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Increased expression of preproneuropeptide Y and preprosomatostatin mRNA in striatum after selective serotoninergic lesions in rats
Neurosciencc Letters, 160 (1993) 197 200 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
197
NSL 09844
Increased expression of preproneuropeptide Y and preprosomatostatin mRNA in striatum after selective serotoninergic lesions in rats C. Bendotti, G. Tarizzo, F. Fumagalli, S. Baldessari and R. Samanin lstituto di Ricerche Farmacologiche 'Mario Negri', Milan ( ltaly) (Received 11 May 1993; Revised version received 30 June 1993; Accepted 30 June 1993)
K~9, words'." Preproneuropeptide Y mRNA; Preprosomatostatin mRNA; 5,7-Dihydroxytryptamine; Serotoninergic lesion; Striatum; Hippocampus; Frontal cortex; Rat The levels of neuropeptide Y and somatostatin may change when serotoninergic neurotransmission is altered in different brain regions. To assess whether serotonin regulates the synthesis of these peptides, we measured the levels of preproneuropeptide Y (ppNPY) and preprosomatostatin (ppSOM) mRNA in different brain regions after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), a selective serotonin neurotoxin. The mRNA of these peptides significantly increased in the striatum but not in hippocampus and frontal cortex. It thus appears that serotonin has an inhibitory effect on the biosynthesis of neuropeptide Y and somatostatin in striatum whereas it probably acts by stimulating the release of these peptides in hippocampus and frontal cortex.
Neuropeptide Y (NPY) is a 36-amino acid peptide first isolated from porcine brain extracts which is highly concentrated and widely distributed in the nervous system of various mammalian species. NPY-immunoreactive neurons in the cerebral cortex, striatum and partially in the hippocampus also contain somatostatin (SOM) immunoreactivity [2, 7, 15]. This population of neurons may be affected in certain neurological and psychiatric disorders [5, 20]. Immunocytochemical investigations have shown that most NPY/SOM neurons in forebrain areas are local interneurons that may receive afferent synapses from different neurotransmitter systems. Levels of NPY and SOM immunoreactivities are sensitive to alterations of serotonin (5-HT) neurotransmission in different brain regions [12]. Although these data suggest that a central serotonergic mechanism plays a role in the regulation of these neuropeptides, it is not clear whether its influence is inhibitory or facilitatory. To investigate this aspect further, we analyzed the biosynthesis of these peptides in different brain regions after selective lesions of the serotoninergic system by measuring the levels of preproneuropeptide Y (ppNPY) mRNA and preprosomatostatin (ppSOM) mRNA. Twenty/.tl of Correspondence: C. Bendotti, Lab. Neuropharmacology, Istituto 'Mario Negri', Via Eritrea 62, 20157 Milano, Italy. Fax: (39) (2) 3546277.
0.1% ascorbic acid containing 150/~g (free base) of the selective 5-HT neurotoxin, 5,7-dihdroxytryptamine (5,7DHT) were injected into the cerebral ventricles of Equithesin (9.7 mg/ml sodium pentobarbital in saline, 42.6 mg/ml chloral hydrate in propylenglycol and 21.2 mg/ml MgSO 4 in ethanol; 3.5 ml/kg, intraperitoneally) anesthetized male Sprague-Dawley rats (200-300 g). Control rats received the vehicle (0.1% ascorbic acid in 0.9% NaC1). Desmethylimipramine (25 mg/kg i.p.), an inhibitor of noradrenaline uptake, was injected 30 min before 5,7-DHT to prevent damage to noradrenergic neurons. Five and 15 days after the lesion the rats were killed by decapitation, and the brains were rapidly removed and dissected out. To assess the extent and specificity of the lesion, 5-HT, dopamine, and noradrenaline levels were measured in the hippocampus, striatum, and frontal cortex of separate groups of animals (five rats for each group) by high performance liquid chromatography with electrochemical detection according to Invernizzi et al. [11]. Other groups of rats (six per group) were used for the extraction of RNA. Total RNA was isolated from hippocampus, striatum and frontal cortex with guanidine isothiocyanate as described by Chirwing et al. [6]. 15 pg of each RNA were then subjected to electrophoresis on 1.2% agarose/formaldehyde gel, transferred to a nylon membrane (Gene Screen-Plus NEN) and fixed by baking at 80°C for 2 h. The membranes were prehybridized for 2 h
198 300
ppSOM mRNA [~ Controls
200
[]
5,7-DHT
•
5,7-DHT 15days
5days
• o100 (.9
~6 o~ v
0
~o t'--
C3
300 _
ppNPY mRNA
(.9
~)
200
100
HIPPOCAMPUS
STRIATUM
FRONTALCORTEX
Fig. 1. Preprosomatostatin (ppSOM) mRNA and preproneuropeptide Y (ppNPY) mRNA levels in rat brain regions at different intervals after intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7DHT). The optical density was evaluated for each band and the values corresponding to the ratio between ppNPY mRNA or ppSOM mRNA and fl-actin mRNA were used for statistical analysis. Data are expressed as a percentage of controls. Each column represents the mean + S.E. (vertical bar) of 4 6 determinations. *P < 0.05, **P < 0.01 vs. controls (Dunnett's test).
at 42°C in a solution containing 50% formamide, 1% SDS (sodium dodecyl sulphate), 1 M sodium chloride and 10% dextran sulphate and 100 #g/ml of denaturated salmon sperm DNA, then hybridized with 32p-labelled ppSOM, ppNPY or fl-actin cDNA probes (0.8-1 x 106 cpm/ml) at 42°C for 16-18 h. cDNA probes were synthesized with [32p]dCTP using a random priming kit (Clontech). The rat NPY cDNA is a 287-bp XbaI-AvaI fragment containing the exon 2 of the NPY gene isolated from the plasmid p-GEM3 [16]. The rat ppSOM cDNA is a 305bp fragment, containing 47 bases of 5' untranslated sequence and the bases coding for the first 86 amino acids of ppSOM, isolated from the plasmid Bluescript [3]. Beta-actin is a 0.8-kb fragment from a human cDNA clone corresponding to the sequence published by Hanokoglu et al. [10]. The blots were washed twice with 2 x SSC (0.3 M NaC1, 0.03 M sodium citrate) and 0.1% SDS at room temperature then twice in 0.1 x SSC and 0.1% SDS at 65°C. The membranes were exposed to X-ray film at -80°C with intensifying screens for the time needed to aquire a signal to be in a linear range for quantification.
The autoradiograms were scanned and quantitatcd using a computerized image analysis system (IBAS, KontronZeiss). The optical density integrated for the area of each band was calculated. The signal associated with the presence of/~-actin mRNA was used as internal standard to normalize the ppNPY and ppSOM mRNA expression. Five and 15 days after intracerebroventricular injection of 5,7-DHT, 5-HT levels were markedly reduced in all brain regions, with the peak effect {more than 90% depletion) in the hippocampus at both times (Table I). No significant changes were observed in the levels of dopamine and noradrenaline in any of the brain regions examined (data not shown). Northern blot analysis of the ppNPY mRNA and ppSOM mRNA revealed single bands of approximately 0.8 and 0.7 kb, respectively, in all brain regions, ppNPY mRNA levels were markedly increased in the striatum of 5,7-DHT-treated rats, in comparison to controls, at both intervals after the lesion (Fig. 1) whereas ppSOM m R N A contents were significantly increased in this area only on day 5 (Fig. 1). No changes of ppNPY and ppSOM mRNA were observed in the hippocampus or frontal cortex (Fig. 1). A widespread increase in SOM levels has been reported after intracerebroventricular injection of 5,7DHT [12]. A reduction in 5-HT transmission may lead to an increase of SOM levels by different mechanisms. If 5-HT exerts an inhibitory action on SOM synthesis, one would expect the mRNA levels of the peptide to increase after 5,7-DHT injection, as in the striatum. On the other hand, if the increase of SOM levels depends on inhibition of SOM release from nerve terminals then mRNA levels might not change or could even decrease as feed-back inibition in response to the accumulation of SOM. The fact that 5,7-DHT raises the levels of SOM, but not its TABLE 1
EFFECT OF 5,7-DIHYDROXYTRYPTAMINE (5,7-DHT) ON 5HT IN HIPPOCAMPUS (HIP), STRIATUM (ST) A N D FRONTAL CORTEX (FCX) OF RATS Each value is the mean of 4-5 rats 5,7-DHT was injected intracerebroventricularly at the dose of 150 #g/20 ,ul (free base) 30 min after desmethylimipramine 25 mg/kg i.p. Control rats were injected with an equal volume of 0.1% ascorbic acid dissolved in saline. Treatment
Vehicle 5,7-DHT
Days after 5-HT levels (ng/g tissue) treatment Mean _+ S.E.
5 15
*P < 0.01 Dunnett's test.
HIP
ST
FCX
210.5 _+ 11.2 15.2 + 1.9* 14.3_+ 1.7"
306.3 _+40 88.0 _+ 11.6* 54.3_+ 38.4*
401.0 -+ 6.1 125.7 _+ 5.8* 105.0 _+ 5.1"
199 m R N A , in the h i p p o c a m p u s and cortex m a y be explained on the basis of a stimulatory effect o f 5-HT on S O M release in these regions. This would be consistent with previous evidence o f enhanced S O M release induced by 5-HT in synaptosomes o f cerebral cortex and h y p o t h a l a m u s [4]. However, we cannot exclude the possibility that different time course o f changes in m R N A expression occur in these brain regions c o m p a r e d to striatum or that changes are limited to discrete cell populations in these regions and they can be missed by N o r t h ern blot analysis. Similarly to p p S O M m R N A , the levels o f p p N P Y m R N A rose in the striatum but not in h i p p o c a m p u s and frontal cortex. The effect o f 5-HT lesion on the striatal p p N P Y m R N A was greater and lasted longer than that on p p S O M m R N A , suggesting that the increased gene expression of these peptides under these conditions m a y involve different mechanisms. Previous studies found no changes in the levels o f N P Y in striatum after an intracerebral injection o f 5,7-DHT [12]. However, since the dose o f 5,7-DHT was three times lower than we used, the level of 5-HT depletion in that study m a y not have been sufficient to change the striatal N P Y contents. The same study, though, reported that repeated treatment with pchlorophenylalanine, a selective inhibitor o f tryptophan hydroxylase, significantly raised the levels o f N P Y immunoreactivity in different brain regions including the striatum [12]. These and our data indicate that a decrease in 5-HT transmission stimulates the biosynthesis o f N P Y in the striatum. Since N P Y and SOM colocalize in the same interneurons in the striatum [2], it appears clear from our data that the removal o f 5-HT efferents to this region changes the activity o f these neurons. It is not known, however, whether this effect is mediated directly by the serotonergic system or is secondary to the effects on other systems. There is evidence that the nigrostriatal dopaminergic system has a regulatory influence on N P Y and S O M neurons although it is not completely clear whether dopamine (DA) plays an inhibitory or facilitatory role in these neuropeptidergic systems [13, 17, 18]. Lesions o f the dopaminergic nigrostriatal pathway by 6-hyd r o x y d o p a m i n e have been reported to either raise or lower the levels o f p p S O M m R N A [17, 18] and to raise the levels o f N P Y and its m R N A in the striatum [13, 17], whereas ~-methyl-p-tyrosine, an inhibitor o f tyrosine hydroxylase, or the dopaminergic antagonist haloperidol reduced the levels o f both peptides [1, 14]. The present study found no changes in striatal D A after 5,7-DHT, thus excluding the possibility that aspecific d a m a g e to dopaminergic neurons might contribute to the increased expression o f p p N P Y and p p S O M m R N A . However, 5-HT neurons m a y influence the activity of the
dopaminergic nigrostriatal system by changing the synthesis, release and turnover o f this neurotransmitter [8, 9, 19]. Therefore, changes in dopaminergic neurotransmission secondary to 5-HT lesions might well be involved in the changes in p p N P Y and p p S O M m R N A observed in the present study. In conclusion, whatever the mechanism involved, serotonin neurotransmission seems to play an inhibitory role on the biosynthesis o f N P Y and SOM in the striaturn, whereas it probably acts by stimulating the release o f these peptides in h i p p o c a m p u s and cortex. We wish to thank Dr. H a k a n Persson o f the K a r o linska Institute, Stockholm, Sweden, for kindly providing the rat N P Y c D N A . This work was supported by the Convenzione Psicofarmacologia of National Research Council (CNR), Rome. l Beal, M.F., Chattha, G.K. and Martin, J.B., A comparison of regional somatostatin and neuropeptide Y distribution in rat striatum and brain, Brain Res., 377 (1986) 240-245. 2 Beal, M.F. and Martin, J.B., Effects of neuroleptic drugs on brain somatostatin-like immunoreactivity, Neurosci. Lett., 47 (1984) 125 130. 3 Bendotti, C., Hohmann, C., Forloni, G., Reeves, R., Coyle, J.T. and Oster-Granite, M.L., Developmental expression of somatostatin in mouse brain. II. In situ hybridization, Dev. Brain Res., 53 (1990) 26-39. 4 Bennett, G.W., Edwardson, J.A., Marcano de Cotte, D., Berelowitz, M., Pimstone, B.L. and Kronheim, S., Release of somatostatin from rat brain synaptosomes, J. Neurochem., 32 (1979) 1127 1130. 5 Bissette,G. and Myers, B., Somatostatin in AIzheimer's disease and depression, Life Sci., 51 (1992) 1389-1410. 6 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 7 Chronwall, B.M., Chase, T.N. and O'Donohue, T.L., Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons, Neurosci. Lett., 52 (1984) 213- 217. 8 Ennis, C., Kemp, J.D. and Cox, B., Characterisation of inhibitory 5-hydroxytryptamine receptors that modulate dopamine release in the striatum, J. Neurochem., 36 (1981) 1515 1520. 9 Giambalvo, C.T. and Snodgrass S.R., Effect ofp-chloroamphetamine and 5,7-dihydroxytryptamine on rotation and dopamine turnover, Brain Res., 149 (1978) 453-467. 10 Hanokoglu, I., Tanese, N. and Fuchs, E., Complementary DNA sequence of human cytoplasmic actin. Interspecies divergence of 3' non coding regions, J. Mol. Biol, 163 (1983) 673 678. 11 Invernizzi, R., Fracasso, C., Caccia, S., Di Clemente, A., Garattini, S. and Samanin, R., Effect of L-cysteineon the long-term depletion of brain indoles caused by p-chloroamphetamine and d-fenfluramine in rats: relation to brain drug concentrations, Eur. J. Pharmacol., 163 (1989) 77-83. 12 Kakigi, T. and Maeda K., Effect of serotonergic agents on regional concentrations of somatostatin- and neuropeptide Y-like immunoreactivities in rat brain, Brain Res, 599 (1992) 45 50. 13 Kerkerian, L., Bosler, O., Pelletier, G. and Nieoullon, A., Striatal neuropeptide Y neurons are under the influence of the nigrostriatal
200
dopammergic pathway: immunohistochemical evidence, Neurosci. Lett., 66 (1986) 106 112. 14 Kerkerian, L., Salin, It and Nieoullon, A., Pharmacological characterization of dopaminergic influence on expression of neuropeptide Y immunoreaclivily by rat striatal neurons, Neuroscience, 26 (1988) 809 817.
15 Kohler, C., Eriksson, L.G., Davies, S. and Chan-Palay, V., "Colocalization of neuropeptide tyrosine and somatostatin immunoreactivity in neurons of individual subfields of the rat hippocampal region, Neurosci. Lett., 78 (1987) 1 6. 16 Larhammar, D., Ericsson, A. and Persson, H., Structure and expression of the rat neuropeptide Y gene (gene family/polymorphism/mRNA), Proc. Natl. Acad. Sci. USA, 84 (1987) 2068-2072. 17 Lindefors, N., Brene, S., Herrera-Marschitz, M. and Persson, H.,
Neuropeptide gene expression in brain is differentially regulated by midbrain dopamme neurons. Exp. Brain. Res.. 80 (1990)489 500. 18 Soghomonian, J.J. and Chesselet, M.F., Lesions of the dopaminergic nigrostriatal pathway alter preprosomatostatin messenger RN A levels in the striatum, the entopeduncular nucleus and the lateral hypothalamus of the rat, Neuroscience. 42 (1991) 49 59. 19 Spampinato, U., Esposito, E. and Samanin, R.. Serotonin agonist reduce dopamine synthesis in the striatum only when impulse flow of nigro-striatal neurons is intact, J. Neurochem., 45 (1985) 98(-) 982. 20 Wahlestedt, C., Ekman, R. and Widerlov, E.. Neuropeptide Y (NPY) and the central nervous system: distribution, effects and possible relationship to neurological and psychiatric disorders, Prog. Neuropsychopharmacol. Biol. Psychiat., 13 (1989) 31 54.
197
NSL 09844
Increased expression of preproneuropeptide Y and preprosomatostatin mRNA in striatum after selective serotoninergic lesions in rats C. Bendotti, G. Tarizzo, F. Fumagalli, S. Baldessari and R. Samanin lstituto di Ricerche Farmacologiche 'Mario Negri', Milan ( ltaly) (Received 11 May 1993; Revised version received 30 June 1993; Accepted 30 June 1993)
K~9, words'." Preproneuropeptide Y mRNA; Preprosomatostatin mRNA; 5,7-Dihydroxytryptamine; Serotoninergic lesion; Striatum; Hippocampus; Frontal cortex; Rat The levels of neuropeptide Y and somatostatin may change when serotoninergic neurotransmission is altered in different brain regions. To assess whether serotonin regulates the synthesis of these peptides, we measured the levels of preproneuropeptide Y (ppNPY) and preprosomatostatin (ppSOM) mRNA in different brain regions after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), a selective serotonin neurotoxin. The mRNA of these peptides significantly increased in the striatum but not in hippocampus and frontal cortex. It thus appears that serotonin has an inhibitory effect on the biosynthesis of neuropeptide Y and somatostatin in striatum whereas it probably acts by stimulating the release of these peptides in hippocampus and frontal cortex.
Neuropeptide Y (NPY) is a 36-amino acid peptide first isolated from porcine brain extracts which is highly concentrated and widely distributed in the nervous system of various mammalian species. NPY-immunoreactive neurons in the cerebral cortex, striatum and partially in the hippocampus also contain somatostatin (SOM) immunoreactivity [2, 7, 15]. This population of neurons may be affected in certain neurological and psychiatric disorders [5, 20]. Immunocytochemical investigations have shown that most NPY/SOM neurons in forebrain areas are local interneurons that may receive afferent synapses from different neurotransmitter systems. Levels of NPY and SOM immunoreactivities are sensitive to alterations of serotonin (5-HT) neurotransmission in different brain regions [12]. Although these data suggest that a central serotonergic mechanism plays a role in the regulation of these neuropeptides, it is not clear whether its influence is inhibitory or facilitatory. To investigate this aspect further, we analyzed the biosynthesis of these peptides in different brain regions after selective lesions of the serotoninergic system by measuring the levels of preproneuropeptide Y (ppNPY) mRNA and preprosomatostatin (ppSOM) mRNA. Twenty/.tl of Correspondence: C. Bendotti, Lab. Neuropharmacology, Istituto 'Mario Negri', Via Eritrea 62, 20157 Milano, Italy. Fax: (39) (2) 3546277.
0.1% ascorbic acid containing 150/~g (free base) of the selective 5-HT neurotoxin, 5,7-dihdroxytryptamine (5,7DHT) were injected into the cerebral ventricles of Equithesin (9.7 mg/ml sodium pentobarbital in saline, 42.6 mg/ml chloral hydrate in propylenglycol and 21.2 mg/ml MgSO 4 in ethanol; 3.5 ml/kg, intraperitoneally) anesthetized male Sprague-Dawley rats (200-300 g). Control rats received the vehicle (0.1% ascorbic acid in 0.9% NaC1). Desmethylimipramine (25 mg/kg i.p.), an inhibitor of noradrenaline uptake, was injected 30 min before 5,7-DHT to prevent damage to noradrenergic neurons. Five and 15 days after the lesion the rats were killed by decapitation, and the brains were rapidly removed and dissected out. To assess the extent and specificity of the lesion, 5-HT, dopamine, and noradrenaline levels were measured in the hippocampus, striatum, and frontal cortex of separate groups of animals (five rats for each group) by high performance liquid chromatography with electrochemical detection according to Invernizzi et al. [11]. Other groups of rats (six per group) were used for the extraction of RNA. Total RNA was isolated from hippocampus, striatum and frontal cortex with guanidine isothiocyanate as described by Chirwing et al. [6]. 15 pg of each RNA were then subjected to electrophoresis on 1.2% agarose/formaldehyde gel, transferred to a nylon membrane (Gene Screen-Plus NEN) and fixed by baking at 80°C for 2 h. The membranes were prehybridized for 2 h
198 300
ppSOM mRNA [~ Controls
200
[]
5,7-DHT
•
5,7-DHT 15days
5days
• o100 (.9
~6 o~ v
0
~o t'--
C3
300 _
ppNPY mRNA
(.9
~)
200
100
HIPPOCAMPUS
STRIATUM
FRONTALCORTEX
Fig. 1. Preprosomatostatin (ppSOM) mRNA and preproneuropeptide Y (ppNPY) mRNA levels in rat brain regions at different intervals after intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7DHT). The optical density was evaluated for each band and the values corresponding to the ratio between ppNPY mRNA or ppSOM mRNA and fl-actin mRNA were used for statistical analysis. Data are expressed as a percentage of controls. Each column represents the mean + S.E. (vertical bar) of 4 6 determinations. *P < 0.05, **P < 0.01 vs. controls (Dunnett's test).
at 42°C in a solution containing 50% formamide, 1% SDS (sodium dodecyl sulphate), 1 M sodium chloride and 10% dextran sulphate and 100 #g/ml of denaturated salmon sperm DNA, then hybridized with 32p-labelled ppSOM, ppNPY or fl-actin cDNA probes (0.8-1 x 106 cpm/ml) at 42°C for 16-18 h. cDNA probes were synthesized with [32p]dCTP using a random priming kit (Clontech). The rat NPY cDNA is a 287-bp XbaI-AvaI fragment containing the exon 2 of the NPY gene isolated from the plasmid p-GEM3 [16]. The rat ppSOM cDNA is a 305bp fragment, containing 47 bases of 5' untranslated sequence and the bases coding for the first 86 amino acids of ppSOM, isolated from the plasmid Bluescript [3]. Beta-actin is a 0.8-kb fragment from a human cDNA clone corresponding to the sequence published by Hanokoglu et al. [10]. The blots were washed twice with 2 x SSC (0.3 M NaC1, 0.03 M sodium citrate) and 0.1% SDS at room temperature then twice in 0.1 x SSC and 0.1% SDS at 65°C. The membranes were exposed to X-ray film at -80°C with intensifying screens for the time needed to aquire a signal to be in a linear range for quantification.
The autoradiograms were scanned and quantitatcd using a computerized image analysis system (IBAS, KontronZeiss). The optical density integrated for the area of each band was calculated. The signal associated with the presence of/~-actin mRNA was used as internal standard to normalize the ppNPY and ppSOM mRNA expression. Five and 15 days after intracerebroventricular injection of 5,7-DHT, 5-HT levels were markedly reduced in all brain regions, with the peak effect {more than 90% depletion) in the hippocampus at both times (Table I). No significant changes were observed in the levels of dopamine and noradrenaline in any of the brain regions examined (data not shown). Northern blot analysis of the ppNPY mRNA and ppSOM mRNA revealed single bands of approximately 0.8 and 0.7 kb, respectively, in all brain regions, ppNPY mRNA levels were markedly increased in the striatum of 5,7-DHT-treated rats, in comparison to controls, at both intervals after the lesion (Fig. 1) whereas ppSOM m R N A contents were significantly increased in this area only on day 5 (Fig. 1). No changes of ppNPY and ppSOM mRNA were observed in the hippocampus or frontal cortex (Fig. 1). A widespread increase in SOM levels has been reported after intracerebroventricular injection of 5,7DHT [12]. A reduction in 5-HT transmission may lead to an increase of SOM levels by different mechanisms. If 5-HT exerts an inhibitory action on SOM synthesis, one would expect the mRNA levels of the peptide to increase after 5,7-DHT injection, as in the striatum. On the other hand, if the increase of SOM levels depends on inhibition of SOM release from nerve terminals then mRNA levels might not change or could even decrease as feed-back inibition in response to the accumulation of SOM. The fact that 5,7-DHT raises the levels of SOM, but not its TABLE 1
EFFECT OF 5,7-DIHYDROXYTRYPTAMINE (5,7-DHT) ON 5HT IN HIPPOCAMPUS (HIP), STRIATUM (ST) A N D FRONTAL CORTEX (FCX) OF RATS Each value is the mean of 4-5 rats 5,7-DHT was injected intracerebroventricularly at the dose of 150 #g/20 ,ul (free base) 30 min after desmethylimipramine 25 mg/kg i.p. Control rats were injected with an equal volume of 0.1% ascorbic acid dissolved in saline. Treatment
Vehicle 5,7-DHT
Days after 5-HT levels (ng/g tissue) treatment Mean _+ S.E.
5 15
*P < 0.01 Dunnett's test.
HIP
ST
FCX
210.5 _+ 11.2 15.2 + 1.9* 14.3_+ 1.7"
306.3 _+40 88.0 _+ 11.6* 54.3_+ 38.4*
401.0 -+ 6.1 125.7 _+ 5.8* 105.0 _+ 5.1"
199 m R N A , in the h i p p o c a m p u s and cortex m a y be explained on the basis of a stimulatory effect o f 5-HT on S O M release in these regions. This would be consistent with previous evidence o f enhanced S O M release induced by 5-HT in synaptosomes o f cerebral cortex and h y p o t h a l a m u s [4]. However, we cannot exclude the possibility that different time course o f changes in m R N A expression occur in these brain regions c o m p a r e d to striatum or that changes are limited to discrete cell populations in these regions and they can be missed by N o r t h ern blot analysis. Similarly to p p S O M m R N A , the levels o f p p N P Y m R N A rose in the striatum but not in h i p p o c a m p u s and frontal cortex. The effect o f 5-HT lesion on the striatal p p N P Y m R N A was greater and lasted longer than that on p p S O M m R N A , suggesting that the increased gene expression of these peptides under these conditions m a y involve different mechanisms. Previous studies found no changes in the levels o f N P Y in striatum after an intracerebral injection o f 5,7-DHT [12]. However, since the dose o f 5,7-DHT was three times lower than we used, the level of 5-HT depletion in that study m a y not have been sufficient to change the striatal N P Y contents. The same study, though, reported that repeated treatment with pchlorophenylalanine, a selective inhibitor o f tryptophan hydroxylase, significantly raised the levels o f N P Y immunoreactivity in different brain regions including the striatum [12]. These and our data indicate that a decrease in 5-HT transmission stimulates the biosynthesis o f N P Y in the striatum. Since N P Y and SOM colocalize in the same interneurons in the striatum [2], it appears clear from our data that the removal o f 5-HT efferents to this region changes the activity o f these neurons. It is not known, however, whether this effect is mediated directly by the serotonergic system or is secondary to the effects on other systems. There is evidence that the nigrostriatal dopaminergic system has a regulatory influence on N P Y and S O M neurons although it is not completely clear whether dopamine (DA) plays an inhibitory or facilitatory role in these neuropeptidergic systems [13, 17, 18]. Lesions o f the dopaminergic nigrostriatal pathway by 6-hyd r o x y d o p a m i n e have been reported to either raise or lower the levels o f p p S O M m R N A [17, 18] and to raise the levels o f N P Y and its m R N A in the striatum [13, 17], whereas ~-methyl-p-tyrosine, an inhibitor o f tyrosine hydroxylase, or the dopaminergic antagonist haloperidol reduced the levels o f both peptides [1, 14]. The present study found no changes in striatal D A after 5,7-DHT, thus excluding the possibility that aspecific d a m a g e to dopaminergic neurons might contribute to the increased expression o f p p N P Y and p p S O M m R N A . However, 5-HT neurons m a y influence the activity of the
dopaminergic nigrostriatal system by changing the synthesis, release and turnover o f this neurotransmitter [8, 9, 19]. Therefore, changes in dopaminergic neurotransmission secondary to 5-HT lesions might well be involved in the changes in p p N P Y and p p S O M m R N A observed in the present study. In conclusion, whatever the mechanism involved, serotonin neurotransmission seems to play an inhibitory role on the biosynthesis o f N P Y and SOM in the striaturn, whereas it probably acts by stimulating the release o f these peptides in h i p p o c a m p u s and cortex. We wish to thank Dr. H a k a n Persson o f the K a r o linska Institute, Stockholm, Sweden, for kindly providing the rat N P Y c D N A . This work was supported by the Convenzione Psicofarmacologia of National Research Council (CNR), Rome. l Beal, M.F., Chattha, G.K. and Martin, J.B., A comparison of regional somatostatin and neuropeptide Y distribution in rat striatum and brain, Brain Res., 377 (1986) 240-245. 2 Beal, M.F. and Martin, J.B., Effects of neuroleptic drugs on brain somatostatin-like immunoreactivity, Neurosci. Lett., 47 (1984) 125 130. 3 Bendotti, C., Hohmann, C., Forloni, G., Reeves, R., Coyle, J.T. and Oster-Granite, M.L., Developmental expression of somatostatin in mouse brain. II. In situ hybridization, Dev. Brain Res., 53 (1990) 26-39. 4 Bennett, G.W., Edwardson, J.A., Marcano de Cotte, D., Berelowitz, M., Pimstone, B.L. and Kronheim, S., Release of somatostatin from rat brain synaptosomes, J. Neurochem., 32 (1979) 1127 1130. 5 Bissette,G. and Myers, B., Somatostatin in AIzheimer's disease and depression, Life Sci., 51 (1992) 1389-1410. 6 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 7 Chronwall, B.M., Chase, T.N. and O'Donohue, T.L., Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons, Neurosci. Lett., 52 (1984) 213- 217. 8 Ennis, C., Kemp, J.D. and Cox, B., Characterisation of inhibitory 5-hydroxytryptamine receptors that modulate dopamine release in the striatum, J. Neurochem., 36 (1981) 1515 1520. 9 Giambalvo, C.T. and Snodgrass S.R., Effect ofp-chloroamphetamine and 5,7-dihydroxytryptamine on rotation and dopamine turnover, Brain Res., 149 (1978) 453-467. 10 Hanokoglu, I., Tanese, N. and Fuchs, E., Complementary DNA sequence of human cytoplasmic actin. Interspecies divergence of 3' non coding regions, J. Mol. Biol, 163 (1983) 673 678. 11 Invernizzi, R., Fracasso, C., Caccia, S., Di Clemente, A., Garattini, S. and Samanin, R., Effect of L-cysteineon the long-term depletion of brain indoles caused by p-chloroamphetamine and d-fenfluramine in rats: relation to brain drug concentrations, Eur. J. Pharmacol., 163 (1989) 77-83. 12 Kakigi, T. and Maeda K., Effect of serotonergic agents on regional concentrations of somatostatin- and neuropeptide Y-like immunoreactivities in rat brain, Brain Res, 599 (1992) 45 50. 13 Kerkerian, L., Bosler, O., Pelletier, G. and Nieoullon, A., Striatal neuropeptide Y neurons are under the influence of the nigrostriatal
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