Brain Research, 538 (1991) 187-192 Elsevier
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Antiparkinson-like effects of neurotensin in 6-hydroxydopamine lesioned rats Francois B. Jolicoeur 1, Robert Rivest 1, Serge St.-Pierre 1'2 and Andrea Drumheller 1 1Departments of Psychiatry and Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que. J1H 5N4 (Canada) and 2 1NRS, Pointe Claire, Que. (Canada) (Accepted 7 August 1990) Key words: Neurotensin; Parkinson's disease; Hypokinesia; Muscle rigidity; Tremor; Animal model
The aim of the present study was to examine the effects of neurotensin in an animal model of Parkinson's disease (PD). Bilateral administration of 6-OHDA in the medial forebrain bundle at the level of the posterolateral hypothalamus of rats resulted in the appearance of the 3 principal neurological signs of PD: hypokinesia, rigidity and tremor. These symptoms were accompanied by severe losses of dopamine and its main metabolites in terminal regions of well-known dopamine pathways. Norepinephrine concentrations were also decreased in several regions but to a lesser extent than dopamine. Intracerebroventricular administration of neurotensin, in doses ranging from 7.5 to 120.0 ag, resulted in dose related attenuations of both muscular rigidity and tremors of lesioned animals. However, hypokinesia, defined as decreased motor activity, was not significantly affected by the peptide. Administration of 120.0/~g of [AIa]NT, an inactive analogue of neurotensin, failed to alter any of the 3 neurological signs. Together, these results reveal selective antiparkinson-like effects of neurotensin in an animal model. The theoretical significance of these findings is discussed.
INTRODUCTION The interaction between the tridecapeptide neurotensin and central dopaminergic systems is now well established (see refs. 14, 22 for reviews). The evidence substantiating such an interaction includes the presence of high concentrations of neurotensin-like immunoreactivity (NTLI) as well as putative receptors of the peptide in many areas containing dopamine cell bodies or terminals 5'2s. A particularly dense population of neurotensin receptors is found in the substantia nigra of a variety of mammalian species, including man 17'25'31. These receptors appear to be located directly on dopaminergic neurons since chemical lesions of the nigrostriatal pathway with 6-hydroxydopamine ( 6 - O H D A ) results in a substantial loss of nigral neurotensin receptors in rodents 9'21'27. Reductions of neurotensin receptors in the substantia nigra were also noted in primates following degeneration of nigrostriatal dopaminergic fibers with 1-metbyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) 35. More importantly, a marked decrease in the density of neurotensin receptors in the substantia nigra of patients afflicted with Parkinson's disease (PD) has been reported 3°'33'34. Although the actual concentrations of N T L I are not lowered in the substantia nigra of Parkin-
son patients 2, the severe loss of nigral neurotensin receptors suggests that the peptide might be implicated in the etiology and/or symptomatology of this disease. The aim of the present study was to examine the effects of neurotensin in an animal model of PD. We have shown recently that bilateral posterolateral hypothalamic injections of 6 - O H D A produce, in rats, a syndrome consisting of the 3 cardinal symptoms of Parkinson's disease: hypokinesia, muscular rigidity and tremors 16. Using this model, we examined the dose-related effects of intracerebroventricular administration of the peptide on the neurological signs produced by such lesions in rats. In order to verify the specificity of the findings obtained with neurotensin, the effects of an inactive analogue of the peptide, [Alall]NT, were also assessed. MATERIALS AND METHODS Animals Male hooded rats obtained from Canadian Breeding Farm (St.-Constant, Quebec) and weighing between 250-300 g were used. They were housed in individual cages situated in a temperature controlled room having a 12 h light/dark cycle. Animals were anaesthetized with a xylazine/ketamine mixture and 4 /~1 of a 6-OHDA solution (6.5/~g per ~1 of distilled water containing 0.04% ascorbic acid) was injected bilaterally into the hypothalamus according to the following coordinates: A.P: 5.0 mm, L: 2.0 mm and
Correspondence: EB. Jolicoeur, Department of Psychiatry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada. 0006-8993/91/$03.50 ~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
188 V: 8.0 mm from dura 3. The 6-OHDA solutions were prepared fresh immediately prior to each injection in order to minimize oxidation. Sham-operated animals were injected with isovolumetric solutions of ascorbic acid. Solutions were administered by means of 30 ga needles at a rate of 1 pl per min after which injection needles were kept in place for 4 min to allow for complete diffusion. Immediately after the lesions, animals were implanted with a 23 ga guide cannula aimed at the left cerebroventricle, according to a previously published procedure from this laboratory ~5. Following surgery, because 6-OHDA results in aphagia and adipsia, animals were intubated daily with 8 ml of a liquid diet containing 25 g sucrose, 1.8 ml Polyvisol vitamins, 2 eggs, 30 ml Kaopectate, 125 ml water and 400 ml evaporated milk.
TABLE I
Effects of neurotensin (120.Ol~g) on catalepsy and akinesia induced by hypothalamic 6-OHDA lesions Groups
Catalepsy
Motor activity
Sham-operated 6-OHDA + 0.9% NaC1 6-OHDA + neurotensin
23.1 + 6.1 360.0 + 0.0"* 360.0 + 0.0"*
171.0 + 24.5 34.5 + 6.2** 24.8 + 8.8**
Values represent means + S.E.M. of total scores obtained in the 6 post-injection test periods. Significant differences between the sham-operated group as revealed by Dunnett tests are indicated by asterisks (**P < 0.01).
Procedure Forty-eight hours following surgery, behavioral testing was initiated. In all experiments the investigator was unaware of treatment conditions. Spontaneous motor activity was measured for 1 min by means of a photocell activity apparatus (Lehigh Valley Electronics). The presence and intensity of catalepsy were determined by placing an animal's front paws on a horizontal wooden bar (1 cm in width) suspended 10 cm above the testing table surface. Time spent in that position, up to a maximum of 1 min, was recorded. Muscular rigidity was assessed in two tests. In the grasping test, a rat was suspended by its front paws grasping a metal rod (diameter: 0.5 cm) which was held by the experimenter about 50 cm above the table. The time the animal remained on the bar (maximum 1 min) was noted. In the tail rigidity test, the animal's tail was raised approx. 5 cm from the table with a metal rod (diameter: 0.5 cm) positioned 2 cm from the end of the tail. The time the tail stayed on the rod was recorded (maximum 30 s). For tremors, an animal was lifted by the tail so that the hind quarters were suspended approx. 10 cm above the table, with the forelimbs still resting on the surface. The animal was kept in that position for a period of 10 s and the presence or absence of body or leg tremors was noted. For all tests, the observer was unaware of the treatment conditions. Among animals displaying all 3 symptoms of PD, as revealed by the above tests, 7 groups of 8 animals each were constituted. These groups received either 0.9% NaCI, 7.5, 30.0, 60.0, 90.0 or 120.0/~g of neurotensin or 120/~g of [Alala]NT. Injections were made into the lateral ventricle by means of 30 ga injection needles. An additional group of 8 sham-operated rats was administered 0.9% NaCI. Animals were then submitted to the battery of neurobehavioral tests at 15, 30, 45, 60, 90 and 120 min following injections.
rated using a linear gradient of 0-8% methanol over 8 min, starting 4 min after injection.
Chemicals Neurotensin and [Alalt]NT were synthesized by the solid phase technique according to our previously described procedure 32. 6-OHDA hydrochloride, ascorbic acid, amines and metabolites were purchased from Sigma Chemical Co.; sodium acetate and citric acid from J.T. Baker; HCIO 4 and HPLC grade methanol from Fisher Scientific; sodium octyl sulphate from ICN.
Data analysis Results obtained on activity, catalepsy, grasping and tail rigidity were analyzed by means of ANOVAs for independent samples. When significant main effects were found, comparisons between controls and neurotensin or [Alall]NT treated groups were carried out by means of Dunnett tests. Total observations of tremors constituted non-parametric data and were analyzed by Fisher exact probability tests. Differences between sham-operated and 6OHDA-treated animals in regional concentrations of an amine or a given metabolite were analyzed by means of t tests for independent samples.
60-
Biochemical analyses A separate group of 6 animals, also displaying all symptoms of PD, and a group of 6 sham-operated rats were sacrificed 48 h following 6-OHDA administration and their brains rapidly removed and placed on a frozen dissection block. The following regions were excised according to a previously published procedureS: corpus striatum, nucleus accumbens, amygdala, hypothalamus and substantia nigra, All regions were homogenized in 1.0 M perchloric acid (HCIO4). Homogenization volume for the amygdala was 1.0 ml and 400/zl for all other regions. Regions were centrifuged at 15,000 r.p.m, for 15 min and the supernatants removed and filtered. Separation and quantification of dopamine (DA), its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as of norepinephrine (NE) were then performed using HPLC coupled with electrochemical detection according to our previously described method 4. Briefly, amines and metabolites were separated on a 4 mm x 15 cm ultrasphere 5/~m column using gradient elution (HPLC apparatus by Beckman Instr.). The column eluate was monitored by an LC 2A electrochemical detector (Bioanalytical Systems) equipped with a glassy carbon electrode and operating at an applied potential of 0.65 V. The primary eluting solvent was a mixture of 0.1 M sodium acetate and 0.02 M citric acid, pH 4.2, containing 0.3 mM EDTA and 0.05 mM sodium octyl sulfate. The secondary solvent was methanol. Amines were sepa-
s0 v
40
o-,oo 0
15
30
45
TIME
60
90
120
(min)
Fig. 1. Grasping response time in seconds obtained with 0.9% NaCI (continuous line), 7.5 (open squares), 30.0 (open triangles), and 90.0/ag (dark squares) neurotensin as well as with 120.0/~g (dark triangles) of [Alall]NT presented as a function of time. Injections were performed immediately following baseline measurements at t = 0. Significant differences from saline treated animals as revealed by Dunnett tests are indicated by asterisks (*P < 0.05).
189 TABLE II
Regional brain concentrations of NE, DA, DO PA C and HVA expressed in ng/mg wet weight Regions Corpus striatum Sham 6-OHDA N. accumbens Sham 6-OHDA Amygdala Sham 6-OHDA Hypothalamus Sham 6-OHDA Substantia nigra Sham 6-OHDA
NE
DA
DO PA C
HVA
0.08 + 0.02 0.05 + 0.01'
7.02 + 0.52 0.85 _+ 0.20**
1.66 _+0.25 0.27 + 0.06**
0.63 _+0.09 0.06 + 0.02
0.52 _+0.08 0.21 + 0.10"*
5.80 _+0.56 1.16 + 0.52**
2.62 _+0.52 0.62 + 0.14
0.82 + 0.15 0.15 + 0.08**
0.48 _+0.05 0.24 _+0.08**
0.46 + 0.11 0.17 + 0.03**
0.11 _+0.005 0.04 + 0.009**
0.08 + 0.01 0.03 + 0.008**
1.47 + 0.31 0.13 + 0.05**
0.27 + 0.05 0.16 + 0.06*
0.15 _+0.015 0.10 _+0.02*
0.06 + 0.01 0.05 + 0.007
0.26 + 0.06 0.22 + 0.05
0.66 _+0.15 1.72 + 1.65
0.30 _+0.09 0.32 _+0.16
0.17 _+ 0.06 0.15 + 0.05
Values represent means + S . D . of each group (n = 6). Significant differences as revealed by t-tests for independent samples are indicated by asterisks (**P < 0.01; *P < 0.05).
RESULTS
Neurobehavioral data As expected, bilateral hypothalamic administration of 6-OHDA resulted in a profound catalepsy and a strong reduction in spontaneous activity of animals. None of the doses of neurotensin nor 120.0/tg of [Alall]NT affected these two effects of the lesion. These findings are summarized in Table I where total post-injection scores of catalepsy and motor activity obtained in shamoperated animals and in lesioned rats treated with either 0.9% NaC1 or the largest dose of neurotensin adminis-
30,
tered (120.0 /~g) are presented. On the other hand, starting with 30.0 /~g of neurotensin, the prolonged grasping time of lesioned animals was significantly reduced. The inhibitory effect already noted at 15 min, was generally maximal at 45 min, and gradually dissipated during the following post-injection test periods. The administration of 120.0 gg of the analogue [AlaI1]NT did not affect significantly the grasping response. These results are illustrated in Fig. 1 where mean grasping time in sec obtained in lesioned animals receiving either 0.9% NaCl, 120.0 #g of the analogue or representative doses of neurotensin is presented as a function of time. The tail rigidity of lesioned animals was also significantly attenuated by neurotensin. This effect was seen with all doses
30U) n-
O
20.
:i
40-
o m
30-
UJ n,. Iu.
>.
n-
20. uJ U} m
o 10, i-
0
1;
3~)
415 TIME
6~l
9~)
12=0
(rain)
Fig. 2. Tail suspension time in seconds obtained with 0.9% NaCI (continuous line), 7.5 (open squares), 30.0 (open triangles), and 90.0 gg (dark squares) neurotensin presented as a function of time. Injections were performed immediately following baseline measurements at t = 0. Significant differences from saline-treated animals as revealed by Dunnett tests are indicated by asterisks (*P < 0.05).
0.0
7.5
30.0
60.0
30.0
120.0
DOSE (PO) Fig. 3. Total observations of tremors during the 6 post-injection test periods are presented for animals treated with either 0.9% NaCI (open column), individual doses of neurotensin (striped columns) or 120/tg of [Alall]NT (dark column). Significant differences from saline treated animals as revealed by Fisher exact probability tests are indicated by asterisks (*P < 0.05).
190 of the peptide and, again, was already observed at 15 min and most prominent at 45 min following injections. Administration of 120.0/~g [AIaI~]NT did not alter tail rigidity. These results are summarized in Fig. 2 where mean tail suspension time of lesioned rats administered 0.9% NaCI or representative doses of neurotensin is given for each test period. Finally, neurotensin significantly decreased tremor of lesioned animals. Tremor was reduced first with 30.0/~g and then decreased in a linear fashion with larger doses of neurotensin. As was the case for muscle rigidity, the inhibitory effect was obtained in the first post-injection test period and was most evident at 45 min following administration. Tremor was not affected by the analogue of neurotensin. These results are illustrated in Fig. 3 where total observations of tremors obtained during the 6 post-injection test periods are presented for each dose of neurotensin administered and for 120.0/~g [Alan]NT.
Biochemical data Bilateral administration of 6 - O H D A resulted in significant reductions in D A and its metabolites D O P A C and HVA in all regions examined except the substantia nigra where no significant changes were found. Significant reductions in regional NE concentrations were also observed in treated animals. These effects are presented in Table II where regional brain concentrations of NE, DA, D O P A C and HVA, expressed in ng per mg wet weight, are presented for both sham-operated and lesioned animals. DISCUSSION As expected from our previous work 16, bilateral administration of 6-OHDA in the medial forebrain bundle at the level of the posterolateral hypothalamus resulted in the appearance of the 3 principal neurological signs of PD: hypokinesia, rigidity and tremor. These symptoms were accompanied by severe losses of D A and its main metabolites in terminal regions of both the nigrostriatal and mesolimbic dopamine pathways (Table II). Not surprisingly, pronounced reductions were also noted in the hypothalamus, the site of 6-OHDA injection. Degeneration of both nigrostriatal and mesolimbic dopaminergic systems as well as a marked decrease in hypothalamic D A concentrations has been documented in PD 1. In contrast to PD, levels of D A were not significantly affected in the substantia nigra of lesioned animals. However, the large standard deviation of mean D A concentrations in this region of lesioned animals indicates the wide variability of D A levels. In fact, examination of the raw data revealed that in some instances substantia nigral levels of D A were dramatically
increased, while in others there was either no change or a slight decrease. This is probably due to the fact that neurochemical determinations were performed 48 h after lesioning, and that the retrograde degeneration process was not completed in all animals. In a related study it was shown that the toxin significantly reduced tyrosine hydroxylase and D O P A decarboxylase activity in the SN and the CP. However, in that study, the dose and route of administration were different from those used here ~s. Nonetheless, these neurochemical findings suggest that reductions in D A in terminal regions of dopaminergic fibers are sufficient, at least in rats, to induce the 3 cardinal symptoms of PD. Regional concentrations of NE were also lowered significantly in the same regions where changes in DA were found (Table II). However, similarly to what has been reported in PD ~'7, the decreases in NE concentrations were less pronounced than the reductions in DA levels. The results of the present study demonstrate highly selective effects of neurotensin on the neurological signs of PD in animals. These effects cannot be ascribed to a non-specific action of peptide administration since they were not obtained with [Alan]NT, an inactive analogue of neurotensin n. Neurotensin markedly decreased muscular rigidity and tremor of lesioned animals without affecting hypokinesia as attested by the lack of effect of the peptide on the catalepsy and the reduced motor activity of lesioned animals (Table I). That neurotensin failed to affect these two components of hypokinesia could be seen as an obvious outcome considering the reported akinetic effect of the peptide in normal animals m'2°. However, under normal circumstances, the dose-related effects of neurotensin on activity are biphasic and the 3 largest doses of the peptide administered in the present study (60, 90 and 120/~g), do not affect motor activity nor induce catalepsy in naive animals m. Therefore the observed differential effects of neurotensin on hypokinesia, rigidity and tremor are meaningful and they suggest that distinct neural mechanisms or different DA receptor subtypes underlie these 3 neurological signs in lesioned animals. The attenuation of muscular rigidity by neurotensin was seen in both the grasping and tail rigidity tests with more potent and efficacious effects obtained in the latter test (Figs. 1 and 2). Whereas 30.0 #g of neurotensin was needed to decrease the grasping response time of lesioned animals, the smallest dose administered, 7.5 ktg, was sufficient to attenuate tail rigidity. Furthermore, although significantly reduced, the grasping time of lesioned was still longer than that of sham-operated animals, even with the largest dose of neurotensin. On the other hand, with the two largest doses of the peptide, tail rigidity was completely abolished (data not shown).
191 It is noteworthy that neurotensin, even in large doses, does not affect muscular tone in naive animals as evaluated by the grasping test ~°. Therefore the attenuating effect observed in the present study points to a genuine interaction of the peptide with the neural mechanisms underlying the muscular rigidity of lesioned animals. Neurotensin also significantly reduced tremors in lesioned animals (Fig. 3). Significant effects were obtained first with 30.0 /zg when total frequency of tremor, recorded as an all-or-none symptom, was examined statistically. However, subjective evaluations of the neurotensin-treated animals suggested that the intensity of tremors was also attenuated by the peptide and that this effect was already evident with 7.5/zg. The exact mechanisms by which neurotensin exerted its antiparkinson-like effects in animals remain to be elucidated. Anticholinergic agents are used in the treatment of PD, mostly in the early stages of the disease 28. We have shown recently that neurotensin can decrease potently physostigmine-induced yawning responses in animals, thus suggesting that the peptide might possess central anticholinergic properties 29. Whether or not the observed antiparkinson-like effects can be attributed partially if not totally to an anticholinergic action of neurotensin will have to be determined in future pharmacological studies. As mentioned in the introduction, the substantia nigra in various mammalian species, including man, contains a very dense population of NT receptors 17"25'31. The density of these receptors is reduced markedly following degeneration of the nigrostriatal pathway after chemical lesioning in experimental animals and following the degenerative process in PD 9'3°'33'35. Obviously, a loss of nigral NT receptors in our lesioned animals cannot be assumed. Actually, the fact that concentrations of D A and its metabolites were unchanged in the substantia nigra strongly suggests that nigral dopaminergic cells remained intact in these animals and that density of neurotensin receptors in this region was unaltered. Therefore a stimulatory action of the peptide on dopaminergic cells of the substantia nigra and a consequential activation of the nigrostriatal pathway could underlie the REFERENCES 1 Agid, Y. and Javoy-Agid, E, Peptides and Parkinson's disease, Trends Neurosci., 8 (1985) 30--35. 2 Bissette G., Nemeroff, C.B., Decker, M.W., Kizer, J.S., Agid, Y. and Javoy-Agid, E, Alterations in regional brain concentrations of neurotensin and bombesin in Parkinson's disease, Ann. Neurol., 17 (1985) 324-328. 3 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. R. Neth. Acad. Sci., 52 (1959) 1-38. 4 Drumheller, A.L., St.-Pierre, S. and Jolicoeur, EB., Effects of neurotensin on regional concentrations of norepinephrine in rat brain, Brain Res. Bull., 16 (1986) 755-758.
effects of the peptide on muscle rigidity and tremors of lesioned animals. In accordance with this possibility, it has been shown that micro-application of neurotensin depolarizes nigral dopamine neurons 24 and that intranigral injection of the peptide results in prolonged increases in D A in both striatum and globus pallidus 19. However, the lack of effect of neurotensin on catalepsy and akinesia would be difficult to reconcile with a stimulatory action of the peptide on nigral dopamine neurons. Obviously, a systematic study of the effects of local injections of neurotensin in the substantia nigra, as well as in other regions of the basal ganglia, on the neurological signs of 6 - O H D A lesioned animals would help to specify the mechanisms of action of the peptide in its differential antiparkinson-like properties. An important implication of the present results pertains to the much discussed dopamine-neurotensin interaction 14'22'26. An inhihitory influence on dopaminergic systems is generally ascribed to neurotensin 2°'22'31. Indeed, several studies have shown that the peptide can significantly decrease behaviors induced by pharmacological stimulation of dopamine 6'12'15. In contrast, the present results demonstrate that the peptide can attenuate behavioral manifestations induced by a hypo-dopaminergic condition. Also, we have observed recently that neurotensin can markedly inhibit behaviors produced by small doses of dopamine agonists which are thought to activate the so-called inhibitory dopamine autoreceptors 13'29. Thus it appears that neurotensin can modulate behaviors mediated by both pharmacological stimulation and inhibition of dopaminergic transmission. In summary, the results of the present study reveal selective antiparkinson-like effects of neurotensin in an animal model. Although the mechanisms underlying these effects remain to be ascertained, future investigation, including structure-activity studies, could lead to the development of neurotensin derivatives that could prove to be valuable alternatives or adjuncts to the present pharmacotherapy of PD. Acknowledgement. This work was supported by a Development Grant No. DG-284 from the Medical Research Council of Canada.
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Antiparkinson-like effects of neurotensin in 6-hydroxydopamine lesioned rats Francois B. Jolicoeur 1, Robert Rivest 1, Serge St.-Pierre 1'2 and Andrea Drumheller 1 1Departments of Psychiatry and Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que. J1H 5N4 (Canada) and 2 1NRS, Pointe Claire, Que. (Canada) (Accepted 7 August 1990) Key words: Neurotensin; Parkinson's disease; Hypokinesia; Muscle rigidity; Tremor; Animal model
The aim of the present study was to examine the effects of neurotensin in an animal model of Parkinson's disease (PD). Bilateral administration of 6-OHDA in the medial forebrain bundle at the level of the posterolateral hypothalamus of rats resulted in the appearance of the 3 principal neurological signs of PD: hypokinesia, rigidity and tremor. These symptoms were accompanied by severe losses of dopamine and its main metabolites in terminal regions of well-known dopamine pathways. Norepinephrine concentrations were also decreased in several regions but to a lesser extent than dopamine. Intracerebroventricular administration of neurotensin, in doses ranging from 7.5 to 120.0 ag, resulted in dose related attenuations of both muscular rigidity and tremors of lesioned animals. However, hypokinesia, defined as decreased motor activity, was not significantly affected by the peptide. Administration of 120.0/~g of [AIa]NT, an inactive analogue of neurotensin, failed to alter any of the 3 neurological signs. Together, these results reveal selective antiparkinson-like effects of neurotensin in an animal model. The theoretical significance of these findings is discussed.
INTRODUCTION The interaction between the tridecapeptide neurotensin and central dopaminergic systems is now well established (see refs. 14, 22 for reviews). The evidence substantiating such an interaction includes the presence of high concentrations of neurotensin-like immunoreactivity (NTLI) as well as putative receptors of the peptide in many areas containing dopamine cell bodies or terminals 5'2s. A particularly dense population of neurotensin receptors is found in the substantia nigra of a variety of mammalian species, including man 17'25'31. These receptors appear to be located directly on dopaminergic neurons since chemical lesions of the nigrostriatal pathway with 6-hydroxydopamine ( 6 - O H D A ) results in a substantial loss of nigral neurotensin receptors in rodents 9'21'27. Reductions of neurotensin receptors in the substantia nigra were also noted in primates following degeneration of nigrostriatal dopaminergic fibers with 1-metbyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) 35. More importantly, a marked decrease in the density of neurotensin receptors in the substantia nigra of patients afflicted with Parkinson's disease (PD) has been reported 3°'33'34. Although the actual concentrations of N T L I are not lowered in the substantia nigra of Parkin-
son patients 2, the severe loss of nigral neurotensin receptors suggests that the peptide might be implicated in the etiology and/or symptomatology of this disease. The aim of the present study was to examine the effects of neurotensin in an animal model of PD. We have shown recently that bilateral posterolateral hypothalamic injections of 6 - O H D A produce, in rats, a syndrome consisting of the 3 cardinal symptoms of Parkinson's disease: hypokinesia, muscular rigidity and tremors 16. Using this model, we examined the dose-related effects of intracerebroventricular administration of the peptide on the neurological signs produced by such lesions in rats. In order to verify the specificity of the findings obtained with neurotensin, the effects of an inactive analogue of the peptide, [Alall]NT, were also assessed. MATERIALS AND METHODS Animals Male hooded rats obtained from Canadian Breeding Farm (St.-Constant, Quebec) and weighing between 250-300 g were used. They were housed in individual cages situated in a temperature controlled room having a 12 h light/dark cycle. Animals were anaesthetized with a xylazine/ketamine mixture and 4 /~1 of a 6-OHDA solution (6.5/~g per ~1 of distilled water containing 0.04% ascorbic acid) was injected bilaterally into the hypothalamus according to the following coordinates: A.P: 5.0 mm, L: 2.0 mm and
Correspondence: EB. Jolicoeur, Department of Psychiatry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada. 0006-8993/91/$03.50 ~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
188 V: 8.0 mm from dura 3. The 6-OHDA solutions were prepared fresh immediately prior to each injection in order to minimize oxidation. Sham-operated animals were injected with isovolumetric solutions of ascorbic acid. Solutions were administered by means of 30 ga needles at a rate of 1 pl per min after which injection needles were kept in place for 4 min to allow for complete diffusion. Immediately after the lesions, animals were implanted with a 23 ga guide cannula aimed at the left cerebroventricle, according to a previously published procedure from this laboratory ~5. Following surgery, because 6-OHDA results in aphagia and adipsia, animals were intubated daily with 8 ml of a liquid diet containing 25 g sucrose, 1.8 ml Polyvisol vitamins, 2 eggs, 30 ml Kaopectate, 125 ml water and 400 ml evaporated milk.
TABLE I
Effects of neurotensin (120.Ol~g) on catalepsy and akinesia induced by hypothalamic 6-OHDA lesions Groups
Catalepsy
Motor activity
Sham-operated 6-OHDA + 0.9% NaC1 6-OHDA + neurotensin
23.1 + 6.1 360.0 + 0.0"* 360.0 + 0.0"*
171.0 + 24.5 34.5 + 6.2** 24.8 + 8.8**
Values represent means + S.E.M. of total scores obtained in the 6 post-injection test periods. Significant differences between the sham-operated group as revealed by Dunnett tests are indicated by asterisks (**P < 0.01).
Procedure Forty-eight hours following surgery, behavioral testing was initiated. In all experiments the investigator was unaware of treatment conditions. Spontaneous motor activity was measured for 1 min by means of a photocell activity apparatus (Lehigh Valley Electronics). The presence and intensity of catalepsy were determined by placing an animal's front paws on a horizontal wooden bar (1 cm in width) suspended 10 cm above the testing table surface. Time spent in that position, up to a maximum of 1 min, was recorded. Muscular rigidity was assessed in two tests. In the grasping test, a rat was suspended by its front paws grasping a metal rod (diameter: 0.5 cm) which was held by the experimenter about 50 cm above the table. The time the animal remained on the bar (maximum 1 min) was noted. In the tail rigidity test, the animal's tail was raised approx. 5 cm from the table with a metal rod (diameter: 0.5 cm) positioned 2 cm from the end of the tail. The time the tail stayed on the rod was recorded (maximum 30 s). For tremors, an animal was lifted by the tail so that the hind quarters were suspended approx. 10 cm above the table, with the forelimbs still resting on the surface. The animal was kept in that position for a period of 10 s and the presence or absence of body or leg tremors was noted. For all tests, the observer was unaware of the treatment conditions. Among animals displaying all 3 symptoms of PD, as revealed by the above tests, 7 groups of 8 animals each were constituted. These groups received either 0.9% NaCI, 7.5, 30.0, 60.0, 90.0 or 120.0/~g of neurotensin or 120/~g of [Alala]NT. Injections were made into the lateral ventricle by means of 30 ga injection needles. An additional group of 8 sham-operated rats was administered 0.9% NaCI. Animals were then submitted to the battery of neurobehavioral tests at 15, 30, 45, 60, 90 and 120 min following injections.
rated using a linear gradient of 0-8% methanol over 8 min, starting 4 min after injection.
Chemicals Neurotensin and [Alalt]NT were synthesized by the solid phase technique according to our previously described procedure 32. 6-OHDA hydrochloride, ascorbic acid, amines and metabolites were purchased from Sigma Chemical Co.; sodium acetate and citric acid from J.T. Baker; HCIO 4 and HPLC grade methanol from Fisher Scientific; sodium octyl sulphate from ICN.
Data analysis Results obtained on activity, catalepsy, grasping and tail rigidity were analyzed by means of ANOVAs for independent samples. When significant main effects were found, comparisons between controls and neurotensin or [Alall]NT treated groups were carried out by means of Dunnett tests. Total observations of tremors constituted non-parametric data and were analyzed by Fisher exact probability tests. Differences between sham-operated and 6OHDA-treated animals in regional concentrations of an amine or a given metabolite were analyzed by means of t tests for independent samples.
60-
Biochemical analyses A separate group of 6 animals, also displaying all symptoms of PD, and a group of 6 sham-operated rats were sacrificed 48 h following 6-OHDA administration and their brains rapidly removed and placed on a frozen dissection block. The following regions were excised according to a previously published procedureS: corpus striatum, nucleus accumbens, amygdala, hypothalamus and substantia nigra, All regions were homogenized in 1.0 M perchloric acid (HCIO4). Homogenization volume for the amygdala was 1.0 ml and 400/zl for all other regions. Regions were centrifuged at 15,000 r.p.m, for 15 min and the supernatants removed and filtered. Separation and quantification of dopamine (DA), its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as of norepinephrine (NE) were then performed using HPLC coupled with electrochemical detection according to our previously described method 4. Briefly, amines and metabolites were separated on a 4 mm x 15 cm ultrasphere 5/~m column using gradient elution (HPLC apparatus by Beckman Instr.). The column eluate was monitored by an LC 2A electrochemical detector (Bioanalytical Systems) equipped with a glassy carbon electrode and operating at an applied potential of 0.65 V. The primary eluting solvent was a mixture of 0.1 M sodium acetate and 0.02 M citric acid, pH 4.2, containing 0.3 mM EDTA and 0.05 mM sodium octyl sulfate. The secondary solvent was methanol. Amines were sepa-
s0 v
40
o-,oo 0
15
30
45
TIME
60
90
120
(min)
Fig. 1. Grasping response time in seconds obtained with 0.9% NaCI (continuous line), 7.5 (open squares), 30.0 (open triangles), and 90.0/ag (dark squares) neurotensin as well as with 120.0/~g (dark triangles) of [Alall]NT presented as a function of time. Injections were performed immediately following baseline measurements at t = 0. Significant differences from saline treated animals as revealed by Dunnett tests are indicated by asterisks (*P < 0.05).
189 TABLE II
Regional brain concentrations of NE, DA, DO PA C and HVA expressed in ng/mg wet weight Regions Corpus striatum Sham 6-OHDA N. accumbens Sham 6-OHDA Amygdala Sham 6-OHDA Hypothalamus Sham 6-OHDA Substantia nigra Sham 6-OHDA
NE
DA
DO PA C
HVA
0.08 + 0.02 0.05 + 0.01'
7.02 + 0.52 0.85 _+ 0.20**
1.66 _+0.25 0.27 + 0.06**
0.63 _+0.09 0.06 + 0.02
0.52 _+0.08 0.21 + 0.10"*
5.80 _+0.56 1.16 + 0.52**
2.62 _+0.52 0.62 + 0.14
0.82 + 0.15 0.15 + 0.08**
0.48 _+0.05 0.24 _+0.08**
0.46 + 0.11 0.17 + 0.03**
0.11 _+0.005 0.04 + 0.009**
0.08 + 0.01 0.03 + 0.008**
1.47 + 0.31 0.13 + 0.05**
0.27 + 0.05 0.16 + 0.06*
0.15 _+0.015 0.10 _+0.02*
0.06 + 0.01 0.05 + 0.007
0.26 + 0.06 0.22 + 0.05
0.66 _+0.15 1.72 + 1.65
0.30 _+0.09 0.32 _+0.16
0.17 _+ 0.06 0.15 + 0.05
Values represent means + S . D . of each group (n = 6). Significant differences as revealed by t-tests for independent samples are indicated by asterisks (**P < 0.01; *P < 0.05).
RESULTS
Neurobehavioral data As expected, bilateral hypothalamic administration of 6-OHDA resulted in a profound catalepsy and a strong reduction in spontaneous activity of animals. None of the doses of neurotensin nor 120.0/tg of [Alall]NT affected these two effects of the lesion. These findings are summarized in Table I where total post-injection scores of catalepsy and motor activity obtained in shamoperated animals and in lesioned rats treated with either 0.9% NaC1 or the largest dose of neurotensin adminis-
30,
tered (120.0 /~g) are presented. On the other hand, starting with 30.0 /~g of neurotensin, the prolonged grasping time of lesioned animals was significantly reduced. The inhibitory effect already noted at 15 min, was generally maximal at 45 min, and gradually dissipated during the following post-injection test periods. The administration of 120.0 gg of the analogue [AlaI1]NT did not affect significantly the grasping response. These results are illustrated in Fig. 1 where mean grasping time in sec obtained in lesioned animals receiving either 0.9% NaCl, 120.0 #g of the analogue or representative doses of neurotensin is presented as a function of time. The tail rigidity of lesioned animals was also significantly attenuated by neurotensin. This effect was seen with all doses
30U) n-
O
20.
:i
40-
o m
30-
UJ n,. Iu.
>.
n-
20. uJ U} m
o 10, i-
0
1;
3~)
415 TIME
6~l
9~)
12=0
(rain)
Fig. 2. Tail suspension time in seconds obtained with 0.9% NaCI (continuous line), 7.5 (open squares), 30.0 (open triangles), and 90.0 gg (dark squares) neurotensin presented as a function of time. Injections were performed immediately following baseline measurements at t = 0. Significant differences from saline-treated animals as revealed by Dunnett tests are indicated by asterisks (*P < 0.05).
0.0
7.5
30.0
60.0
30.0
120.0
DOSE (PO) Fig. 3. Total observations of tremors during the 6 post-injection test periods are presented for animals treated with either 0.9% NaCI (open column), individual doses of neurotensin (striped columns) or 120/tg of [Alall]NT (dark column). Significant differences from saline treated animals as revealed by Fisher exact probability tests are indicated by asterisks (*P < 0.05).
190 of the peptide and, again, was already observed at 15 min and most prominent at 45 min following injections. Administration of 120.0/~g [AIaI~]NT did not alter tail rigidity. These results are summarized in Fig. 2 where mean tail suspension time of lesioned rats administered 0.9% NaCI or representative doses of neurotensin is given for each test period. Finally, neurotensin significantly decreased tremor of lesioned animals. Tremor was reduced first with 30.0/~g and then decreased in a linear fashion with larger doses of neurotensin. As was the case for muscle rigidity, the inhibitory effect was obtained in the first post-injection test period and was most evident at 45 min following administration. Tremor was not affected by the analogue of neurotensin. These results are illustrated in Fig. 3 where total observations of tremors obtained during the 6 post-injection test periods are presented for each dose of neurotensin administered and for 120.0/~g [Alan]NT.
Biochemical data Bilateral administration of 6 - O H D A resulted in significant reductions in D A and its metabolites D O P A C and HVA in all regions examined except the substantia nigra where no significant changes were found. Significant reductions in regional NE concentrations were also observed in treated animals. These effects are presented in Table II where regional brain concentrations of NE, DA, D O P A C and HVA, expressed in ng per mg wet weight, are presented for both sham-operated and lesioned animals. DISCUSSION As expected from our previous work 16, bilateral administration of 6-OHDA in the medial forebrain bundle at the level of the posterolateral hypothalamus resulted in the appearance of the 3 principal neurological signs of PD: hypokinesia, rigidity and tremor. These symptoms were accompanied by severe losses of D A and its main metabolites in terminal regions of both the nigrostriatal and mesolimbic dopamine pathways (Table II). Not surprisingly, pronounced reductions were also noted in the hypothalamus, the site of 6-OHDA injection. Degeneration of both nigrostriatal and mesolimbic dopaminergic systems as well as a marked decrease in hypothalamic D A concentrations has been documented in PD 1. In contrast to PD, levels of D A were not significantly affected in the substantia nigra of lesioned animals. However, the large standard deviation of mean D A concentrations in this region of lesioned animals indicates the wide variability of D A levels. In fact, examination of the raw data revealed that in some instances substantia nigral levels of D A were dramatically
increased, while in others there was either no change or a slight decrease. This is probably due to the fact that neurochemical determinations were performed 48 h after lesioning, and that the retrograde degeneration process was not completed in all animals. In a related study it was shown that the toxin significantly reduced tyrosine hydroxylase and D O P A decarboxylase activity in the SN and the CP. However, in that study, the dose and route of administration were different from those used here ~s. Nonetheless, these neurochemical findings suggest that reductions in D A in terminal regions of dopaminergic fibers are sufficient, at least in rats, to induce the 3 cardinal symptoms of PD. Regional concentrations of NE were also lowered significantly in the same regions where changes in DA were found (Table II). However, similarly to what has been reported in PD ~'7, the decreases in NE concentrations were less pronounced than the reductions in DA levels. The results of the present study demonstrate highly selective effects of neurotensin on the neurological signs of PD in animals. These effects cannot be ascribed to a non-specific action of peptide administration since they were not obtained with [Alan]NT, an inactive analogue of neurotensin n. Neurotensin markedly decreased muscular rigidity and tremor of lesioned animals without affecting hypokinesia as attested by the lack of effect of the peptide on the catalepsy and the reduced motor activity of lesioned animals (Table I). That neurotensin failed to affect these two components of hypokinesia could be seen as an obvious outcome considering the reported akinetic effect of the peptide in normal animals m'2°. However, under normal circumstances, the dose-related effects of neurotensin on activity are biphasic and the 3 largest doses of the peptide administered in the present study (60, 90 and 120/~g), do not affect motor activity nor induce catalepsy in naive animals m. Therefore the observed differential effects of neurotensin on hypokinesia, rigidity and tremor are meaningful and they suggest that distinct neural mechanisms or different DA receptor subtypes underlie these 3 neurological signs in lesioned animals. The attenuation of muscular rigidity by neurotensin was seen in both the grasping and tail rigidity tests with more potent and efficacious effects obtained in the latter test (Figs. 1 and 2). Whereas 30.0 #g of neurotensin was needed to decrease the grasping response time of lesioned animals, the smallest dose administered, 7.5 ktg, was sufficient to attenuate tail rigidity. Furthermore, although significantly reduced, the grasping time of lesioned was still longer than that of sham-operated animals, even with the largest dose of neurotensin. On the other hand, with the two largest doses of the peptide, tail rigidity was completely abolished (data not shown).
191 It is noteworthy that neurotensin, even in large doses, does not affect muscular tone in naive animals as evaluated by the grasping test ~°. Therefore the attenuating effect observed in the present study points to a genuine interaction of the peptide with the neural mechanisms underlying the muscular rigidity of lesioned animals. Neurotensin also significantly reduced tremors in lesioned animals (Fig. 3). Significant effects were obtained first with 30.0 /zg when total frequency of tremor, recorded as an all-or-none symptom, was examined statistically. However, subjective evaluations of the neurotensin-treated animals suggested that the intensity of tremors was also attenuated by the peptide and that this effect was already evident with 7.5/zg. The exact mechanisms by which neurotensin exerted its antiparkinson-like effects in animals remain to be elucidated. Anticholinergic agents are used in the treatment of PD, mostly in the early stages of the disease 28. We have shown recently that neurotensin can decrease potently physostigmine-induced yawning responses in animals, thus suggesting that the peptide might possess central anticholinergic properties 29. Whether or not the observed antiparkinson-like effects can be attributed partially if not totally to an anticholinergic action of neurotensin will have to be determined in future pharmacological studies. As mentioned in the introduction, the substantia nigra in various mammalian species, including man, contains a very dense population of NT receptors 17"25'31. The density of these receptors is reduced markedly following degeneration of the nigrostriatal pathway after chemical lesioning in experimental animals and following the degenerative process in PD 9'3°'33'35. Obviously, a loss of nigral NT receptors in our lesioned animals cannot be assumed. Actually, the fact that concentrations of D A and its metabolites were unchanged in the substantia nigra strongly suggests that nigral dopaminergic cells remained intact in these animals and that density of neurotensin receptors in this region was unaltered. Therefore a stimulatory action of the peptide on dopaminergic cells of the substantia nigra and a consequential activation of the nigrostriatal pathway could underlie the REFERENCES 1 Agid, Y. and Javoy-Agid, E, Peptides and Parkinson's disease, Trends Neurosci., 8 (1985) 30--35. 2 Bissette G., Nemeroff, C.B., Decker, M.W., Kizer, J.S., Agid, Y. and Javoy-Agid, E, Alterations in regional brain concentrations of neurotensin and bombesin in Parkinson's disease, Ann. Neurol., 17 (1985) 324-328. 3 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. R. Neth. Acad. Sci., 52 (1959) 1-38. 4 Drumheller, A.L., St.-Pierre, S. and Jolicoeur, EB., Effects of neurotensin on regional concentrations of norepinephrine in rat brain, Brain Res. Bull., 16 (1986) 755-758.
effects of the peptide on muscle rigidity and tremors of lesioned animals. In accordance with this possibility, it has been shown that micro-application of neurotensin depolarizes nigral dopamine neurons 24 and that intranigral injection of the peptide results in prolonged increases in D A in both striatum and globus pallidus 19. However, the lack of effect of neurotensin on catalepsy and akinesia would be difficult to reconcile with a stimulatory action of the peptide on nigral dopamine neurons. Obviously, a systematic study of the effects of local injections of neurotensin in the substantia nigra, as well as in other regions of the basal ganglia, on the neurological signs of 6 - O H D A lesioned animals would help to specify the mechanisms of action of the peptide in its differential antiparkinson-like properties. An important implication of the present results pertains to the much discussed dopamine-neurotensin interaction 14'22'26. An inhihitory influence on dopaminergic systems is generally ascribed to neurotensin 2°'22'31. Indeed, several studies have shown that the peptide can significantly decrease behaviors induced by pharmacological stimulation of dopamine 6'12'15. In contrast, the present results demonstrate that the peptide can attenuate behavioral manifestations induced by a hypo-dopaminergic condition. Also, we have observed recently that neurotensin can markedly inhibit behaviors produced by small doses of dopamine agonists which are thought to activate the so-called inhibitory dopamine autoreceptors 13'29. Thus it appears that neurotensin can modulate behaviors mediated by both pharmacological stimulation and inhibition of dopaminergic transmission. In summary, the results of the present study reveal selective antiparkinson-like effects of neurotensin in an animal model. Although the mechanisms underlying these effects remain to be ascertained, future investigation, including structure-activity studies, could lead to the development of neurotensin derivatives that could prove to be valuable alternatives or adjuncts to the present pharmacotherapy of PD. Acknowledgement. This work was supported by a Development Grant No. DG-284 from the Medical Research Council of Canada.
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