide evidence for possible dysfunction in the septo-hippocampal system in IDPN-treated rats. Serge Przedborski is Research Assistant for the National Fund for Scientific Research (Belgium) and is supported, as well as Mia Wright, by a Fellowship from the Parkinson's disease Foundation. 1 Beckstead,R.M., Domesick,V.B. and Nauta, W.J.H., Efferentconnections of the substantia nigra and ventral tegmental area in the rat, Brain Res., 175 (1979) 191-217. 2 Brown, L.L. and Lorden, J.F., Regional cerebral glucoseutilization reveals widespread abnormalities in the motor system of the rat mutant dystonic, J. Neurosci., 9 (1989) 40334041. 3 Cadet, J.L., The iminodipropionitrile (IDPN)-induceddyskinetic syndrome:behavioral and biochemical pharmacology,Neurosci. Biobehav. Rev., 13 (1989) 3945. 4 Cadet, J.L., Della Puppa, A. and London, E., Involvementof nigrotecto-reticulospinal pathways in the iminodipropionitrile (IDPN) model of spasmodic dyskinesias: a 2-deoxy-o[l-14C]glucosestudy in the rat, Brain Res., 484 (1989) 57~64. 5 Cadet, J.L., Jackson-Lewis,V. and Fahn, S., The iminodipropionitrile (IDPN)-induceddyskinetic syndrome: regional serotonin metabolism in rat brain, Brain Res., 456 (1988) 371-374. 6 Cadet, J.L., Kuyatt, B., Fahn, S. and De Souza, E.B., Differential changes in ~25I-LSD-labeled5-HT-2 serotonin receptors in discrete regions of brain in the rat model of persistent dyskinesias induced by iminodipropionitrile (IDPN): evidencefrom autoradiographic studies, Brain Res., 437 (1987) 383-386. 7 Cadet, J.L. and Rothman, R.B., Increased cortical serotonin-2 (5-HT-2) receptors in the iminodipropionitrile (IDPN)-modelof persistent dyskinesia in the rat, Neuropeptides, 10 (1987) 175 180. 8 Cole, S.O., Brain mechanismsof amphetamine-induced anorexia, locomotionand stereotypy:a review, Neurosci. Biobehav. Rev., 2 (1978) 89-100. 9 Diamond, B.I., Sethi, K. and Borison, R.L., Serotonin modulation of hyperkinesia and phasic neck
57 dystonia induced by fl-ff-iminodipropionitrile (IDPN) in rats, Neurology, 36 (1986) 341. 10 Grantyn, A. and Grantyn, R., Axonal patterns and sites of termination of cat superior colliculus neurons projecting in the tecto-bulbo-spinal tract. Exp. Brain Res., 46 (1982) 243-256. 11 Graybiel, A.M., Organization of the nigrotectal connection: an experimental tracer study in the cat, Brain Res., 143 (1978) 339-348. 12 Green, A.R., 5-HT-mediated behavior. Animal studies, Neuropharmacology, 23 (1984) 1521- 1528. 13 Hjorth, S., Carlsson, A., Lindberg, P., Sanchez, D., Wikstrom, H., Arvidsson, L.E., Hacksell, U. and Nilsson, L.J.G., 8-Hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT, a potent and selective simplified ergo congener with central 5-HT-receptor stimulating activity, J. Neural Transm., 55 (1982) 169188. 14 Isaacson, R.L., The Limbic System, 2nd edn., Plenum, New York, 1982, pp. 137-168. 15 Langlais, P.J., Juang, P.C. and Gabay, S., Regional neurochemical studies on the effects offl,ff-iminodipropionitrile (IDPN) in the rat, J. Neurosci. Res., 1 (1975) 419425. 16 Lucki, I. and Frazer, A., Prevention of the serotonin syndrome in rats by repeated administration of monoamine oxidase inhibitors, but not tricyclic antidepressants, Psychopharmacology, 77 (1982) 205 211. 17 Pazos, A. and Palacios, J.M., Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. serotonin-1 receptors, Brain Res., 346 (1985) 205-230. 18 Peroutka, S.J. 5-Hydroxytryptamine receptor subtypes, Annu. Rev. Neurosci., 11 (1988) 45~50. 19 Peroutka, S.J., Lebovitz, R,M. and Snyder, S.H., Two distinct central serotonin receptors with different physiological functions, Science, 212 (1981) 827-829. 20 Przedborski, S., Wright, M., Fahn, S. and Cadet, J.L., Regional changes in brain 5-HTIA serotonin receptors in the rat model of persistent spasmodic dyskinesias induced by iminodipropionitrile, Brain Res., 504 (1989) 311-314. 21 Pycock, C.J., Turning behavior in animals, Neuroscience, 5 (1980) 461-514. 22 Tricklebank, M.D., Forler, C. and Fozard, J.R., The involvement of subtypes of the 5-HT~ receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat, Eur. J. Pharmacol., 106 (1985) 271-282.
51
Elsevier Scientific Publishers Ireland Ltd.
NSL 07046
Quantitative autoradiographic changes in 5-[3H]HT labeled 5-HT1 serotonin receptors in discrete regions of brain in the rat model of persistent dyskinesias induced by iminodipropionitrile (IDPN) Serge Przedborski, Mia Wright, Stanley F a h n and Jean L u d Cadet Neurological Institute, College of Physicians and Surgeons, Columbia University, New York, N Y 10032 (U.S.A.) (Received 2 March 1990; Revised version received 15 April 1990; Accepted 19 April 1990)
Key words." Iminodipropionitrile; Dyskinesias; Serotonin receptor; Autoradiographic; Frontal cortex; Striatum; Hippocampus; Septum; Superior Colliculus Chronic injections of iminodipropionitrile (IDPN) to rat cause a persistent motor hyperactivity, lateral and vertical sustained twisting movement of the neck, random circling and increased startle response. These abnormalities are similar to those observed after the acute administration of serotonin (5-HT) agonists in rodents. Significant changes in 5-HT concentration and in 5-HT2 receptor density in several motorrelated brain regions have been observed in IDPN-treated rats. The present quantitative autoradiographic study was undertaken to assess the possibility that IDPN may also affect 5-HTt receptors in rat brain. IDPN caused significant increases of 5-[3H]HT binding in the oriens and pyramidal layers of the CA3 field of hippocampus. In contrast, there were significant decreases of 5-[3H]HT binding in the frontal and cingulate cortices, the olfactory tubercle, the ventromedial aspect of the caudate-putamen, the nucleus accumbens, the superior colliculus, and the lateral septal nuclei. These results provide further evidence for the involvement of the 5-HT system in the development of the IDPN-induced dyskinetic syndrome.
Chronic treatment with the neurotoxin iminodipropionitrile (IDPN) induced in rodents and in nonhuman primate a complex and persistent behavioral syndrome characterized by motor hyperactivity, sustained twisting lateral (laterocollis) and vertical (retrocollis) movements of the neck and increased startle response (see ref. 3 for review). These behavioral abnormalities are analogous to those induced by acute injections of serotonin (5-HT) agonists [12]. Because of the similarities between the IDPNand the 5-HT-induced syndromes, we have undertaken a series of studies in order to investigate the possible involvement of the 5-HT system in the behavior abnormal-
Correspondence.. S. Przedborski, Neurological Institute, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
52 ities observed in IDPN-treated rats [3]. Significant increases in 5-HT concentrations were found in the nucleus accumbens and in the caudate-putamen nucleus in IDPNtreated rats [5]. Homogenate [7] and autoradiographic [6] receptor binding experiments have revealed significant changes in 5-HT receptor subtype 2 (5-HT2) density in various brain regions in similarly treated animals. Although it was initially thought that the activation of the 5-HT2 receptors mediated most of the 5-HT-induced behavioral abnormalities [12, 19], recent evidence has implicated 5-HTj receptors in the manifestation of these behavioral changes [16, 22]. For example, the administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT), a potent 5-HTI agonist, elicited most of the 5-HT-induced manifestations such as motor hyperactivity, head weaving and forepaw treading [13, 22]. Similarly, in the IDPN model recent data also support the possible involvement of the 5-HTI receptors in some of the behavioral abnormalities [12]. Moreover, the administration of 8-OHDPAT to rat has been reported to inhibit the IDPN-induced dystonic movements of the neck and backward locomotion [9]. The following quantitative autoradiographic study was thus carried out in order to determine whether chronic treatment with IDPN would cause specific topographical changes in 5-[3H]HT-labeled 5-HT1 receptors in rat brain. Adult male Sprague-Dawley rats weighing 220-250 g at the beginning of the experiment were maintained on a 12-h light,lark cycle and were provided with food and water ad libitum. Rats were divided into two groups; one group received daily i.p. injections of IDPN (100 mg/kg) until the behavioral syndrome developed (5-7 days) and a second group received daily i.p. injections of saline vehicle (1 ml/kg). All rats treated with IDPN developed the abnormalities which persisted without any need for further injections. Four weeks after the last injection, 5 IDPN-treated and 5 control animals were killed by decapitation. This time period was chosen according to our previous behavior and biochemical studies [3]. The brains were rapidly removed, frozen on dry ice and stored at -70°C. Sections (12/~m) were cut at -20°C with a cryostat, thaw-mounted onto chrome alum/gelatin-subbed glass slides and stored at - 70'~C until use. The sections were labeled in vitro for receptor binding assay autoradiography according to the method described by Pazos and Palacios [17]. Sections were preincubated (25°C; 30 min) in 50 mM Tris-HCl (pH 7.6) containing 4 mM CaCI2 and 0.01% ascorbic acid before being incubated (25°C; 60 min) in the same buffer with 2 nM 5-[3H]HT (30 Ci/mmol, New England Nuclear). Non-specific binding was determined by incubating adjacent sections in the presence of I/~M unlabeled 5-HT and represented 5-10% of total binding. After incubation, the sections were washed in fresh ice-cold buffer for 5 min, dipped in ice-cold distilled water and dried rapidly with cold air. Dried sections were placed in an X-ray cassette with plastic standards ([3H]-micro-scales, Amersham) and were apposed to LKB 3H-Ultrafilm for 8 weeks. After the films were developed, the sections were stained with cresyl violet for histological reference. The developed autoradiographic films were quantified using a Loats PC-based computerized image analysis system (Amersham). The film optical densities were converted to fmol of 5-[3H]HT bound per mg tissue using a standard curve generated by the [3HI-micro-scales. Statistical differences between the
53
TABLE I E F F E C T O F C H R O N I C A D M I N I S T R A T I O N OF I D P N O N T H E B I N D I N G O F 2 n M 5-[3H]HT TO 5-HTI R E C E P T O R S IN T H E R A T B R A I N Brain regions
Cerebral cortex Prefrontal Frontal, layersI-III layers IV-VI Parietal Temporal Cingulate Entorhinal Olfactory tubercle Caudate-putamen Dorsolateral Ventromedial Nucleus Accumbens Globus Pallidus Claustrum Hippocampus Dentate C y r u s CA4 (hilus) CAI CA2 CA3, oriens layer pyramidal layer molecular layer Subiculum Interpeduncular nucleus Substantia nigra Pars compacta Pars reticulata Pars lateralis Ventral tegmental area Centralgrey Superior colliculus Septal nucleus Dorsal lateral Ventral lateral
Specific binding (fmol/mg tissue + S.E.M.)
Control
IDPN-treated
P-values
225.66__+ 9.88 154.50__+ 7.40 233.55+ 7.13 149.50+ 7.90 190.75+ 8.31 284.13 + 8.66 286.87 ___i 1.36 389.15 ___24.31
228.75___ 8.16 154.00+ 6.54 212.50+ 4.76 149.83+ 7.23 192.13__+ 10.97 246.00___ 9.88 294.00 + 12.82 277.50 + 20.45
n.s. n.s. 0.045 n.s. n.s. 0.0085 n.s. 0.0012
189.60+ 6.46 204.25+ 4.21 311.00__+ 15.50 499.25 ___13.69 307.83 -+ 10.68
176.66+ 4.07 178.00_+ 3.79 269.00+ 13.09 472.50___ 10.23 299.50___ 8.25
n.s. 0.019 0.045 n.s. n.s.
576.13 + 15.07 399.62 -+ 23.48 376.50+ 8.36 415.40___ 11.45 403.16-+27.59 201.25 __+12.79 586.50___ 23.93 494.38 + I 1.78 415.00+ 9.50
535.25 __+19.51 362.50-+ 21.64 396.00+__ 19.58 406.13___ 11.58 571.10+25.88 281.86 __+13.46 592.83 + 26.31 467.25-+ 14.67 424.00+10.71
n.s. n.s. n.s. n.s. 0.0013 0.0036 n.s. n.s. n.s.
189.00 ___I 1.62 594.75 ___12.73 276.35+ 15.93 179.50+ 5.88 320.66+ 9.26 339.63 _+ 10.12
201.83 ___13.31 588.63 + 11.34 237.25 + 18.52 161.13+ 8.24 301.60+ 6.06 276.13 + 11.78
n.s. n.s. n.s. n.s. n.s. 0.012
580.17 + 16.05 475.80__+ 21.69
483.33 ___21.45 336.33 + 11.96
0.003 0.002
The values represent the means + the standard error of the mean (SEM) of 5-[3H]HT bound for 5 control (saline-treated) and 5 IDPN-treated rats. Non-specific binding was defined by 1 g M unlabeled 5-HT and was subtracted from all readings.
54
55 two groups were determined with Student's t-test. The null hypothesis was rejected at the 0.05 level. The distribution of 5-[3H]HT binding was comparable to what has been previously reported [17] and is summarized.in Table I. Chronic injections of I D P N were associated with significant increases of 5-[3H]HT binding in the oriens (+41%) and in the pyramidal ( + 40 %) layers of the CA3 field of hippocampus (Table I). In contrast, as figure 1 shows, there were significant decreases in 5-[3H]HT binding in layers IVVI of the frontal cortex ( - 10%), the cingulate cortex ( - 14%), the olfactory tubercle ( - 29 %), the ventromedial part of the caudate-putamen nucleus ( - 13 %), the nucleus accumbens ( - 14%), the superior colliculus ( - 19%) and in the dorsal lateral ( - 17%) and ventral lateral ( - 29 %) septal nuclei (see Table I for specific values of binding). Since biochemical studies have indicated that 5-[3H]HT binds to 5-HT1 receptors [17, 18], the present quantitative autoradiographic study indicates that chronic administration of IDPN causes significant changes in 5-HT1 receptors in several discrete regions of the brain of treated rats. The decreases in cortical 5-[3H]HT binding in the brain of IDPN-treated rats are in contrast to the increases previously observed in both 5-HTIA [20] and 5-HT2 [6, 7] receptors in similarly treated animals. Since the majority of 5-[3H]HT-labeled sites in rat frontal cortex are of 5-HTIB subtype [17], it is likely that the decreases in cortical 5-[3H]HT binding reflect changes in 5-HT1B receptors which are thought to be located on presynaptic terminals [18]. This idea is supported by the finding of significant decreases in cortical 5-HT concentration after chronic administration of IDPN [15]. These observations raise the possibility that chronic treatment with IDPN may result in loss of cortical 5-HT nerve terminals. Immunohistochemical studies may help to test that assumption. Since both the binding of [125I]lysergic acid diethylamide-labeled 5-HT2 [6] and 5[3H]HT-labeled 5-HTI receptors are significantly decreased in the nucleus accumbens and the ventromedial part of the striatum in IDPN-treated rats, it is likely that 5-HTz and 5-HTI receptors are regulated in a similar fashion in these regions of rat brain. It is also possible that the changes observed in those receptors are related to compensatory down-regulation secondary to the sustained increase of 5-HT concentration found in the striatum and the nucleus accumbens of similarly treated animals [5]. From a behavioral perspective, it is likely that these changes play a role in the manifestation in the IDPN-induced syndrome since these brain regions have been implicated in motor phenomena such as increased locomotion [8] and circling behavior [21] which are prominent aspects of the IDPN-induced abnormalities. Although it
Fig. 1. Representativedark-field transform autoradiograms illustrating the effectof chronic administration of IDPN on 5-13H]HTbinding to 5-HT-1 receptors in rat brain. 5-[3H]HTbinding in hemicoronal sections of saline-treated rats (A-C) and IDPN-treated rats (D-F). Autoradiograms were generated as described in the text. Fr, frontal cortex; Cg, cingulatecortex; CPu, caudate-putamennucleus;Acb, nucleus accumbens; OTu, olfactory tubercle; LS, lateral septal nucleus; HIP, hippocampus; SuG, superficialgrey layer of the superior colliculus; CG, central grey; SN, substantia nigra; IP, interpeduncularnucleus. Bar = 140/tin.
56 remains to be determined how I D P N might have led to these abnormalities in the rat brain, the decreases in 5-[3H]HT binding in the superior colliculus might also be related to some aspects of the behavioral syndrome. This idea is supported by neuroanatomical studies which have identified projections from the substantia nigra pars reticulata to the superior collicutus [1, 1 1] which, in turn, sends efferents to cell bodies in the brainstem [10]. This interpretation is also supported by 2-deoxy-D-[l-lnC]glucose studies which have revealed abnormalities in glucose utilization in the superior colliculus of IDPN-treated animals [4] and in a genetic model of dystonia (rat mutant dystonic) [2]. I D P N caused increases in 5-[3H]HT binding in the oriens and pyramidal layers of the CA3 field of the hypocampus. These changes probably reflect increases in 5-HT-IB receptors since we reported previously decreases in 8-OH[3H]DPAT-Iabeled 5-HTIA receptors in these regions [20]. The overexcitability observed in I D P N treated animals [3] may be related to these changes since lesion of the septo-hippocampal axis is associated with a similar behavioral pattern [14]. In summary, the present study provides further evidence for the involvement of the 5-HT system in the IDPN-induced behavioral syndrome. Most of the changes observed in 5-HTI and 5-HT2 receptors can be explained by reciprocal alterations in receptor numbers in response to sustained increases in 5-HT levels in the same regions of the brain. The alternative explanation that some of those changes may be due to neuronal death is not plausible in view of the absence of any neuropathological studies which have been able to document cell death in these brain regions (reviewed in ref. 3). These studies also provide evidence for possible dysfunction in the septo-hippocampal system in IDPN-treated rats. Serge Przedborski is Research Assistant for the National Fund for Scientific Research (Belgium) and is supported, as well as Mia Wright, by a Fellowship from the Parkinson's disease Foundation. 1 Beckstead,R.M., Domesick,V.B. and Nauta, W.J.H., Efferentconnections of the substantia nigra and ventral tegmental area in the rat, Brain Res., 175 (1979) 191-217. 2 Brown, L.L. and Lorden, J.F., Regional cerebral glucoseutilization reveals widespread abnormalities in the motor system of the rat mutant dystonic, J. Neurosci., 9 (1989) 40334041. 3 Cadet, J.L., The iminodipropionitrile (IDPN)-induceddyskinetic syndrome:behavioral and biochemical pharmacology,Neurosci. Biobehav. Rev., 13 (1989) 3945. 4 Cadet, J.L., Della Puppa, A. and London, E., Involvementof nigrotecto-reticulospinal pathways in the iminodipropionitrile (IDPN) model of spasmodic dyskinesias: a 2-deoxy-o[l-14C]glucosestudy in the rat, Brain Res., 484 (1989) 57~64. 5 Cadet, J.L., Jackson-Lewis,V. and Fahn, S., The iminodipropionitrile (IDPN)-induceddyskinetic syndrome: regional serotonin metabolism in rat brain, Brain Res., 456 (1988) 371-374. 6 Cadet, J.L., Kuyatt, B., Fahn, S. and De Souza, E.B., Differential changes in ~25I-LSD-labeled5-HT-2 serotonin receptors in discrete regions of brain in the rat model of persistent dyskinesias induced by iminodipropionitrile (IDPN): evidencefrom autoradiographic studies, Brain Res., 437 (1987) 383-386. 7 Cadet, J.L. and Rothman, R.B., Increased cortical serotonin-2 (5-HT-2) receptors in the iminodipropionitrile (IDPN)-modelof persistent dyskinesia in the rat, Neuropeptides, 10 (1987) 175 180. 8 Cole, S.O., Brain mechanismsof amphetamine-induced anorexia, locomotionand stereotypy:a review, Neurosci. Biobehav. Rev., 2 (1978) 89-100. 9 Diamond, B.I., Sethi, K. and Borison, R.L., Serotonin modulation of hyperkinesia and phasic neck
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