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Intracerebroventricular administration of substance P increases dopamine content in the brain of 6-hydroxydopamine-lesioned rats
113 Substance P increases dopamine content in 6-OHDA-lesioned brain Neuroscience Vol. 95, No. 1, pp. 113–117, 2000
Pergamon PII: S0306-4522(99)00400-5
Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
INTRACEREBROVENTRICULAR ADMINISTRATION OF SUBSTANCE P INCREASES DOPAMINE CONTENT IN THE BRAIN OF 6-HYDROXYDOPAMINELESIONED RATS I. N. KRASNOVA,*† E. R. BYCHKOV,* V. I. LIOUDYNO,‡ O. E. ZUBAREVA‡ and S. A. DAMBINOVA* *Laboratory of Molecular Neurobiology, Institute of the Human Brain, Russian Academy of Sciences, 9 Acad. Pavlov Street, St Petersburg 197376, Russia ‡Pavlov Department of Physiology, Institute for Experimental Medicine, Russian Academy of Medical Sciences, 12 Acad. Pavlov Street, St Petersburg 197376, Russia
Abstract—The interactions existing between substance P- and dopamine-positive neurons, notably in the basal ganglia, suggest that substance P may have therapeutic use in treatment of Parkinson’s disease characterized by impaired dopaminergic transmission. The effects of intracerebroventricularly administered substance P were tested on the levels of dopamine and its metabolites in the striatum, nucleus accumbens and frontal cortex of 6-hydroxydopamine-lesioned rats. Intracerebroventricular injection of 6hydroxydopamine decreased the levels of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the brain structures under investigation. Administration of substance P in low dose (0.35 nmol/kg) had no effect on the 6-hydroxydopamine-induced reduction of the dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid contents in the brain. However, treatment with substance P in higher dose (3.5 nmol/kg) increased the concentrations of dopamine and its metabolites in the striatum, nucleus accumbens and frontal cortex relative to saline-treated group. Additionally, 6-hydroxydopamine lesions significantly increased 3,4dihydroxyphenylacetic acid/dopamine and homovanillic acid/dopamine ratios in the striatum and nucleus accumbens. Substance P (3.5 nmol/kg) partially reversed lesion-induced increases in 3,4-dihydroxyphenylacetic acid/dopamine and homovanillic acid/ dopamine ratios in the striatum, but did not alter these ratios in nucleus accumbens. To test whether substance P fragmentation is responsible for this phenomenon, substance P5–11, which is one of the main substance P fragments in rat CNS, was administered in equimolar dose. Substance P5–11 was found to have no effect on the content of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the striatum and nucleus accumbens. In the frontal cortex, substance P5–11 produced decreases in dopamine levels and increases in homovanillic acid/dopamine ratio. The results of this study suggest that substance P helps to restore dopamine deficit in the brain in an animal model of Parkinson’s disease, with the positive effects being more prominent on the nigrostriatal than on the mesocorticolimbic dopaminergic system, but substance P5–11 is not responsible for this effect. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: dopamine, 6-hydroxydopamine, substance P, striatum, nucleus accumbens.
Parkinson’s disease is a slowly progressive movement disorder which presents clinically with tremor, rigidity and akinesia. This clinical picture is thought to result from the marked degeneration of dopaminergic neurons in the substantia nigra and, to a lesser extent, of those in the ventral tegmental area (VTA). This degeneration of dopamine (DA)-ergic neurons is associated with 50–70% decrease in tyrosine hydroxylase activity as was demonstrated in the VTA. 1,19 In addition, DA content has been found to be decreased in the neostriatum, the nucleus accumbens and frontal cortex of parkinsonian brains. 4,34,40 Since L-DOPA treatment was introduced in the 1960s, it has played a significant role in the treatment of patients with Parkinson’s disease. However, a large number of patients fail to maintain a good response to L-DOPA and become more symptomatic. Moreover, various problems attributable to long-term treatment with L-DOPA have been found; these
include, for example, “wearing-off” and “on-off” phenomena, dyskinesia, and psychotic symptoms. 2,30 More recently, it has been suggested that it might be more advantageous to use neuroprotective therapy to preserve the integrity and function of vulnerable neurons. This approach might help slow down the progressively disabling course of the disease. 15,23 One such approach might involve manipulation of the substance P (SP)-ergic system. For example, it has been reported that SP can enhance neural growth in vitro 18 and to counteract the effects of neurotoxins administered to animals. 29,39 Neurons containing SP show close anatomical interactions with the nigrostriatal and mesolimbic DA-ergic systems. 11,28,33,44 SP exerts a stimulatory influence on DA-ergic neurons: both peripheral injection of the neuropeptide in freely moving animals 8 or direct application to the substantia nigra or VTA 6,36 can increase DA levels in the striatum or nucleus accumbens. In numerous studies it was found that N- and Cterminal fragments of neuropeptide induced the same effects as SP. 7,21,38 It was also reported that DA-dependent decrease in SP-like immunoreactivity level was found in the substantia nigra, striatum and nucleus accumbens of post mortem parkinsonian brains. 3,14,42 Thus, the aim of the present study is to examine the possibility that intracerebroventricular administration of SP could restore the normal levels of DA in the brains in an animal model of Parkinson’s disease. We also tested
†To whom correspondence should be addressed at: Molecular Neuropsychiatry Section, Division of Intramural Research, NIH/NIDA, PO Box 5180, Baltimore, MD 21224, U.S.A. Tel.: 1 1-410-550-1512; fax: 1 1-410-550-2745. E-mail address: [email protected] (I. N. Krasnova) Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetra-acetate; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; NK, neurokinin; NMDA, N-methyl-d-aspartate; 6-OHDA, 6-hydroxydopamine; SP, substance P; VTA, ventral tegmental area. 113
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Table 1. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the striatum of 6-hydroxydopamine-lesioned rats Group
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
7.384 ^ 0.323 (7)
0.790 ^ 0.049 (7)
0.424 ^ 0.035 (7)
0.107 ^ 0.003 (7)
0.057 ^ 0.004 (7)
1.244 ^ 0.268* (6) 1.632 ^ 0.375 (6)
0.193 ^ 0.032* (6) 0.231 ^ 0.050 (6)
0.166 ^ 0.022* (6) 0.157 ^ 0.023 (6)
0.162 ^ 0.020* (6) 0.125 ^ 0.015 (6)
0.141 ^ 0.017* (6) 0.115 ^ 0.016 (6)
4.139 ^ 0.340† (4)
0.499 ^ 0.036† (4)
0.343 ^ 0.049† (4)
0.121 ^ 0.004† (4)
0.083 ^ 0.008† (4)
1.045 ^ 0.137 (6)
0.172 ^ 0.025 (6)
0.169 ^ 0.020 (6)
0.164 ^ 0.009 (6)
0.162 ^ 0.010 (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
whether the C-terminal fragment SP5–11 was responsible for this effect. EXPERIMENTAL PROCEDURES
Chemicals SP, SP5–11, DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), ascorbic acid, citric acid, sodium dihydrophosphate and sodium octylsulfate were purchased from Sigma Chemical Co.; perchloric acid and high-performance liquid chromatography (HPLC) grade methanol from Fluka, 6-hydroxydopamine (6OHDA) hydrochloride from Serva. Animals and 6-hydroxydopamine lesions Male Wistar rats weighing 220–250 g were maintained in a temperature- (20 ^ 28C) and light- (lights on between 0800 and 2000) controlled environment. Food and water were available ad libitum. All animal use procedures were according to the Russian Academy of Sciences Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Rats were anesthetized with hexenal (200 mg/kg) and positioned in a stereotaxic frame. A stainless steel guide cannula was implanted at the following coordinates: 32 posterior 0.6 mm, lateral ^1.2 mm, ventral 23.5 mm and anchored to the skull with dental cement. 6-OHDA was dissolved in 0.15 M NaCl solution containing 0.02% ascorbic acid and injected intracerebroventricularly in a dose of 1.5 mg/kg 15 days prior to the experiment. Control animals were injected with saline. The 6-OHDA solutions were prepared fresh immediately prior to injection in order to minimize oxidation. Solutions were administered at a rate of 2 ml per min, after each injection needles were kept in place for 2 min to allow for complete diffusion. The animals were given intraperitoneal injections of desipramine (25 mg/kg) 30 min prior to 6-OHDA to prevent the uptake of the neurotoxin into noradrenergic nerve terminals or saline infusions in the treated and control rats, respectively. Experimental protocol On the day of the experiment groups of animals received the following treatment: (i) unlesioned controls, treated with saline; (ii) 6OHDA-lesioned controls, treated with saline; (iii) 6-OHDA-lesioned rats, treated with SP, 0.35 nmol/kg; (iv) 6-OHDA-lesioned rats, treated with SP, 3.5 nmol/kg and (v) 6-OHDA-lesioned rats, treated with SP5–11, 3.5 nmol/kg. SP and SP5–11 were dissolved in saline and injected in a volume of 10 ml with a 10-ml Hamilton microsyringe connected to a 30-gauge injector which protruded 1 mm beyond the tip of the cannula guide and was placed into the lateral ventricle. Thirty minutes later animals were decapitated with guillotine, the brains were rapidly removed from the skulls and placed on an icecooled glass plate. Striatum, nucleus accumbens and frontal cortex were dissected from the brain. Tissues were weighed, frozen in liquid nitrogen and stored at 2708C until extraction. The tissues obtained from each animal were homogenized in 0.1 M perchloric acid and centrifuged at 25,000 g for 10 min.
Determination of the concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid DA, DOPAC and HVA contents in supernatants of the brain structures were measured by HPLC with electrochemical detection. 24 The analytical column was Partisil 5 ODS 3 (5 mkm, 4.6 × 250.0 mm) from Whatman Chemical Separation. The mobile phase was 0.01 M sodium dihydrogenphosphate, 0.01 M citric acid, 2 mM sodium EDTA, 1 mM sodium octylsulfate, 10% methanol, pH 3.5 at flow rate 1.0 ml/min and temperature 258C. The installation consisted of 305 Gilson piston pump, 805 Gilson manometric module and Bioanalytical Systems LC-4B amperometric detector. The glassy carbon electrode was used at a potential of 0.75 V. Peak areas and sample concentrations were calculated with a Gilson 714 chromatography data system. Contents of DA, DOPAC and HVA were calculated as ng/mg of tissue weight. The ratios DOPAC/DA and HVA/DA were used as indices of transmitter metabolism. Statistical analysis Statistical analysis was performed by means of analysis of variance followed by a two-sided Student’s t-test. Differences were considered significant if the probability of error was less than 5%. All results were expressed as the mean ^ S.E.M., and the sample size n represents the number of individual samples analysed. RESULTS
Effects of 6-hydroxydopamine, substance P and substance P5–11 in the striatum The changes in the concentrations of DA and its metabolites in striatum of experimental groups of animals are presented in Table 1. A significant depletion of DA to 17%, DOPAC to 24% and HVA to 39% was found in the 6-OHDA-lesioned rats compared to the sham-operated group injected with saline. The experiment showed that administration of SP in low dose (0.35 nmol/kg) had no significant effect, while injection of SP in higher dose (3.5 nmol/kg) caused a large increase in the striatal DA, DOPAC and HVA levels. The concentration of DA was 3.3 times higher (DOPAC, 2.6 times; HVA, 2.1 times) than those in 6-OHDA-treated animals, which received saline injections. C-terminal fragment SP5–11 (3.5 nmol/kg) produced no effect on concentrations of substances under investigation. The turnover of DA was assessed by calculating the ratios of DOPAC/DA and HVA/DA. These ratios were significantly increased by 6-OHDA lesion in the striatum (Table 1). SP (0.35 nmol/kg) and SP5–11 (3.5 nmol/kg) had no effect, but SP (3.5 nmol/kg) decreased DOPAC/DA and HVA/DA ratios in the striatum by 25% and 41%, respectively, relative to 6-OHDA-lesioned subjects.
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Substance P increases dopamine content in 6-OHDA-lesioned brain
Table 2. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the nucleus accumbens of 6-hydroxydopamine-lesioned rats Group Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
6.060 ^ 0.257 (7)
0.838 ^ 0.040 (7)
0.352 ^ 0.019 (7)
0.139 ^ 0.007 (7)
0.058 ^ 0.003 (7)
1.538 ^ 0.233* (6)
0.262 ^ 0.032* (6)
0.139 ^ 0.023* (6)
0.174 ^ 0.010* (6)
0.091 ^ 0.010* (6)
1.884 ^ 0.390 (6)
0.290 ^ 0.048 (6)
0.162 ^ 0.017 (6)
0.167 ^ 0.010 (6)
0.095 ^ 0.011 (6)
4.378 ^ 0.475† (4)
0.872 ^ 0.267† (4)
0.332 ^ 0.054† (4)
0.195 ^ 0.051 (4)
0.070 ^ 0.010 (4)
1.245 ^ 0.138 (6)
0.204 ^ 0.026 (6)
0.135 ^ 0.015 (6)
0.162 ^ 0.005 (6)
0.110 ^ 0.006† (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
Table 3. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the frontal cortex of 6-hydroxydopamine-lesioned rats Group Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
0.166 ^ 0.028 (7)
0.034 ^ 0.003 (7)
0.032 ^ 0.005 (7)
0.231 ^ 0.029 (7)
0.218 ^ 0.035 (7)
0.065 ^ 0.004* (6)
0.014 ^ 0.001* (6)
0.019 ^ 0.002* (6)
0.217 ^ 0.020 (6)
0.284 ^ 0.032 (6)
0.076 ^ 0.015 (6)
0.016 ^ 0.003 (6)
0.018 ^ 0.002 (6)
0.229 ^ 0.022 (6)
0.267 ^ 0.049 (6)
0.156 ^ 0.061† (4)
0.029 ^ 0.008† (4)
0.034 ^ 0.007† (4)
0.209 ^ 0.023 (4)
0.270 ^ 0.068 (4)
0.043 ^ 0.08† (6)
0.011 ^ 0.002 (6)
0.016 ^ 0.003 (6)
0.254 ^ 0.014 (6)
0.426 ^ 0.071† (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
Effects of 6-hydroxydopamine, substance P and substance P5–11 in the nucleus accumbens and frontal cortex 6-OHDA produced a significant decrease in the concentrations of DA, DOPAC and HVA in the nucleus accumbens compared to the sham-operated rats (Table 2). Injection of SP at 0.35 nmol/kg didn’t change the 6-OHDA-induced reduction of DA and its metabolite contents in the nucleus accumbens, however SP at 3.5 nmol/kg significantly increased contents of DA, DOPAC and HVA. SP5–11 (3.5 nmol/kg) had no effect on the DA and its metabolite contents compared to the saline-treated group. DOPAC/DA and HVA/DA ratios were significantly increased by 6-OHDA lesion in the nucleus accumbens (Table 2). In these ratios, no differences were observed between saline- or SP-treated (0.35 and 3.5 nmol/kg) groups. SP5–11 (3.5 nmol/kg) caused an increase in HVA/DA ratio versus the saline-treated group. In the frontal cortex 6-OHDA caused a significant reduction of DA, DOPAC and HVA concentrations compared to sham-operated rats injected with saline (Table 3). In 6OHDA-treated rats there were no significant differences between saline- and SP-treated in low dose (0.35 nmol/kg) animals. However, SP at 3.5 nmol/kg significantly increased the contents of DA, DOPAC and HVA. Administration of SP5–11 (3.5 nmol/kg) caused a decrease in DA concentration relative to 6-OHDA-treated subjects, but had no effect on DOPAC and HVA contents. 6-OHDA had no effect on the DOPAC/DA and HVA/DA ratios in the frontal cortex (Table 3). Injections of SP (0.35 and 3.5 nmol/kg) produced no significant changes in DOPAC/DA and HVA/DA ratios as
compared to those in saline-treated 6-OHDA animals, SP5–11 (3.5 nmol/kg) caused increase in HVA/DA ratio in the frontal cortex. DISCUSSION
The objective of the present study was to investigate the effects of central administration of SP and the carboxy terminal sequence SP5–11 on DA, DOPAC and HVA content in the striatum, nucleus accumbens and frontal cortex of 6-OHDA-lesioned rats. Administration of 6-OHDA in our study resulted in a marked reduction in the DA, DOPAC and HVA contents in the striatum, nucleus accumbens and frontal cortex. The most significant destruction of the DA transmission was registered in the striatum and nucleus accumbens. These decreases are consistent with previously published data. 12,31 In addition, there were significant increases in DOPAC/DA and HVA/ DA ratios in the striatum and nucleus accumbens. This is probably related to compensatory increase of functional activity of DA-ergic system. 10,46 Administration of low dose SP (0.35 nmol/kg) had no effect, but SP at a higher dose (3.5 nmol/kg) caused significant increases in the concentrations of DA, DOPAC and HVA in the striatum, nucleus accumbens and frontal cortex of 6-OHDA-treated rats. These results indicate that SP has exerted a significant reversal of the total effects of 6-OHDA in these brain regions. Interestingly, in the striatum the DOPAC/DA and HVA/DA ratios were less increased after SP. It seems that a lesser degree of DA depletion might
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have made a compensatory increase in DA-ergic activity unnecessary. These data demonstrate that SP at the dose used here had the most significant effect on the terminals of nigrostriatal projections with lesser effects on mesocorticolimbic system. Previous work has shown that administration of SP or stable analogs in similar doses in substantia nigra or VTA to freely moving rats increased DA levels in the striatum and nucleus accumbens, respectively. 6,36 Intraperitoneal injection of the neuropeptide also increased DA release in nucleus accumbens and striatum. 8 The time of decapitation chosen in our experiment (30 min after SP administration) corresponds to the time of significant increase of extracellular DA in the striatum after intranigral injection of neuropeptide to freely moving rats 36 and maximal movement recovery in open field test of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rats after intranasal inhalation of SP. 25 The mechanisms underlying the stimulatory effects of SP on DA-ergic neurons are still under investigation. SP shows highest affinity for neurokinin 1 (NK1) receptors, although the naturally occurring tachykinins have a high affinity for, and act as full agonists on, the three receptor types: NK1, NK2 and NK3. 35 Therefore, the type of the receptors involved in the effect observed in the striatum and nucleus accumbens may differ. Previous works have shown that SP may increase the release of DA in the striatum by activation of tachykinin NK1 receptors, 21,36,45 NK3 receptors 26 or NK1 and NK3 receptors. 16 Also, it was demonstrated that both NK1 and NK3 receptors in the striatum are postsynaptic with respect to DA-ergic neurons, whereas nigral NK3 receptors are located on dopaminergic neurons. 43 It was concluded that there were direct NK3-mediated interactions between SP and DA-ergic neurons in the substantia nigra, but the DA-ergic effect of SP within the striatum was indirect and may be modulated through cholinergic link. 9,22 It seems that the latter way plays a more important role in DA-denervated nigrostriatal system. In addition to its effects on DA release, SP may also cause an increase in DA synthesis. This could occur through an indirect effect of SP on the release of glutamate which has been shown to increase tyrosine hydroxylase activity via stimulation of N-methyl-d-aspartate (NMDA) receptors 5 which are found on striatal DA-ergic terminals. 20 This idea is supported by the observation that SP can cause an increase in glutamate release in the striatum. 37 The recent demonstration that administration of NMDA, an agonist at NMDA-type
glutamate receptors, can increase DA synthesis in the striatum 47 also supports this contention. Although it has been shown that tachykinins excite mesocorticolimbic DA-ergic neurons, the identity of the receptor subtypes that regulate the excitability of these neurons remains to be elucidated. Some studies have implicated predominantly NK1 tachykinin receptors, 13 whereas others have suggested that NK3 26 but not NK1 or NK2 receptors 41 mediate the principal excitatory influence of SP. To our knowledge, there are, at present, no reports on the regulation of DA synthesis or tyrosine hydroxylase activity within the mesocorticolimbic system itself, however, it is very likely that SP might exert effects similar to those reported for the striatum, as discussed above. The enzymatic conversion into fragments with agonistic or antagonistic properties has been proposed to be a general principle in the regulation and expression of SP action. 17,38 In our study no effect was observed with C-terminal heptapeptide SP5–11 in the striatum and nucleus accumbens, whereas SP in equimolar dose increased DA, DOPAC and HVA content. In the frontal cortex a decrease in the DA content was registered with subsequent increase in HVA/ DA ratio versus 6-OHDA-lesioned control, who received saline injections. Boix and co-workers 7,8 found that C-terminal analog of SP, [pGlu 5, MePhe 8, Sar 9]SP5–11, and SP1–7 do not change the DA release in the striatum, but SP1–11 increases DA release in this structure. If this effect of SP on striatal DA is related to its facilitatory action on recovery after unilateral nigrostriatal lesions, 27 we may suppose on the basis of the previous studies and present results, that a full molecule is necessary to obtain such an effect; neither C- nor the N-terminal fragments would facilitate recovery. CONCLUSIONS
The results of the present experiment suggest that SP can increase DA levels in the DA depleted brain by activation of nigrostriatal and mesocorticolimbic DA-ergic neurons. These data suggest that SP analogs might be of potential use in the treatment armamentorium against Parkinson’s disease. Acknowledgements—The authors are very grateful to Dr J. L. Cadet for his continuous support and editorial comments. Also we would like to thank Ms Y. V. Tcherkas for her kind help in preparation of manuscript.
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(1997) Pretreatment with neurokinin substance P but not with cholecystokinin-8S can alleviate functional deficit of partial nigrostriatal 6-hydroxydopamine lesion. Peptides 18, 1161–1168. 30. Ogawa N. (1994) Levodopa and dopamine agonists in the treatment of Parkinson’s disease: advantages and disadvantages. Eur. Neurol. Suppl. 3, 20–28. 31. Onn S. P., Berger T. W., Stricker E. M. and Zigmond M. J. (1986) Effects of the intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: histochemical and neurochemical analysis. Brain Res. 376, 8–19. 32. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. 2nd edn. Academic, Florida. 33. Pickel V. M., Jon T. H. and Chan J. (1988) Substance P in the rat nucleus accumbens: ultrastructural localization in axon terminals and their relationship to dopaminergic afferents. Brain Res. 444, 247–264. 34. Price K. S., Farley I. J. and Hornykiewicz O. (1978) Neurochemistry of Parkinson’s disease: relation between striatal and limbic dopamine. In Advances in Biochemical Psychopharmacology (eds Riberts P. J. and Woodruff G. N.), Vol. 19, pp. 293–300. Raven, New York. 35. Regoli D., Boudon A. and Fauchere J.-L. (1994) Receptors and antagonists for substance P and related peptides. Pharmac. Rev. 46, 551–599. 36. Reid M. S., Herrera-Marschtz M., Hokfelt T., Ohlin M., Valentino K. L. and Ungerstedt U. (1990) Effects of intranigral substance P and neurokinin A on striatal dopamine release. Interactions with substance P antagonists. Neuroscience 36, 643–658. 37. Reid M. S., Herrera-Marschtz M., Kehr J. and Ungerstedt U. (1990) Striatal dopamine and glutamate release: effects of intranigral injections of substance P. Acta physiol. scand. 140, 527–537. 38. Reid M. S., Herrera-Marschtz M., Terenius L. and Ungerstedt U. (1990) Intranigral substance P modulation of striatal dopamine: interaction with N-terminal and C-terminal substance P fragments. Brain Res. 526, 228–234. 39. Sanberg P. R., Emerich D. F., Aebischer P., Amisetti S. M., Ouellette W., Koutouzis T. K., Cahill D. W. and Norman A. B. (1993) Substance P containing polymer implants protect against striatal excitotoxicity. Brain Res. 628, 327–329. 40. Scatton B., Javoy-Agid F., Rouquier L. and Agid Y. (1983) Reduction of cortical dopamine, noradrenaline, serotonon and their metabolites in Parkinson’s disease. Brain Res. 275, 321–328. 41. Seabrook G. R., Bowery B. J. and Hill R. G. (1995) Pharmacology of tachykinin receptors on neurones in the ventral tegmental area of rat brain slices. Eur. J. Pharmac. 273, 113–119. 42. Sivam S. P. (1991) Dopamine dependent decrease in enkephalin and substance P levels in basal ganglia regions of postmortem parkinsonian brains. Neuropeptides 18, 201–207. 43. Stoessl A. J. (1994) Localisation of striatal and nigral tachykinin receptors in the rat. Brain Res. 646, 13–18. 44. Tamiya R., Hanada M., Kawai Y., Inagaki S. and Takagi H. (1990) Substance P afferents have synaptic contacts with dopaminergic neurons in the ventral tegmental area of the rat. Neurosci. Lett. 110, 11–15. 45. Tang F. I., Chiu T. H. and Wang Y. (1998) Electrochemical studies of the effects of substance P on dopamine terminals in the rat striatum. Expl Neurol. 152, 41–49. 46. Zigmond M. J., Abercombie E. D., Grace A. A. and Stricker E. M. (1990) Compensations after lesions of central dopaminergic neurons: some clinical and basic implications. Trends Neurosci. 13, 290–296. 47. Zigmond M. J., Castro S. L., Keefe K. A., Abercombie E. D. and Sved A. F. (1998) Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum. Amino Acids 14, 57–62. (Accepted 26 July 1999)
Pergamon PII: S0306-4522(99)00400-5
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INTRACEREBROVENTRICULAR ADMINISTRATION OF SUBSTANCE P INCREASES DOPAMINE CONTENT IN THE BRAIN OF 6-HYDROXYDOPAMINELESIONED RATS I. N. KRASNOVA,*† E. R. BYCHKOV,* V. I. LIOUDYNO,‡ O. E. ZUBAREVA‡ and S. A. DAMBINOVA* *Laboratory of Molecular Neurobiology, Institute of the Human Brain, Russian Academy of Sciences, 9 Acad. Pavlov Street, St Petersburg 197376, Russia ‡Pavlov Department of Physiology, Institute for Experimental Medicine, Russian Academy of Medical Sciences, 12 Acad. Pavlov Street, St Petersburg 197376, Russia
Abstract—The interactions existing between substance P- and dopamine-positive neurons, notably in the basal ganglia, suggest that substance P may have therapeutic use in treatment of Parkinson’s disease characterized by impaired dopaminergic transmission. The effects of intracerebroventricularly administered substance P were tested on the levels of dopamine and its metabolites in the striatum, nucleus accumbens and frontal cortex of 6-hydroxydopamine-lesioned rats. Intracerebroventricular injection of 6hydroxydopamine decreased the levels of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the brain structures under investigation. Administration of substance P in low dose (0.35 nmol/kg) had no effect on the 6-hydroxydopamine-induced reduction of the dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid contents in the brain. However, treatment with substance P in higher dose (3.5 nmol/kg) increased the concentrations of dopamine and its metabolites in the striatum, nucleus accumbens and frontal cortex relative to saline-treated group. Additionally, 6-hydroxydopamine lesions significantly increased 3,4dihydroxyphenylacetic acid/dopamine and homovanillic acid/dopamine ratios in the striatum and nucleus accumbens. Substance P (3.5 nmol/kg) partially reversed lesion-induced increases in 3,4-dihydroxyphenylacetic acid/dopamine and homovanillic acid/ dopamine ratios in the striatum, but did not alter these ratios in nucleus accumbens. To test whether substance P fragmentation is responsible for this phenomenon, substance P5–11, which is one of the main substance P fragments in rat CNS, was administered in equimolar dose. Substance P5–11 was found to have no effect on the content of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the striatum and nucleus accumbens. In the frontal cortex, substance P5–11 produced decreases in dopamine levels and increases in homovanillic acid/dopamine ratio. The results of this study suggest that substance P helps to restore dopamine deficit in the brain in an animal model of Parkinson’s disease, with the positive effects being more prominent on the nigrostriatal than on the mesocorticolimbic dopaminergic system, but substance P5–11 is not responsible for this effect. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: dopamine, 6-hydroxydopamine, substance P, striatum, nucleus accumbens.
Parkinson’s disease is a slowly progressive movement disorder which presents clinically with tremor, rigidity and akinesia. This clinical picture is thought to result from the marked degeneration of dopaminergic neurons in the substantia nigra and, to a lesser extent, of those in the ventral tegmental area (VTA). This degeneration of dopamine (DA)-ergic neurons is associated with 50–70% decrease in tyrosine hydroxylase activity as was demonstrated in the VTA. 1,19 In addition, DA content has been found to be decreased in the neostriatum, the nucleus accumbens and frontal cortex of parkinsonian brains. 4,34,40 Since L-DOPA treatment was introduced in the 1960s, it has played a significant role in the treatment of patients with Parkinson’s disease. However, a large number of patients fail to maintain a good response to L-DOPA and become more symptomatic. Moreover, various problems attributable to long-term treatment with L-DOPA have been found; these
include, for example, “wearing-off” and “on-off” phenomena, dyskinesia, and psychotic symptoms. 2,30 More recently, it has been suggested that it might be more advantageous to use neuroprotective therapy to preserve the integrity and function of vulnerable neurons. This approach might help slow down the progressively disabling course of the disease. 15,23 One such approach might involve manipulation of the substance P (SP)-ergic system. For example, it has been reported that SP can enhance neural growth in vitro 18 and to counteract the effects of neurotoxins administered to animals. 29,39 Neurons containing SP show close anatomical interactions with the nigrostriatal and mesolimbic DA-ergic systems. 11,28,33,44 SP exerts a stimulatory influence on DA-ergic neurons: both peripheral injection of the neuropeptide in freely moving animals 8 or direct application to the substantia nigra or VTA 6,36 can increase DA levels in the striatum or nucleus accumbens. In numerous studies it was found that N- and Cterminal fragments of neuropeptide induced the same effects as SP. 7,21,38 It was also reported that DA-dependent decrease in SP-like immunoreactivity level was found in the substantia nigra, striatum and nucleus accumbens of post mortem parkinsonian brains. 3,14,42 Thus, the aim of the present study is to examine the possibility that intracerebroventricular administration of SP could restore the normal levels of DA in the brains in an animal model of Parkinson’s disease. We also tested
†To whom correspondence should be addressed at: Molecular Neuropsychiatry Section, Division of Intramural Research, NIH/NIDA, PO Box 5180, Baltimore, MD 21224, U.S.A. Tel.: 1 1-410-550-1512; fax: 1 1-410-550-2745. E-mail address: [email protected] (I. N. Krasnova) Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetra-acetate; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; NK, neurokinin; NMDA, N-methyl-d-aspartate; 6-OHDA, 6-hydroxydopamine; SP, substance P; VTA, ventral tegmental area. 113
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Table 1. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the striatum of 6-hydroxydopamine-lesioned rats Group
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
7.384 ^ 0.323 (7)
0.790 ^ 0.049 (7)
0.424 ^ 0.035 (7)
0.107 ^ 0.003 (7)
0.057 ^ 0.004 (7)
1.244 ^ 0.268* (6) 1.632 ^ 0.375 (6)
0.193 ^ 0.032* (6) 0.231 ^ 0.050 (6)
0.166 ^ 0.022* (6) 0.157 ^ 0.023 (6)
0.162 ^ 0.020* (6) 0.125 ^ 0.015 (6)
0.141 ^ 0.017* (6) 0.115 ^ 0.016 (6)
4.139 ^ 0.340† (4)
0.499 ^ 0.036† (4)
0.343 ^ 0.049† (4)
0.121 ^ 0.004† (4)
0.083 ^ 0.008† (4)
1.045 ^ 0.137 (6)
0.172 ^ 0.025 (6)
0.169 ^ 0.020 (6)
0.164 ^ 0.009 (6)
0.162 ^ 0.010 (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
whether the C-terminal fragment SP5–11 was responsible for this effect. EXPERIMENTAL PROCEDURES
Chemicals SP, SP5–11, DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), ascorbic acid, citric acid, sodium dihydrophosphate and sodium octylsulfate were purchased from Sigma Chemical Co.; perchloric acid and high-performance liquid chromatography (HPLC) grade methanol from Fluka, 6-hydroxydopamine (6OHDA) hydrochloride from Serva. Animals and 6-hydroxydopamine lesions Male Wistar rats weighing 220–250 g were maintained in a temperature- (20 ^ 28C) and light- (lights on between 0800 and 2000) controlled environment. Food and water were available ad libitum. All animal use procedures were according to the Russian Academy of Sciences Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Rats were anesthetized with hexenal (200 mg/kg) and positioned in a stereotaxic frame. A stainless steel guide cannula was implanted at the following coordinates: 32 posterior 0.6 mm, lateral ^1.2 mm, ventral 23.5 mm and anchored to the skull with dental cement. 6-OHDA was dissolved in 0.15 M NaCl solution containing 0.02% ascorbic acid and injected intracerebroventricularly in a dose of 1.5 mg/kg 15 days prior to the experiment. Control animals were injected with saline. The 6-OHDA solutions were prepared fresh immediately prior to injection in order to minimize oxidation. Solutions were administered at a rate of 2 ml per min, after each injection needles were kept in place for 2 min to allow for complete diffusion. The animals were given intraperitoneal injections of desipramine (25 mg/kg) 30 min prior to 6-OHDA to prevent the uptake of the neurotoxin into noradrenergic nerve terminals or saline infusions in the treated and control rats, respectively. Experimental protocol On the day of the experiment groups of animals received the following treatment: (i) unlesioned controls, treated with saline; (ii) 6OHDA-lesioned controls, treated with saline; (iii) 6-OHDA-lesioned rats, treated with SP, 0.35 nmol/kg; (iv) 6-OHDA-lesioned rats, treated with SP, 3.5 nmol/kg and (v) 6-OHDA-lesioned rats, treated with SP5–11, 3.5 nmol/kg. SP and SP5–11 were dissolved in saline and injected in a volume of 10 ml with a 10-ml Hamilton microsyringe connected to a 30-gauge injector which protruded 1 mm beyond the tip of the cannula guide and was placed into the lateral ventricle. Thirty minutes later animals were decapitated with guillotine, the brains were rapidly removed from the skulls and placed on an icecooled glass plate. Striatum, nucleus accumbens and frontal cortex were dissected from the brain. Tissues were weighed, frozen in liquid nitrogen and stored at 2708C until extraction. The tissues obtained from each animal were homogenized in 0.1 M perchloric acid and centrifuged at 25,000 g for 10 min.
Determination of the concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid DA, DOPAC and HVA contents in supernatants of the brain structures were measured by HPLC with electrochemical detection. 24 The analytical column was Partisil 5 ODS 3 (5 mkm, 4.6 × 250.0 mm) from Whatman Chemical Separation. The mobile phase was 0.01 M sodium dihydrogenphosphate, 0.01 M citric acid, 2 mM sodium EDTA, 1 mM sodium octylsulfate, 10% methanol, pH 3.5 at flow rate 1.0 ml/min and temperature 258C. The installation consisted of 305 Gilson piston pump, 805 Gilson manometric module and Bioanalytical Systems LC-4B amperometric detector. The glassy carbon electrode was used at a potential of 0.75 V. Peak areas and sample concentrations were calculated with a Gilson 714 chromatography data system. Contents of DA, DOPAC and HVA were calculated as ng/mg of tissue weight. The ratios DOPAC/DA and HVA/DA were used as indices of transmitter metabolism. Statistical analysis Statistical analysis was performed by means of analysis of variance followed by a two-sided Student’s t-test. Differences were considered significant if the probability of error was less than 5%. All results were expressed as the mean ^ S.E.M., and the sample size n represents the number of individual samples analysed. RESULTS
Effects of 6-hydroxydopamine, substance P and substance P5–11 in the striatum The changes in the concentrations of DA and its metabolites in striatum of experimental groups of animals are presented in Table 1. A significant depletion of DA to 17%, DOPAC to 24% and HVA to 39% was found in the 6-OHDA-lesioned rats compared to the sham-operated group injected with saline. The experiment showed that administration of SP in low dose (0.35 nmol/kg) had no significant effect, while injection of SP in higher dose (3.5 nmol/kg) caused a large increase in the striatal DA, DOPAC and HVA levels. The concentration of DA was 3.3 times higher (DOPAC, 2.6 times; HVA, 2.1 times) than those in 6-OHDA-treated animals, which received saline injections. C-terminal fragment SP5–11 (3.5 nmol/kg) produced no effect on concentrations of substances under investigation. The turnover of DA was assessed by calculating the ratios of DOPAC/DA and HVA/DA. These ratios were significantly increased by 6-OHDA lesion in the striatum (Table 1). SP (0.35 nmol/kg) and SP5–11 (3.5 nmol/kg) had no effect, but SP (3.5 nmol/kg) decreased DOPAC/DA and HVA/DA ratios in the striatum by 25% and 41%, respectively, relative to 6-OHDA-lesioned subjects.
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Substance P increases dopamine content in 6-OHDA-lesioned brain
Table 2. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the nucleus accumbens of 6-hydroxydopamine-lesioned rats Group Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
6.060 ^ 0.257 (7)
0.838 ^ 0.040 (7)
0.352 ^ 0.019 (7)
0.139 ^ 0.007 (7)
0.058 ^ 0.003 (7)
1.538 ^ 0.233* (6)
0.262 ^ 0.032* (6)
0.139 ^ 0.023* (6)
0.174 ^ 0.010* (6)
0.091 ^ 0.010* (6)
1.884 ^ 0.390 (6)
0.290 ^ 0.048 (6)
0.162 ^ 0.017 (6)
0.167 ^ 0.010 (6)
0.095 ^ 0.011 (6)
4.378 ^ 0.475† (4)
0.872 ^ 0.267† (4)
0.332 ^ 0.054† (4)
0.195 ^ 0.051 (4)
0.070 ^ 0.010 (4)
1.245 ^ 0.138 (6)
0.204 ^ 0.026 (6)
0.135 ^ 0.015 (6)
0.162 ^ 0.005 (6)
0.110 ^ 0.006† (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
Table 3. Effect of substance P and substance P5–11 administration on dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in the frontal cortex of 6-hydroxydopamine-lesioned rats Group Control (sham-operated rats) 1 saline 6-OHDA, 1.5 mg/kg 1 saline 6-OHDA, 1.5 mg/kg 1 SP, 0.35 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP, 3.5 nmol/kg 6-OHDA, 1.5 mg/kg 1 SP5–11, 3.5 nmol/kg
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Ratio DOPAC/DA
Ratio HVA/DA
0.166 ^ 0.028 (7)
0.034 ^ 0.003 (7)
0.032 ^ 0.005 (7)
0.231 ^ 0.029 (7)
0.218 ^ 0.035 (7)
0.065 ^ 0.004* (6)
0.014 ^ 0.001* (6)
0.019 ^ 0.002* (6)
0.217 ^ 0.020 (6)
0.284 ^ 0.032 (6)
0.076 ^ 0.015 (6)
0.016 ^ 0.003 (6)
0.018 ^ 0.002 (6)
0.229 ^ 0.022 (6)
0.267 ^ 0.049 (6)
0.156 ^ 0.061† (4)
0.029 ^ 0.008† (4)
0.034 ^ 0.007† (4)
0.209 ^ 0.023 (4)
0.270 ^ 0.068 (4)
0.043 ^ 0.08† (6)
0.011 ^ 0.002 (6)
0.016 ^ 0.003 (6)
0.254 ^ 0.014 (6)
0.426 ^ 0.071† (6)
Data are presented as mean ^ S.E.M., (n per group). *Significantly different from control group, P , 0.01; †significantly different from 6-OHDA 1 saline, P , 0.05.
Effects of 6-hydroxydopamine, substance P and substance P5–11 in the nucleus accumbens and frontal cortex 6-OHDA produced a significant decrease in the concentrations of DA, DOPAC and HVA in the nucleus accumbens compared to the sham-operated rats (Table 2). Injection of SP at 0.35 nmol/kg didn’t change the 6-OHDA-induced reduction of DA and its metabolite contents in the nucleus accumbens, however SP at 3.5 nmol/kg significantly increased contents of DA, DOPAC and HVA. SP5–11 (3.5 nmol/kg) had no effect on the DA and its metabolite contents compared to the saline-treated group. DOPAC/DA and HVA/DA ratios were significantly increased by 6-OHDA lesion in the nucleus accumbens (Table 2). In these ratios, no differences were observed between saline- or SP-treated (0.35 and 3.5 nmol/kg) groups. SP5–11 (3.5 nmol/kg) caused an increase in HVA/DA ratio versus the saline-treated group. In the frontal cortex 6-OHDA caused a significant reduction of DA, DOPAC and HVA concentrations compared to sham-operated rats injected with saline (Table 3). In 6OHDA-treated rats there were no significant differences between saline- and SP-treated in low dose (0.35 nmol/kg) animals. However, SP at 3.5 nmol/kg significantly increased the contents of DA, DOPAC and HVA. Administration of SP5–11 (3.5 nmol/kg) caused a decrease in DA concentration relative to 6-OHDA-treated subjects, but had no effect on DOPAC and HVA contents. 6-OHDA had no effect on the DOPAC/DA and HVA/DA ratios in the frontal cortex (Table 3). Injections of SP (0.35 and 3.5 nmol/kg) produced no significant changes in DOPAC/DA and HVA/DA ratios as
compared to those in saline-treated 6-OHDA animals, SP5–11 (3.5 nmol/kg) caused increase in HVA/DA ratio in the frontal cortex. DISCUSSION
The objective of the present study was to investigate the effects of central administration of SP and the carboxy terminal sequence SP5–11 on DA, DOPAC and HVA content in the striatum, nucleus accumbens and frontal cortex of 6-OHDA-lesioned rats. Administration of 6-OHDA in our study resulted in a marked reduction in the DA, DOPAC and HVA contents in the striatum, nucleus accumbens and frontal cortex. The most significant destruction of the DA transmission was registered in the striatum and nucleus accumbens. These decreases are consistent with previously published data. 12,31 In addition, there were significant increases in DOPAC/DA and HVA/ DA ratios in the striatum and nucleus accumbens. This is probably related to compensatory increase of functional activity of DA-ergic system. 10,46 Administration of low dose SP (0.35 nmol/kg) had no effect, but SP at a higher dose (3.5 nmol/kg) caused significant increases in the concentrations of DA, DOPAC and HVA in the striatum, nucleus accumbens and frontal cortex of 6-OHDA-treated rats. These results indicate that SP has exerted a significant reversal of the total effects of 6-OHDA in these brain regions. Interestingly, in the striatum the DOPAC/DA and HVA/DA ratios were less increased after SP. It seems that a lesser degree of DA depletion might
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have made a compensatory increase in DA-ergic activity unnecessary. These data demonstrate that SP at the dose used here had the most significant effect on the terminals of nigrostriatal projections with lesser effects on mesocorticolimbic system. Previous work has shown that administration of SP or stable analogs in similar doses in substantia nigra or VTA to freely moving rats increased DA levels in the striatum and nucleus accumbens, respectively. 6,36 Intraperitoneal injection of the neuropeptide also increased DA release in nucleus accumbens and striatum. 8 The time of decapitation chosen in our experiment (30 min after SP administration) corresponds to the time of significant increase of extracellular DA in the striatum after intranigral injection of neuropeptide to freely moving rats 36 and maximal movement recovery in open field test of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rats after intranasal inhalation of SP. 25 The mechanisms underlying the stimulatory effects of SP on DA-ergic neurons are still under investigation. SP shows highest affinity for neurokinin 1 (NK1) receptors, although the naturally occurring tachykinins have a high affinity for, and act as full agonists on, the three receptor types: NK1, NK2 and NK3. 35 Therefore, the type of the receptors involved in the effect observed in the striatum and nucleus accumbens may differ. Previous works have shown that SP may increase the release of DA in the striatum by activation of tachykinin NK1 receptors, 21,36,45 NK3 receptors 26 or NK1 and NK3 receptors. 16 Also, it was demonstrated that both NK1 and NK3 receptors in the striatum are postsynaptic with respect to DA-ergic neurons, whereas nigral NK3 receptors are located on dopaminergic neurons. 43 It was concluded that there were direct NK3-mediated interactions between SP and DA-ergic neurons in the substantia nigra, but the DA-ergic effect of SP within the striatum was indirect and may be modulated through cholinergic link. 9,22 It seems that the latter way plays a more important role in DA-denervated nigrostriatal system. In addition to its effects on DA release, SP may also cause an increase in DA synthesis. This could occur through an indirect effect of SP on the release of glutamate which has been shown to increase tyrosine hydroxylase activity via stimulation of N-methyl-d-aspartate (NMDA) receptors 5 which are found on striatal DA-ergic terminals. 20 This idea is supported by the observation that SP can cause an increase in glutamate release in the striatum. 37 The recent demonstration that administration of NMDA, an agonist at NMDA-type
glutamate receptors, can increase DA synthesis in the striatum 47 also supports this contention. Although it has been shown that tachykinins excite mesocorticolimbic DA-ergic neurons, the identity of the receptor subtypes that regulate the excitability of these neurons remains to be elucidated. Some studies have implicated predominantly NK1 tachykinin receptors, 13 whereas others have suggested that NK3 26 but not NK1 or NK2 receptors 41 mediate the principal excitatory influence of SP. To our knowledge, there are, at present, no reports on the regulation of DA synthesis or tyrosine hydroxylase activity within the mesocorticolimbic system itself, however, it is very likely that SP might exert effects similar to those reported for the striatum, as discussed above. The enzymatic conversion into fragments with agonistic or antagonistic properties has been proposed to be a general principle in the regulation and expression of SP action. 17,38 In our study no effect was observed with C-terminal heptapeptide SP5–11 in the striatum and nucleus accumbens, whereas SP in equimolar dose increased DA, DOPAC and HVA content. In the frontal cortex a decrease in the DA content was registered with subsequent increase in HVA/ DA ratio versus 6-OHDA-lesioned control, who received saline injections. Boix and co-workers 7,8 found that C-terminal analog of SP, [pGlu 5, MePhe 8, Sar 9]SP5–11, and SP1–7 do not change the DA release in the striatum, but SP1–11 increases DA release in this structure. If this effect of SP on striatal DA is related to its facilitatory action on recovery after unilateral nigrostriatal lesions, 27 we may suppose on the basis of the previous studies and present results, that a full molecule is necessary to obtain such an effect; neither C- nor the N-terminal fragments would facilitate recovery. CONCLUSIONS
The results of the present experiment suggest that SP can increase DA levels in the DA depleted brain by activation of nigrostriatal and mesocorticolimbic DA-ergic neurons. These data suggest that SP analogs might be of potential use in the treatment armamentorium against Parkinson’s disease. Acknowledgements—The authors are very grateful to Dr J. L. Cadet for his continuous support and editorial comments. Also we would like to thank Ms Y. V. Tcherkas for her kind help in preparation of manuscript.
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