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Somatostatin stimulates striatal acetylcholine release by glutamatergic receptors: an in vivo microdialysis study
Neurochemistry International 40 (2002) 269– 275 www.elsevier.com/locate/neuint
Somatostatin stimulates striatal acetylcholine release by glutamatergic receptors: an in vivo microdialysis study Angelina Rakovska a,*, Janos P. Kiss b, Peter Raichev a, Maria Lazarova a, Reni Kalfin a, Kiril Milenov a a
Laboratory ‘Neuropeptides’, Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonche6 Street, bl. 23, 1113 Sofia, Bulgaria b Institute of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, H-1450 Budapest, Hungary Received 19 February 2001; received in revised form 30 April 2001; accepted 4 May 2001
Abstract The modulation of striatal cholinergic neurons by somatostatin (SOM) was studied by measuring the release of acetylcholine (ACh) in the striatum of freely moving rats. The samples were collected via a transversal microdialysis probe. ACh level in the dialysate was mueasured by the high performance liquid chromatography method with an electrochemical detector. Local administration of SOM (0.1, 0.5 and 1 mM) produced a long-lasting and concentration-dependent increase in the basal striatal ACh output. The stimulant effect of SOM was antagonized by the SOM receptor antagonist Cyclo(7-Aminopentanoyl-Phe-D-TrpLys-Thr[BZL]) (1 mM). In a series of experiments, we studied the effect of 6,7-Dinitroquinoxaline-2, 3-dione (DNQX), a selective non-NMDA (N-methyl-D-aspartate) glutamatergic antagonist, on the basal and SOM-induced ACh release from the striatum. DNQX, 2 mM, perfused through the striatum had no effect on the basal ACh output but inhibited the SOM (1 mM)-induced ACh release. The non-NMDA glutamatergic receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylendioxy-5H-2,3- benzodiazepine (GYKI-52466), 10 mM, antagonized the SOM (1 mM)-induced release of ACh in the striatum. Local administration of the NMDA glutamatergic receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV), 100 mM, blocked SOM (1 mM)-evoked ACh release. Local infusion of tetrodotoxin (1 mM) decreased the basal release of ACh and abolished the 1 mM SOM-induced increase in ACh output suggesting that the stimulated release of ACh depends on neuronal firing. The present results are the first to demonstrate a neuromodulatory role of SOM in the regulation of cholinergic neuronal activity of the striatum of freely moving rats. The potentiating effect of SOM on ACh release in the striatum is mediated (i) by SOM receptor located on glutamatergic nerve terminals, and (ii) by NMDA and non-NMDA glutamatergic receptors located on dendrites of cholinergic interneurones of the striatum. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Striatum; Somatostatin; Acetylcholine release; Microdialysis; Glutamatergic receptors
1. Introduction Somatostatin (SOM) is a cyclic tetradecapeptide isolated from bovine hypothalamus (Brazeau et al., 1973). Immunohistochemical studies have demonstrated that in the rat striatum there are SOM-containing mediumsized aspiny interneurons, 10 – 40 mm in diameter (Johansson, et al., 1984) which account for 1 – 2% of the total population of interneurons in this region (Vincent et al., 1983; Kawaguchi et al., 1995). Basal levels of * Corresponding author. Tel.: + 359-2-979-2305; fax: +359-2-719109. E-mail address: [email protected], [email protected] (A. Rakovska).
SOM in the striatum of the freely moving rats was found to be 5–15 fmol (Radke et al., 1993). There are SOM receptors in the striatum (Schindler et al., 1997; Hathway et al., 1999; Schindler et al., 1999) and their number has been found to increase in patients suffering from Parkinson’s disease (Epelbaum, 1986; Reader and Dewar, 1999). SOM interneurons contain neuropeptide Y (Vincent and Johansson, 1983), neuronal nitric oxide synthase and GABA (Kawaguchi et al., 1995; Kiss et al., 1999). SOM can be measured in the extracellular space of the striatum (Radke et al., 1993), hippocampus (Mathe et al., 1993; Vezzani et al., 1993), frontal cortex (Lathinen et al., 1992) and hypothalamus (Takahashi et al., 1994; Cattaneo et al., 1996) of freely moving rats.
0197-0186/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 1 ) 0 0 0 3 7 - 7
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SOM inhibits the release of acetylcholine (ACh) in slices of rat caudate nucleus and this release is mediated by dopaminergic mechanisms (Arneric and Reis, 1986; Leviel, 2001). In the striatum the neuropeptide stimulates the dopamine levels both in vitro (Chesselet and Reisine, 1983) and in vivo (Thermos et al., 1996). In vivo the SOM-stimulated release of dopamine is mediated by a glutamate-dependent action (Hathway et al., 1998). The glutamatergic excitatory input regulates striatal cholinergic interneurons by increasing the release of ACh through N-methyl-D-Aspartate (NMDA) (Scatton and Lehmann, 1982; Ulus et al., 1992; Knauber et al., 1999) and non-NMDA receptor activation (Cotman et al., 1987; Wisden and Seeberg, 1993; Morary et al., 1998). The role of SOM in the regulation of cholinergic neuronal activity in the striatum is still unclear. The aim of the present research was to elucidate the modulation of striatal cholinergic neurons in vivo by SOM. Trans-striatal microdialysis was used in the awake animal to study the effect of local administration of SOM on the release of ACh in the striatum. A preliminary report of this study has been presented (Raichev et al., 2000).
2. Experimental procedures
2.1. Animal housing and surgery Male adult Charles River Wistar rats weighing 250– 300 g were used. They were housed in groups with free access to food and water and kept on a 12 h light/12 h dark cycle. The rats were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic aparatus. Microdialysis tubes (AN 69 membrane; 220 mm i.d. and 310 mm o.d., molecular weight cut-off 15 000 Da; Dasco, Bologna, Italy) were inserted transversally into the striatum, following the procedure described by Giovannini et al. (1994). The microdialysis tube was covered with Super-Epoxy glue along its entire length except for a region corresponding to the brain areas to be studied (two sections of 3.5 mm separated by a glued central zone 2.5 mm long). The coordinates used for the implantation of the microdialysis tubing were: AP 0.0 and H 5 mm from bregma. (Paxinos and Watson, 1982). All coordinates were referred to bregma, with bregma and lambda on horizontal plane. Procedures involving animal and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1 Dec. 12. 1987; NIH Guide for the Care and Use of Laboratory animals, NIH Publication No. 85-23, 1985).
2.2. Microdialysis procedure One day after surgery each rat was placed in a Plexiglas cage. The inlet of the microdialysis probe was connected to a microperfusion pump and perfused with Ringer’s solution (NaCl 147 mM, CaCl2 1.2 mM, KCl 4.0 mM), containing 7 mM physostigmine sulfate at a constant flow rate of 2 ml/min using a microperfusion pump (Carnegie Medicine, Mod. CMA/100, Sweden). After an one hour stabilization period, during which the animals were perfused without collecting the dialysate, samples were collected at 40 min intervals. The content of ACh and choline (Ch) in the dialysate was analyzed by high performance liquid chromatography (HPLC) as described below. After collecting the first three samples to measure the basal outflow, drugs (dissolved in Ringer solution) were administered locally through the dialysis membrane for 80 min (4th and 5th samples). There after the drugs were withdrawn and the membrane was perfused with normal Ringer solution till the end of the experiments.
2.3. Histological control At the end of experiments rats were anaesthetized with urethane (1.2 mg/kg, ip.) and sacrified by decapitation. The brain was rapidly removed and placed in a vial containing 10 ml of 9% formaldehyde solution in phosphate buffer. Two or three days later the brain was frozen with liquid CO2 and coronal slices (50 mm) were cut using a freezing microtome and were examined by light microscopy (Nikon, Labophot-2) to verify the position of the dialysis membrane. Data obtained from rats in which the dialysis membrane was positioned outside the structure were discarded (B 5%). The conditions of the striatal neurons around the probe were checked at the end of the experiment by staining coronal sections of paraformaldehyde-fixed brains with Richardson’s solution (modified Nissal staining).
2.4. In 6itro reco6ery experiment To evaluate the passage of ACh and Ch through the microdialysis tubing, recovery experiments were performed at room temperature both in the absence and in the presence of the drugs used. The recovery of ACh and Ch from the dialysis membrane in the absence of drugs was: 569 1.4 and 5891.9%, respectively (mean9 SEM, n=13). All the drugs tested did not modify ACh and Ch recovery. The ACh and Ch values were not corrected for recovery.
2.5. Assay of acetylcholine and choline in the dialysate ACh and Ch were directly assayed in the dialysate using the HPLC method with post column enzyme
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reactor and electrochemical detector (Damsma et al., 1987). ACh and Ch were separated on a cation exchange column prepared by loading a reverse-phase column (Chromspher 5 C18, Chrompack) with sodium lauryl sulphate (0.5 mg/ml). The mobile phase consisted of 0.2 mM phosphate buffer (pH 8) containing 5 mM KCL, 1 mM tetramethylammonium (TMA) and 0.3 mM EDTA. The flow rate was 0.7 ml/min (injection volume 20 ml). ACh was hydrolyzed by acetylcholine esterase (AChE) to acetate and Ch in a postcolumn enzyme reactor; Ch was oxidized by Ch oxidase to produce betaine and hydrogen peroxide. Hydrogen peroxide was electrochemically detected by an electrochemical detector (Waters 460) equipped with a platinum electrode at + 500 mV. For the quantitative analysis of ACh and Ch, we constructed a calibration curve by spiking the Ringer’s solution with standard ACh (50 ml of 10 mM ACh solution and 50 ml of 10 mM Ch solution) in the concentration range we expected to find in the dialysates. Three or four concentrations for each calibration curve were then injected at the beginning and the end of the analysis and the peaks were then plotted against the concentrations. A regression line was calculated and quantitation of unknown samples was carried out by the method of inverse prediction. Under these experimental conditions the sensitivity limit (s/n ratio = 3/1) was 500 fmol for ACh and 250 fmol for Ch.
2.6. Materials The following chemicals, purchased from Sigma Chemical Co. (St. Louis, MO, USA) were used: AChE (E.C. 3.1.1.7., grade VI-S), Ch oxidase, Ch-oxidase (E.C.1.1.3.17), paraformaldehyde, physostigmine sulfate, homoserine, OPA, SOM-14, cyclo(7-Aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]), known to be a SOM receptor antagonist and tetrodotoxin (TTX). Glutamatergic receptor antagonists 6,7-Dinitroquinoxaline2,3-dione (DNQX) and D,L-2-amino-5-phosphonopentanoic acid (APV) were purchased from Research Biochemical Inc., RBI (Natick, MA, USA). The nonNMDA glutamatergic antagonist 1-(4-aminophenyl)-4methyl-7, 8-methylendioxil-5H-2,3- benzodiazepine (GYKI-52466) was supplied by the Institute for Drug Research, Budapest, Hungary.
2.7. Statistical analysis ACh outflow rates were expressed either as fmol/ml or as percentage variation over basal output (i.e. the mean of three samples taken before drug administration). Data are means of 9SEM. Significance of differences among experimental groups was evaluated comparing Areas Under the Curves (AUC) by analysis of variance one-way ANOVA followed by Newman-
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Keuls multiple comparison test (**P B 0.01; ***P B 0.001 vs. all other groups).
3. Results
3.1. Effects of somatostatin, cyclo(7 -aminopentanoylPhe-D -Trp-Lys-Thr[BZL]) and tetrodotoxin on the release of acetylcholine Basal ACh outflow from the striatum of freely moving rats was 24.2692.56 pmol/40 min (mean9SEM; 32 rats).The basal release of ACh remained relatively constant throughout the experiment (\6 h, Fig. 1). The initial efflux of Ch from the striatum was 125.369 10.36 pmol/40 min (mean9 SEM; 32 rats) and was unaffected by any of the tested drugs. SOM, applied for 80 min locally in the striatum induced an increase of extracellular ACh level (Fig. 1), which began about 40 min after its administration and lasted for over 3 h (Fig. 1). No behavioral activation was observed during SOM perfusion. Four concentrations of SOM (0.1, 0.5, 1 and 10 mM) were used to study its effects on ACh release which were found to be 1209 13, 145915, 2009 18 and 189913%, respectively (n= 6 for each dose, data are means9 SEM). As shown in Fig. 1, the effect was concentration-dependent with a maximum reached with 1 mM of SOM. In order to understand whether the effect of SOM on the release of ACh in the striatum was mediated by SOM receptor, the effect of the SOM receptor antagonist cyclo(7-aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]) on the release of ACh was investigated. The administration of 1 mM cyclo(7-aminopentanoyl-Phe-D-Trp-LysThr[BZL]) to the striatum did not change the basal release of ACh (variations ranging 915%; insignificant (n.s.) compared to controls). At the same concentration the SOM antagonist administered together with SOM (1 mM, 80 min) abolished the stimulant effect of SOM on the ACh release (89918% vs. SOM: 196 913%, n = 6 for each group, PB0.001) (Fig. 1). Local application of the Na+ channel blocker TTX (1 mM, dissolved in Ringer’s solution) via the dialysis probe led to a gradual decrease of ACh basal levels (Fig. 1). At the same concentration TTX completely prevented the 1 mM SOM-evoked release of ACh (Fig. 1) (n=6 ***P B 0.001). Changes in behavior of the rats were not observed during perfusion of 1 mM TTX in the striatum.
3.2. Effects of glutamatergic antagonists GYKI-52466, DNQX and APV on somatostatin induced acetylcholine release To determine whether the SOM induced ACh release was mediated via non-NMDA glutamatergic receptor,
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the effects of 1-(4-aminophenyl)-4-methyl-7, 8-methylendioxyl-5H-2,3.benzodiazepine (GYKI-52466), selective AMPA/kainate receptor antagonist and DNQX, competitive quisqualate/kainate (non-NMDA) receptor antagonist on the action of SOM (1 mM) were investigated. Local administration of GYKI-52466 (10 mM) was followed by an insignificant decrease in the basal ACh output from the striatum and was returned upon
Fig. 1. Effects of SOM on the ACh release from the striatum in freely moving rats in the absence and in the presence of TTX and cyclo(7aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]). All drugs were administered locally to the striatum via the dialysis membrane after collection of three samples, as shown by the horizontal bar. ACh output is expressed as percentage of changes over the mean of the first three samples taken to be controls (mean 9 SEM of at least six different rats in each group are presented). Dialysate samples were collected for 40 min. Data points represent basal release of ACh-controls (open circles), 0.1 mM SOM (open diamonds), 0.5 mM SOM (filled squares), 1 mM SOM (filled circles) and 1 mM SOM+ 1 mM TTX (open triangles); 1 mM SOM+ 1 mM cyclo(7-aminopentanoyl-Phe-D-TrpLys-Thr[BZL]) (crosses). The bar shown in the inset represents the mean percentage of changes calculated from 160 to 400 min: controls (open), 0.1 mM SOM (vertical), 0.5 mM SOM (diagonal crosshatched), 1 mM SOM (rising to right), 1 mM SOM+ 1 mM TTX (filled); Significant differences among the six experimental groups in the inset were evaluated comparing Area Under Curve (AUC) by one-way ANOVA, for SOM+TTX (F =21.51; PB 0.001) followed by Newman-Keuls multiple comparison test (***PB 0.001 vs. all other groups).
Fig. 2. Effects of SOM on ACh release from the striatum in freely moving rats in the absence and in the presence of 1-(4-aminophenyl)4-methyl-7,8-methyendioxyl-5H-2,3-benzodiazepine hydrochloride (GYKI 52466); 6,7-dinitroquinoxalline-2,3-dione (DNQX) and APV. All drugs were administered locally to the striatum via the dialysis membrane after collection of three samples, as shown by the horizontal bar. ACh output is expressed as percentage of changes over the mean of the first three samples taken to be controls (mean 9SEM of at least six different rats in each group are presented). Dialysate samples were collected for 40 min. Data points represent basal release of ACh-controls (open circles), 1 mM SOM (filled circles), 1 mM SOM+10 mM GYKI 52466 (open diamonds); 1 mM SOM+ 2 mM DNQX (filled triangles) and 1 mM SOM+100 mM APV (crosses). The bars shown in the inset represent the mean percentage changes calculated from 160 to 400 min: controls (open), 1 mM SOM (filled), 1 mM SOM +10 mM GYKI 52466 (horizontal); 1 mM SOM+ 2 mM DNQX (diagonal crosshartched) and 1 mM SOM+100 mM APV (vertical). Significant differences among the five experimental groups in the inset were evaluated comparing Area Under Curve (AUC) by one-way ANOVA (F =11.76; PB 0.001) followed by Newman-Keuls multiple comparison test (**PB0.01 vs. all other groups).
washout (data not shown). When SOM (1 mM) was administered together with GYKI- 52466 (10 mM) no increase in ACh output was observed (1039 21% vs. SOM: 1909 21%, n=6 for each group, n.s., PB0.001) (Fig. 2). When DNQX (2 mM), another non-NMDA receptor antagonist, was administered through the dialysis membrane a decrease in the basal release of ACh was found which however was not significant (data not shown). The stimulant effect of SOM (1 mM) was
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completely antagonized by concomitant administration of DNQX (2 mM) (SOM +DNQX: 89 9 3.9% vs. SOM: 190921%, PB0.001, n =6 for each group) (Fig. 2). The effect of APV, NMDA glutamatergic antagonist was also investigated. APV (100 mM) slightly decreased the basal ACh concentrations. At the same concentration APV completely block the ability of 1 mM SOM to increase ACh release (SOM alone: 189918%, vs. SOM +APV: 99 9 6%, P B0.001) (Fig. 2). All antagonists were administered together with SOM for 80 min through the dialysis probe.
4. Discussion The activity of striatal cholinergic interneurones is regulated by a delicate balance between afferent glutamatergic and dopaminergic pathways, and recurrent collaterals of descending GABAergic axons and intrinsic GABAergic interneurons (Graybiel, 1990; Harsing et al., 2000). In the striatum the neocortical afferents are thought to use glutamate as their excitatory neurotransmitter (Reubi and Cuenod, 1979; Fonnum et al., 1981; Wilson et al., 1990) which acts distally at receptors located on dendrites of striatal cholinergic interneurons causing depolarization and subsequent action potential propagation along the axon to release ACh from the nerve terminals (Lehmann and Scatton, 1982). Using immunocytochemical methods, Lapper and Bolam (1992) have demonstrated that cholinergic neurons of the striatum receive very few synaptic contacts from corticostriatal glutamatergic fibres and a massive synaptic input from the excitatory thalamostriatal pathway. The corticostriatal and the thalamostriatal synapses can be the site where glutamatergic receptors affect striatal cholinergic activity. On the other hand, the presence of SOM interneurons has been found in the striatum (Vincent et al., 1983; Kawaguchi et al., 1995) The axons of SOMpositive cells in the striatum may extend up to 1 mm from the cell body, which allows these cells to influence other neurotransmissions and the flow of blood in a larger space that do the other striatal interneurones (Johansson et al., 1984; Vuillet et al., 1989, 1992; Kawaguchi et al., 1995). SOM was found to enhance ACh-induced excitations in vivo in rat hippocampus and cortex (Mancillas et al., 1986). In our experiments it was found that SOM dose-dependently stimulated the extracellular level of ACh in the striatum. The present results are the first to demonstrate a stimulant effect of SOM on ACh release in the striatum in freely moving rats. The basal ACh level was decreased ( \ 50%) by TTX, a toxin that blocks voltage-sensitive sodium channels, suggesting that the cholinergic neurons are spontaneously active. TTX (1 mM) abolished the SOM-induced increase in ACh release indicating that action potentials mediate the stimulated release of ACh
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measured in vivo by microdialysis.The present results also showed that SOM-evoked release of ACh was probably mediated indirectly by stimulation of excitatory amino acid release through activation of NMDA and non-NMDA glutamatergic receptors. Indeed, in our experiments, the SOM-elicited actylcholine release was antagonized by glutamatergic NMDA (APV) and nonNMDA-receptor antagonists (DNQX and GYKI52466). Because GYKI 52466 is a selective AMPA receptor antagonist (Vizi et al., 1996), these data indicate that both NMDA and AMPA receptors are involved in the regulation of ACh release by SOM. Taken togther, these findings could suggest that the stimulant effect of SOM in the striatum is probably realised via SOM receptor located on the terminals of the glutamatergic corticostriatal projection neurons. In fact, in our experiments the stimulant effect of SOM on the release of ACh was antagonized by the SOM receptor antagonist Cyclo(7-Phe-D-Trp-Lys-Thr[BZL]). Supporting our data are also the hypothesis of Hathway et al. (1998, 1999) according which the receptor that mediates the effects of SOM in the striatum is located on the terminals of cortico-striatal projection neurones which contain glutamate. A schematic summary of the proposed mechanisms for the effects of SOM on the release of ACh in striatum is shown in Fig. 3.
Fig. 3. Scheme of the possible interactions between SOM and the glutamatergic-cholinergic system in the striatum. Glutamate (Glu) released from the cortico-striatal axon terminals tonically controls the release of ACh from the cholinergic interneurons. SOM released from its own neurons activates SOM receptor located on the glutamatergic nerve terminals producting the release of glutamate which in turn increases the release of ACh through activation of NMDA and non-NMDA glutamatergic receptors located on dendrites of cholinergic interneurones of the striatum.
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Our findings about the effect of SOM on ACh release in the striatum are at odds with those reported by Hathway et al. (1998) who have found that SOM stimulates in vivo striatal dopamine and GABA release by a glutamate-dependent action but they failed to observe an effect of SOM on the release of ACh in the striatum. A possible explanation of this discrepancy could be the fact that in the experiments of Hathway et al. (1998) SOM has been perfused through a vertical probe, while we administered the neuropeptide SOM through a transversal probe so it could activate the cholinergic neurons in the two striata. Furthermore, Hathway et al. (1998) used 15 min sample collection while we collected samples for 40 min. The maximal effect of SOM was long-lasting and persisted for more than two hours after its administration. In conclusion, the activity of the striatal cholinergic neurotransmission would be modulated by SOM too. SOM receptors located on the glutamatergic terminals mediate the release of glutamate through activation of NMDA and non-NMDA (probably AMPA) receptors which in turn increase the release of ACh, i.e. the increase of glutamate levels appears to be a target for the action of SOM in the striatum of awake and freely moving rats.
Acknowledgements This work was supported by grants from National Fund ‘Scientific Research’ (L-802/98), Bulgaria, International Society of Neurochemistry (ISN), Hungarian Research Fund (T-032789), Hungarian Medical Research Council (ETT 283/2000). J.P.K. is a Janos Bolyi Research Fellow. The authors are grateful to Dr M.G. Giovannini and C. Susini (Department of Pharmacology, University of Florence, Italy) for the valuable help in introducing the microdialysis method and performing HPLC analysis of ACh.
References Arneric, S.P., Reis, D., 1986. Somatostatin and cholecystokinin octapeptide differentially modulate the release of 3H-acetylcholinefrom caudate nucleus but not cerebral cortex: role of dopamine receptor activation. Brain Res. 374, 153 –161. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R., 1973. Hypothalamic polypeptide that inhibits secretion on immunoreactive pituitary growth hormone. Science 179, 77 – 79. Cattaneo, L., Muller, E., Cocchi, D., 1996. In vivo microdialysis of the hypothalamus: a suitable method to study the function of hypophysiotropic neurons in the rat. J. Neuroendocrinol. 8, 31 – 33. Chesselet, M., Reisine, T., 1983. Somatostatin regulates dopaminergic release in striatal slices and cat caudate nuclei. J. Neurosci. 3, 232– 236.
Cotman, C.W., Monaghan, D.T., Ottresen, O.P., Storm-Mathisen, J., 1987. Anatomical organization of excitatory amino acid receptors and their pathways. Trends Neurosci. 10, 273 – 280. Damsma, G., Lammerts, V.B.D., Van Bueren, D., Westerink, B.H.C., Horn, A.S., 1987. Determination of acetylcholine in the femtomole range by means of HPLC, a post-column enzyme reactor, and electro-chemical detection. Chromatographia 24, 827 – 831. Epelbaum, J., 1986. Somatostatin in the central nervous system: physiological and pathological modifications. Prog. Neurobiol. 27, 63 – 100. Fonnum, F.F., Storm-Mathisen, J., Divac, I., 1981. Biochemical evidence for glutamate as the neurotransmitter in corticostriatal and corticothalamic fibers in rat brain. Neuroscience 6, 863 –873. Giovannini, M.G., Mutolo, D., Bianchi, L., Micheassi, A., Pepeu, G., 1994. NMDA receptor antagonists decrease GABA outflow from the septum and increase acetylcholine outflow from the hippocampus: microdialysis study. J. Neurosci. 14, 1358 –1365. Graybiel, A.M., 1990. Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci. 13, 244 – 254. Harsing, L.G. Jr., Csillik-Perczel, V., Ling, I., Solyom, S., 2000. Negative allosteric modulators of AMPA-preffering receptors inhibit [(3)]GABA release in at striatum. Neurochem. Int. 37 (1), 33 – 45. Hathway, G., Emson, P., Humphrey, P., Kendrick, K., 1998. Somatostatin potently stimulates in vivo striatal dopamine and g-aminobutyric acid release by a glutamate-dependent action. J. Neurochem. 70 (4), 1740 – 1749. Hathway, G., Humphrey, P., Kendrick, K., 1999. Evidence that somatostatin ss2 receptors mediate striatal dopamine release. Br. J. Pharmacol. 128, 1346 – 1352. Johansson, O., Hokfelt, T., Elde, R.P., 1984. Immunochistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13, 265 – 339. Kawaguchi, Y., Wilson, C.J., Augood, S.J., Emson, P.S., 1995. Striatal interneurones: chemical, physiological and morphological characterization. Trends Neurosci. 18, 527 – 535. Kiss, J.P., Hennings, E.C., Zsilla, G., Vizi, E.S., 1999. A possible role of nitric oxide in the regulation of dopamine transporter function in the striatum. Neurochem. Int. 34 (4), 345 – 350. Knauber, J., Kishka, U., Roth, M., Schmidt, W.J., Hennerici, M., Fasbende, K., 1999. Modulation of striatal acetylcholine concentrations by NMDA and the competitive NMDA receptor-antagonist AP-5: an in vivo microdialysis study. J. Neural. Transm. 106 (1), 35 – 45. Lathinen, H., Brankack, J., Koivisto, E., Riekinen, P., 1992. Somatostatin release in rat neocortex during gamma-hydroxybutirateprovoked seizures: microdialysis combined with EEG recording. Brain Res. Bull 29, 837 – 841. Lapper, S.R., Bolam, J.P., 1992. Input from the frontal cortex and the parafascicular nucleus to cholinergic interneurons in the dorsal striatum of the rat. Neuroscience 51, 533 – 545. Lehmann, J., Scatton, B., 1982. Characterization of the excitatory amino acid receptor-mediated release of 3H-acetylcholine from rat striatal slices. Brain Res. 252, 77 – 89. Leviel, V., 2001. The reverse transport of DA, what physiological significance? Neurochem. Int. 38 (2), 83 – 106. Mathe, A., Nomikos, G., Svensson, T., 1993. In vivo release of somatostatin from rat hippocampus and striatum. Neurosci. Lett. 12 (149), 201 – 204. Mancillas, J.R., Siggins, G.R., Bloom, F.E., 1986. Somatostatin selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex. Proc. Natl. Acad. Sci. 83, 7518 –7521. Morary, M., Sbrenna, S., Marti, M., Caliari, F., Bianchi, C., Beani, L., 1998. NMDA and non-NMDA ionotropic glutamate receptors modulate striatal acetylcholine release via pre- and postsynaptic mechanisms. J. Neurochem. 71, 2006 – 2017.
A. Rako6ska et al. / Neurochemistry International 40 (2002) 269–275 Paxinos, G., Watson, G., 1982. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Radke, J.M., Spyraki, C., Thermos, K., 1993. Neuronal release of somatostatin in the rat striatum: an in vivo microdialysis study. Neuroscience 54 (2), 493 –498. Raichev, P., Lazarova, M., Milenov, K., 2000. Somatostatin modulation of acetylcholine release in rat striatum: a microdialysis study. C. R. Acad. Bulg. Sci. 53 (8), 69 –72. Reader, T.A., Dewar, K.M., 1999. Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinson’s disease. Neurochem. Int. 34, 1 – 21. Reubi, J.C., Cuenod, M., 1979. Glutamate release in vitro from corticostriatal terminals. Brain Res. 176, 185 –188. Scatton, B., Lehmann, J., 1982. N-methyl-D-aspartate-type receptors mediate striatal 3H-acetylcholine release evoked by excitatory amino acids. Nature 297, 422 –424. Schindler, M., Sellers, L., Humphrey, P., Emson, P., 1997. Immunohistochemical localization of the somatostatin sst2 (A) receptor in the rat brain and spinal cord. Neuroscience 76, 225 –240. Schindler, M., Humpphrey, P.P., Lohrke, S., Frauf, E., 1999. Immunochystochemical localization of the somatostatin sst2 (B) receptor slice variant in the rat central nervous system. Neuroscience 90, 859 – 874. Takahashi, H., Shintani, Y., Okauchi, T., Ishikawa, M., Bando, H., Azekawa, T., Morita, Y., Saito, S., 1994. Measurement of somatostatin release in rat brain by microdialysis. J. Neurosci. Meth. 52, 33 – 38. Thermos, K., Radke, J., Kastellakis, A., Anagnostakis, Y., Spyraki, C., 1996. Dopamine-somatostatin interactions in the rat striatum: an in vivo microdialysis study. Synapse 22, 209 –216. Ulus, I.H., Buyukuysal, R.L., Wurtman, R.J., 1992. N-methyl-D-aspartate increases acetylcholine release from rat striatum and cor-
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tex: its effect is augmented by choline. J. Pharmacy Exp. Ther. 261, 1122 – 1128. Vezzani, A., Ruiz, R., Monno, A., Rizzi, M., Lindefors, N., Samanin, R., Brodin, E., 1993. Extracellular somatostatin measured by microdialysis in the hippocampus of freely moving rats: evidence for neuronal release. J. Neurochem. 60, 671 – 677. Vincent, S., Johansson, O., 1983. Striatal neurons containing both somatostatin- and avianpancreatic polypeptide (APP)-like immunoreactivities and NADPH-diaphorase activity: a light and electronic microscopiic study. J. Comp. Neurol. 217, 264 –270. Vincent, S.R., Staines, W.A., Fibiger, H.C., 1983. Histochemical demonstration of separate populations of somatostatin and cholinergic neurons in the rat striatum. Neurosci. Lett. 35, 111 – 114. Vizi, E.S., Mike, A., Tarnava, I., 1996. 2,3-Benzodiazepines (GYKI 52466 and analogs): Negative allosteric modulators of AMPA receptors. CNS Drug Reviews, Branford, Connecticut: Neva Press, 2(1), 91 – 126. Vuillet, J., Dimova, R., Nieoullon, A., Kerkerian-Le Golf, L., 1992. Ultrastructural relationships between choine acetyltransferaseand neuropeptide Y-containing neurons in the rat striatum. Neuroscience 46, 351 – 360. Vuillet, J., Kerkerion, L., Kachidian, P., Boster, O., Nieullon, A., 1989. Ultrastructural correlates of functional relationships bethween nigral dopaminergic or cortical afferent fibers and neuropeptide-Y-containing neurons in the rat striatum. Neurosci. Lett. 100, 99 – 104. Wilson, C.J., Chang, H.T., Kitai, S.T., 1990. Firing patterns and synaptic potentials of identified giant aspiny interneurons in the rat neostriatum. J. Neurosci. 10, 508 – 519. Wisden, W., Seeberg, P.H., 1993. A complex mosaic of the affinity kainate receptors in rat brain. J. Neurosci. 13 (8), 3582 – 3598.
Somatostatin stimulates striatal acetylcholine release by glutamatergic receptors: an in vivo microdialysis study Angelina Rakovska a,*, Janos P. Kiss b, Peter Raichev a, Maria Lazarova a, Reni Kalfin a, Kiril Milenov a a
Laboratory ‘Neuropeptides’, Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonche6 Street, bl. 23, 1113 Sofia, Bulgaria b Institute of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, H-1450 Budapest, Hungary Received 19 February 2001; received in revised form 30 April 2001; accepted 4 May 2001
Abstract The modulation of striatal cholinergic neurons by somatostatin (SOM) was studied by measuring the release of acetylcholine (ACh) in the striatum of freely moving rats. The samples were collected via a transversal microdialysis probe. ACh level in the dialysate was mueasured by the high performance liquid chromatography method with an electrochemical detector. Local administration of SOM (0.1, 0.5 and 1 mM) produced a long-lasting and concentration-dependent increase in the basal striatal ACh output. The stimulant effect of SOM was antagonized by the SOM receptor antagonist Cyclo(7-Aminopentanoyl-Phe-D-TrpLys-Thr[BZL]) (1 mM). In a series of experiments, we studied the effect of 6,7-Dinitroquinoxaline-2, 3-dione (DNQX), a selective non-NMDA (N-methyl-D-aspartate) glutamatergic antagonist, on the basal and SOM-induced ACh release from the striatum. DNQX, 2 mM, perfused through the striatum had no effect on the basal ACh output but inhibited the SOM (1 mM)-induced ACh release. The non-NMDA glutamatergic receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylendioxy-5H-2,3- benzodiazepine (GYKI-52466), 10 mM, antagonized the SOM (1 mM)-induced release of ACh in the striatum. Local administration of the NMDA glutamatergic receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV), 100 mM, blocked SOM (1 mM)-evoked ACh release. Local infusion of tetrodotoxin (1 mM) decreased the basal release of ACh and abolished the 1 mM SOM-induced increase in ACh output suggesting that the stimulated release of ACh depends on neuronal firing. The present results are the first to demonstrate a neuromodulatory role of SOM in the regulation of cholinergic neuronal activity of the striatum of freely moving rats. The potentiating effect of SOM on ACh release in the striatum is mediated (i) by SOM receptor located on glutamatergic nerve terminals, and (ii) by NMDA and non-NMDA glutamatergic receptors located on dendrites of cholinergic interneurones of the striatum. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Striatum; Somatostatin; Acetylcholine release; Microdialysis; Glutamatergic receptors
1. Introduction Somatostatin (SOM) is a cyclic tetradecapeptide isolated from bovine hypothalamus (Brazeau et al., 1973). Immunohistochemical studies have demonstrated that in the rat striatum there are SOM-containing mediumsized aspiny interneurons, 10 – 40 mm in diameter (Johansson, et al., 1984) which account for 1 – 2% of the total population of interneurons in this region (Vincent et al., 1983; Kawaguchi et al., 1995). Basal levels of * Corresponding author. Tel.: + 359-2-979-2305; fax: +359-2-719109. E-mail address: [email protected], [email protected] (A. Rakovska).
SOM in the striatum of the freely moving rats was found to be 5–15 fmol (Radke et al., 1993). There are SOM receptors in the striatum (Schindler et al., 1997; Hathway et al., 1999; Schindler et al., 1999) and their number has been found to increase in patients suffering from Parkinson’s disease (Epelbaum, 1986; Reader and Dewar, 1999). SOM interneurons contain neuropeptide Y (Vincent and Johansson, 1983), neuronal nitric oxide synthase and GABA (Kawaguchi et al., 1995; Kiss et al., 1999). SOM can be measured in the extracellular space of the striatum (Radke et al., 1993), hippocampus (Mathe et al., 1993; Vezzani et al., 1993), frontal cortex (Lathinen et al., 1992) and hypothalamus (Takahashi et al., 1994; Cattaneo et al., 1996) of freely moving rats.
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SOM inhibits the release of acetylcholine (ACh) in slices of rat caudate nucleus and this release is mediated by dopaminergic mechanisms (Arneric and Reis, 1986; Leviel, 2001). In the striatum the neuropeptide stimulates the dopamine levels both in vitro (Chesselet and Reisine, 1983) and in vivo (Thermos et al., 1996). In vivo the SOM-stimulated release of dopamine is mediated by a glutamate-dependent action (Hathway et al., 1998). The glutamatergic excitatory input regulates striatal cholinergic interneurons by increasing the release of ACh through N-methyl-D-Aspartate (NMDA) (Scatton and Lehmann, 1982; Ulus et al., 1992; Knauber et al., 1999) and non-NMDA receptor activation (Cotman et al., 1987; Wisden and Seeberg, 1993; Morary et al., 1998). The role of SOM in the regulation of cholinergic neuronal activity in the striatum is still unclear. The aim of the present research was to elucidate the modulation of striatal cholinergic neurons in vivo by SOM. Trans-striatal microdialysis was used in the awake animal to study the effect of local administration of SOM on the release of ACh in the striatum. A preliminary report of this study has been presented (Raichev et al., 2000).
2. Experimental procedures
2.1. Animal housing and surgery Male adult Charles River Wistar rats weighing 250– 300 g were used. They were housed in groups with free access to food and water and kept on a 12 h light/12 h dark cycle. The rats were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic aparatus. Microdialysis tubes (AN 69 membrane; 220 mm i.d. and 310 mm o.d., molecular weight cut-off 15 000 Da; Dasco, Bologna, Italy) were inserted transversally into the striatum, following the procedure described by Giovannini et al. (1994). The microdialysis tube was covered with Super-Epoxy glue along its entire length except for a region corresponding to the brain areas to be studied (two sections of 3.5 mm separated by a glued central zone 2.5 mm long). The coordinates used for the implantation of the microdialysis tubing were: AP 0.0 and H 5 mm from bregma. (Paxinos and Watson, 1982). All coordinates were referred to bregma, with bregma and lambda on horizontal plane. Procedures involving animal and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1 Dec. 12. 1987; NIH Guide for the Care and Use of Laboratory animals, NIH Publication No. 85-23, 1985).
2.2. Microdialysis procedure One day after surgery each rat was placed in a Plexiglas cage. The inlet of the microdialysis probe was connected to a microperfusion pump and perfused with Ringer’s solution (NaCl 147 mM, CaCl2 1.2 mM, KCl 4.0 mM), containing 7 mM physostigmine sulfate at a constant flow rate of 2 ml/min using a microperfusion pump (Carnegie Medicine, Mod. CMA/100, Sweden). After an one hour stabilization period, during which the animals were perfused without collecting the dialysate, samples were collected at 40 min intervals. The content of ACh and choline (Ch) in the dialysate was analyzed by high performance liquid chromatography (HPLC) as described below. After collecting the first three samples to measure the basal outflow, drugs (dissolved in Ringer solution) were administered locally through the dialysis membrane for 80 min (4th and 5th samples). There after the drugs were withdrawn and the membrane was perfused with normal Ringer solution till the end of the experiments.
2.3. Histological control At the end of experiments rats were anaesthetized with urethane (1.2 mg/kg, ip.) and sacrified by decapitation. The brain was rapidly removed and placed in a vial containing 10 ml of 9% formaldehyde solution in phosphate buffer. Two or three days later the brain was frozen with liquid CO2 and coronal slices (50 mm) were cut using a freezing microtome and were examined by light microscopy (Nikon, Labophot-2) to verify the position of the dialysis membrane. Data obtained from rats in which the dialysis membrane was positioned outside the structure were discarded (B 5%). The conditions of the striatal neurons around the probe were checked at the end of the experiment by staining coronal sections of paraformaldehyde-fixed brains with Richardson’s solution (modified Nissal staining).
2.4. In 6itro reco6ery experiment To evaluate the passage of ACh and Ch through the microdialysis tubing, recovery experiments were performed at room temperature both in the absence and in the presence of the drugs used. The recovery of ACh and Ch from the dialysis membrane in the absence of drugs was: 569 1.4 and 5891.9%, respectively (mean9 SEM, n=13). All the drugs tested did not modify ACh and Ch recovery. The ACh and Ch values were not corrected for recovery.
2.5. Assay of acetylcholine and choline in the dialysate ACh and Ch were directly assayed in the dialysate using the HPLC method with post column enzyme
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reactor and electrochemical detector (Damsma et al., 1987). ACh and Ch were separated on a cation exchange column prepared by loading a reverse-phase column (Chromspher 5 C18, Chrompack) with sodium lauryl sulphate (0.5 mg/ml). The mobile phase consisted of 0.2 mM phosphate buffer (pH 8) containing 5 mM KCL, 1 mM tetramethylammonium (TMA) and 0.3 mM EDTA. The flow rate was 0.7 ml/min (injection volume 20 ml). ACh was hydrolyzed by acetylcholine esterase (AChE) to acetate and Ch in a postcolumn enzyme reactor; Ch was oxidized by Ch oxidase to produce betaine and hydrogen peroxide. Hydrogen peroxide was electrochemically detected by an electrochemical detector (Waters 460) equipped with a platinum electrode at + 500 mV. For the quantitative analysis of ACh and Ch, we constructed a calibration curve by spiking the Ringer’s solution with standard ACh (50 ml of 10 mM ACh solution and 50 ml of 10 mM Ch solution) in the concentration range we expected to find in the dialysates. Three or four concentrations for each calibration curve were then injected at the beginning and the end of the analysis and the peaks were then plotted against the concentrations. A regression line was calculated and quantitation of unknown samples was carried out by the method of inverse prediction. Under these experimental conditions the sensitivity limit (s/n ratio = 3/1) was 500 fmol for ACh and 250 fmol for Ch.
2.6. Materials The following chemicals, purchased from Sigma Chemical Co. (St. Louis, MO, USA) were used: AChE (E.C. 3.1.1.7., grade VI-S), Ch oxidase, Ch-oxidase (E.C.1.1.3.17), paraformaldehyde, physostigmine sulfate, homoserine, OPA, SOM-14, cyclo(7-Aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]), known to be a SOM receptor antagonist and tetrodotoxin (TTX). Glutamatergic receptor antagonists 6,7-Dinitroquinoxaline2,3-dione (DNQX) and D,L-2-amino-5-phosphonopentanoic acid (APV) were purchased from Research Biochemical Inc., RBI (Natick, MA, USA). The nonNMDA glutamatergic antagonist 1-(4-aminophenyl)-4methyl-7, 8-methylendioxil-5H-2,3- benzodiazepine (GYKI-52466) was supplied by the Institute for Drug Research, Budapest, Hungary.
2.7. Statistical analysis ACh outflow rates were expressed either as fmol/ml or as percentage variation over basal output (i.e. the mean of three samples taken before drug administration). Data are means of 9SEM. Significance of differences among experimental groups was evaluated comparing Areas Under the Curves (AUC) by analysis of variance one-way ANOVA followed by Newman-
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Keuls multiple comparison test (**P B 0.01; ***P B 0.001 vs. all other groups).
3. Results
3.1. Effects of somatostatin, cyclo(7 -aminopentanoylPhe-D -Trp-Lys-Thr[BZL]) and tetrodotoxin on the release of acetylcholine Basal ACh outflow from the striatum of freely moving rats was 24.2692.56 pmol/40 min (mean9SEM; 32 rats).The basal release of ACh remained relatively constant throughout the experiment (\6 h, Fig. 1). The initial efflux of Ch from the striatum was 125.369 10.36 pmol/40 min (mean9 SEM; 32 rats) and was unaffected by any of the tested drugs. SOM, applied for 80 min locally in the striatum induced an increase of extracellular ACh level (Fig. 1), which began about 40 min after its administration and lasted for over 3 h (Fig. 1). No behavioral activation was observed during SOM perfusion. Four concentrations of SOM (0.1, 0.5, 1 and 10 mM) were used to study its effects on ACh release which were found to be 1209 13, 145915, 2009 18 and 189913%, respectively (n= 6 for each dose, data are means9 SEM). As shown in Fig. 1, the effect was concentration-dependent with a maximum reached with 1 mM of SOM. In order to understand whether the effect of SOM on the release of ACh in the striatum was mediated by SOM receptor, the effect of the SOM receptor antagonist cyclo(7-aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]) on the release of ACh was investigated. The administration of 1 mM cyclo(7-aminopentanoyl-Phe-D-Trp-LysThr[BZL]) to the striatum did not change the basal release of ACh (variations ranging 915%; insignificant (n.s.) compared to controls). At the same concentration the SOM antagonist administered together with SOM (1 mM, 80 min) abolished the stimulant effect of SOM on the ACh release (89918% vs. SOM: 196 913%, n = 6 for each group, PB0.001) (Fig. 1). Local application of the Na+ channel blocker TTX (1 mM, dissolved in Ringer’s solution) via the dialysis probe led to a gradual decrease of ACh basal levels (Fig. 1). At the same concentration TTX completely prevented the 1 mM SOM-evoked release of ACh (Fig. 1) (n=6 ***P B 0.001). Changes in behavior of the rats were not observed during perfusion of 1 mM TTX in the striatum.
3.2. Effects of glutamatergic antagonists GYKI-52466, DNQX and APV on somatostatin induced acetylcholine release To determine whether the SOM induced ACh release was mediated via non-NMDA glutamatergic receptor,
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the effects of 1-(4-aminophenyl)-4-methyl-7, 8-methylendioxyl-5H-2,3.benzodiazepine (GYKI-52466), selective AMPA/kainate receptor antagonist and DNQX, competitive quisqualate/kainate (non-NMDA) receptor antagonist on the action of SOM (1 mM) were investigated. Local administration of GYKI-52466 (10 mM) was followed by an insignificant decrease in the basal ACh output from the striatum and was returned upon
Fig. 1. Effects of SOM on the ACh release from the striatum in freely moving rats in the absence and in the presence of TTX and cyclo(7aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]). All drugs were administered locally to the striatum via the dialysis membrane after collection of three samples, as shown by the horizontal bar. ACh output is expressed as percentage of changes over the mean of the first three samples taken to be controls (mean 9 SEM of at least six different rats in each group are presented). Dialysate samples were collected for 40 min. Data points represent basal release of ACh-controls (open circles), 0.1 mM SOM (open diamonds), 0.5 mM SOM (filled squares), 1 mM SOM (filled circles) and 1 mM SOM+ 1 mM TTX (open triangles); 1 mM SOM+ 1 mM cyclo(7-aminopentanoyl-Phe-D-TrpLys-Thr[BZL]) (crosses). The bar shown in the inset represents the mean percentage of changes calculated from 160 to 400 min: controls (open), 0.1 mM SOM (vertical), 0.5 mM SOM (diagonal crosshatched), 1 mM SOM (rising to right), 1 mM SOM+ 1 mM TTX (filled); Significant differences among the six experimental groups in the inset were evaluated comparing Area Under Curve (AUC) by one-way ANOVA, for SOM+TTX (F =21.51; PB 0.001) followed by Newman-Keuls multiple comparison test (***PB 0.001 vs. all other groups).
Fig. 2. Effects of SOM on ACh release from the striatum in freely moving rats in the absence and in the presence of 1-(4-aminophenyl)4-methyl-7,8-methyendioxyl-5H-2,3-benzodiazepine hydrochloride (GYKI 52466); 6,7-dinitroquinoxalline-2,3-dione (DNQX) and APV. All drugs were administered locally to the striatum via the dialysis membrane after collection of three samples, as shown by the horizontal bar. ACh output is expressed as percentage of changes over the mean of the first three samples taken to be controls (mean 9SEM of at least six different rats in each group are presented). Dialysate samples were collected for 40 min. Data points represent basal release of ACh-controls (open circles), 1 mM SOM (filled circles), 1 mM SOM+10 mM GYKI 52466 (open diamonds); 1 mM SOM+ 2 mM DNQX (filled triangles) and 1 mM SOM+100 mM APV (crosses). The bars shown in the inset represent the mean percentage changes calculated from 160 to 400 min: controls (open), 1 mM SOM (filled), 1 mM SOM +10 mM GYKI 52466 (horizontal); 1 mM SOM+ 2 mM DNQX (diagonal crosshartched) and 1 mM SOM+100 mM APV (vertical). Significant differences among the five experimental groups in the inset were evaluated comparing Area Under Curve (AUC) by one-way ANOVA (F =11.76; PB 0.001) followed by Newman-Keuls multiple comparison test (**PB0.01 vs. all other groups).
washout (data not shown). When SOM (1 mM) was administered together with GYKI- 52466 (10 mM) no increase in ACh output was observed (1039 21% vs. SOM: 1909 21%, n=6 for each group, n.s., PB0.001) (Fig. 2). When DNQX (2 mM), another non-NMDA receptor antagonist, was administered through the dialysis membrane a decrease in the basal release of ACh was found which however was not significant (data not shown). The stimulant effect of SOM (1 mM) was
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completely antagonized by concomitant administration of DNQX (2 mM) (SOM +DNQX: 89 9 3.9% vs. SOM: 190921%, PB0.001, n =6 for each group) (Fig. 2). The effect of APV, NMDA glutamatergic antagonist was also investigated. APV (100 mM) slightly decreased the basal ACh concentrations. At the same concentration APV completely block the ability of 1 mM SOM to increase ACh release (SOM alone: 189918%, vs. SOM +APV: 99 9 6%, P B0.001) (Fig. 2). All antagonists were administered together with SOM for 80 min through the dialysis probe.
4. Discussion The activity of striatal cholinergic interneurones is regulated by a delicate balance between afferent glutamatergic and dopaminergic pathways, and recurrent collaterals of descending GABAergic axons and intrinsic GABAergic interneurons (Graybiel, 1990; Harsing et al., 2000). In the striatum the neocortical afferents are thought to use glutamate as their excitatory neurotransmitter (Reubi and Cuenod, 1979; Fonnum et al., 1981; Wilson et al., 1990) which acts distally at receptors located on dendrites of striatal cholinergic interneurons causing depolarization and subsequent action potential propagation along the axon to release ACh from the nerve terminals (Lehmann and Scatton, 1982). Using immunocytochemical methods, Lapper and Bolam (1992) have demonstrated that cholinergic neurons of the striatum receive very few synaptic contacts from corticostriatal glutamatergic fibres and a massive synaptic input from the excitatory thalamostriatal pathway. The corticostriatal and the thalamostriatal synapses can be the site where glutamatergic receptors affect striatal cholinergic activity. On the other hand, the presence of SOM interneurons has been found in the striatum (Vincent et al., 1983; Kawaguchi et al., 1995) The axons of SOMpositive cells in the striatum may extend up to 1 mm from the cell body, which allows these cells to influence other neurotransmissions and the flow of blood in a larger space that do the other striatal interneurones (Johansson et al., 1984; Vuillet et al., 1989, 1992; Kawaguchi et al., 1995). SOM was found to enhance ACh-induced excitations in vivo in rat hippocampus and cortex (Mancillas et al., 1986). In our experiments it was found that SOM dose-dependently stimulated the extracellular level of ACh in the striatum. The present results are the first to demonstrate a stimulant effect of SOM on ACh release in the striatum in freely moving rats. The basal ACh level was decreased ( \ 50%) by TTX, a toxin that blocks voltage-sensitive sodium channels, suggesting that the cholinergic neurons are spontaneously active. TTX (1 mM) abolished the SOM-induced increase in ACh release indicating that action potentials mediate the stimulated release of ACh
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measured in vivo by microdialysis.The present results also showed that SOM-evoked release of ACh was probably mediated indirectly by stimulation of excitatory amino acid release through activation of NMDA and non-NMDA glutamatergic receptors. Indeed, in our experiments, the SOM-elicited actylcholine release was antagonized by glutamatergic NMDA (APV) and nonNMDA-receptor antagonists (DNQX and GYKI52466). Because GYKI 52466 is a selective AMPA receptor antagonist (Vizi et al., 1996), these data indicate that both NMDA and AMPA receptors are involved in the regulation of ACh release by SOM. Taken togther, these findings could suggest that the stimulant effect of SOM in the striatum is probably realised via SOM receptor located on the terminals of the glutamatergic corticostriatal projection neurons. In fact, in our experiments the stimulant effect of SOM on the release of ACh was antagonized by the SOM receptor antagonist Cyclo(7-Phe-D-Trp-Lys-Thr[BZL]). Supporting our data are also the hypothesis of Hathway et al. (1998, 1999) according which the receptor that mediates the effects of SOM in the striatum is located on the terminals of cortico-striatal projection neurones which contain glutamate. A schematic summary of the proposed mechanisms for the effects of SOM on the release of ACh in striatum is shown in Fig. 3.
Fig. 3. Scheme of the possible interactions between SOM and the glutamatergic-cholinergic system in the striatum. Glutamate (Glu) released from the cortico-striatal axon terminals tonically controls the release of ACh from the cholinergic interneurons. SOM released from its own neurons activates SOM receptor located on the glutamatergic nerve terminals producting the release of glutamate which in turn increases the release of ACh through activation of NMDA and non-NMDA glutamatergic receptors located on dendrites of cholinergic interneurones of the striatum.
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Our findings about the effect of SOM on ACh release in the striatum are at odds with those reported by Hathway et al. (1998) who have found that SOM stimulates in vivo striatal dopamine and GABA release by a glutamate-dependent action but they failed to observe an effect of SOM on the release of ACh in the striatum. A possible explanation of this discrepancy could be the fact that in the experiments of Hathway et al. (1998) SOM has been perfused through a vertical probe, while we administered the neuropeptide SOM through a transversal probe so it could activate the cholinergic neurons in the two striata. Furthermore, Hathway et al. (1998) used 15 min sample collection while we collected samples for 40 min. The maximal effect of SOM was long-lasting and persisted for more than two hours after its administration. In conclusion, the activity of the striatal cholinergic neurotransmission would be modulated by SOM too. SOM receptors located on the glutamatergic terminals mediate the release of glutamate through activation of NMDA and non-NMDA (probably AMPA) receptors which in turn increase the release of ACh, i.e. the increase of glutamate levels appears to be a target for the action of SOM in the striatum of awake and freely moving rats.
Acknowledgements This work was supported by grants from National Fund ‘Scientific Research’ (L-802/98), Bulgaria, International Society of Neurochemistry (ISN), Hungarian Research Fund (T-032789), Hungarian Medical Research Council (ETT 283/2000). J.P.K. is a Janos Bolyi Research Fellow. The authors are grateful to Dr M.G. Giovannini and C. Susini (Department of Pharmacology, University of Florence, Italy) for the valuable help in introducing the microdialysis method and performing HPLC analysis of ACh.
References Arneric, S.P., Reis, D., 1986. Somatostatin and cholecystokinin octapeptide differentially modulate the release of 3H-acetylcholinefrom caudate nucleus but not cerebral cortex: role of dopamine receptor activation. Brain Res. 374, 153 –161. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R., 1973. Hypothalamic polypeptide that inhibits secretion on immunoreactive pituitary growth hormone. Science 179, 77 – 79. Cattaneo, L., Muller, E., Cocchi, D., 1996. In vivo microdialysis of the hypothalamus: a suitable method to study the function of hypophysiotropic neurons in the rat. J. Neuroendocrinol. 8, 31 – 33. Chesselet, M., Reisine, T., 1983. Somatostatin regulates dopaminergic release in striatal slices and cat caudate nuclei. J. Neurosci. 3, 232– 236.
Cotman, C.W., Monaghan, D.T., Ottresen, O.P., Storm-Mathisen, J., 1987. Anatomical organization of excitatory amino acid receptors and their pathways. Trends Neurosci. 10, 273 – 280. Damsma, G., Lammerts, V.B.D., Van Bueren, D., Westerink, B.H.C., Horn, A.S., 1987. Determination of acetylcholine in the femtomole range by means of HPLC, a post-column enzyme reactor, and electro-chemical detection. Chromatographia 24, 827 – 831. Epelbaum, J., 1986. Somatostatin in the central nervous system: physiological and pathological modifications. Prog. Neurobiol. 27, 63 – 100. Fonnum, F.F., Storm-Mathisen, J., Divac, I., 1981. Biochemical evidence for glutamate as the neurotransmitter in corticostriatal and corticothalamic fibers in rat brain. Neuroscience 6, 863 –873. Giovannini, M.G., Mutolo, D., Bianchi, L., Micheassi, A., Pepeu, G., 1994. NMDA receptor antagonists decrease GABA outflow from the septum and increase acetylcholine outflow from the hippocampus: microdialysis study. J. Neurosci. 14, 1358 –1365. Graybiel, A.M., 1990. Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci. 13, 244 – 254. Harsing, L.G. Jr., Csillik-Perczel, V., Ling, I., Solyom, S., 2000. Negative allosteric modulators of AMPA-preffering receptors inhibit [(3)]GABA release in at striatum. Neurochem. Int. 37 (1), 33 – 45. Hathway, G., Emson, P., Humphrey, P., Kendrick, K., 1998. Somatostatin potently stimulates in vivo striatal dopamine and g-aminobutyric acid release by a glutamate-dependent action. J. Neurochem. 70 (4), 1740 – 1749. Hathway, G., Humphrey, P., Kendrick, K., 1999. Evidence that somatostatin ss2 receptors mediate striatal dopamine release. Br. J. Pharmacol. 128, 1346 – 1352. Johansson, O., Hokfelt, T., Elde, R.P., 1984. Immunochistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13, 265 – 339. Kawaguchi, Y., Wilson, C.J., Augood, S.J., Emson, P.S., 1995. Striatal interneurones: chemical, physiological and morphological characterization. Trends Neurosci. 18, 527 – 535. Kiss, J.P., Hennings, E.C., Zsilla, G., Vizi, E.S., 1999. A possible role of nitric oxide in the regulation of dopamine transporter function in the striatum. Neurochem. Int. 34 (4), 345 – 350. Knauber, J., Kishka, U., Roth, M., Schmidt, W.J., Hennerici, M., Fasbende, K., 1999. Modulation of striatal acetylcholine concentrations by NMDA and the competitive NMDA receptor-antagonist AP-5: an in vivo microdialysis study. J. Neural. Transm. 106 (1), 35 – 45. Lathinen, H., Brankack, J., Koivisto, E., Riekinen, P., 1992. Somatostatin release in rat neocortex during gamma-hydroxybutirateprovoked seizures: microdialysis combined with EEG recording. Brain Res. Bull 29, 837 – 841. Lapper, S.R., Bolam, J.P., 1992. Input from the frontal cortex and the parafascicular nucleus to cholinergic interneurons in the dorsal striatum of the rat. Neuroscience 51, 533 – 545. Lehmann, J., Scatton, B., 1982. Characterization of the excitatory amino acid receptor-mediated release of 3H-acetylcholine from rat striatal slices. Brain Res. 252, 77 – 89. Leviel, V., 2001. The reverse transport of DA, what physiological significance? Neurochem. Int. 38 (2), 83 – 106. Mathe, A., Nomikos, G., Svensson, T., 1993. In vivo release of somatostatin from rat hippocampus and striatum. Neurosci. Lett. 12 (149), 201 – 204. Mancillas, J.R., Siggins, G.R., Bloom, F.E., 1986. Somatostatin selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex. Proc. Natl. Acad. Sci. 83, 7518 –7521. Morary, M., Sbrenna, S., Marti, M., Caliari, F., Bianchi, C., Beani, L., 1998. NMDA and non-NMDA ionotropic glutamate receptors modulate striatal acetylcholine release via pre- and postsynaptic mechanisms. J. Neurochem. 71, 2006 – 2017.
A. Rako6ska et al. / Neurochemistry International 40 (2002) 269–275 Paxinos, G., Watson, G., 1982. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Radke, J.M., Spyraki, C., Thermos, K., 1993. Neuronal release of somatostatin in the rat striatum: an in vivo microdialysis study. Neuroscience 54 (2), 493 –498. Raichev, P., Lazarova, M., Milenov, K., 2000. Somatostatin modulation of acetylcholine release in rat striatum: a microdialysis study. C. R. Acad. Bulg. Sci. 53 (8), 69 –72. Reader, T.A., Dewar, K.M., 1999. Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinson’s disease. Neurochem. Int. 34, 1 – 21. Reubi, J.C., Cuenod, M., 1979. Glutamate release in vitro from corticostriatal terminals. Brain Res. 176, 185 –188. Scatton, B., Lehmann, J., 1982. N-methyl-D-aspartate-type receptors mediate striatal 3H-acetylcholine release evoked by excitatory amino acids. Nature 297, 422 –424. Schindler, M., Sellers, L., Humphrey, P., Emson, P., 1997. Immunohistochemical localization of the somatostatin sst2 (A) receptor in the rat brain and spinal cord. Neuroscience 76, 225 –240. Schindler, M., Humpphrey, P.P., Lohrke, S., Frauf, E., 1999. Immunochystochemical localization of the somatostatin sst2 (B) receptor slice variant in the rat central nervous system. Neuroscience 90, 859 – 874. Takahashi, H., Shintani, Y., Okauchi, T., Ishikawa, M., Bando, H., Azekawa, T., Morita, Y., Saito, S., 1994. Measurement of somatostatin release in rat brain by microdialysis. J. Neurosci. Meth. 52, 33 – 38. Thermos, K., Radke, J., Kastellakis, A., Anagnostakis, Y., Spyraki, C., 1996. Dopamine-somatostatin interactions in the rat striatum: an in vivo microdialysis study. Synapse 22, 209 –216. Ulus, I.H., Buyukuysal, R.L., Wurtman, R.J., 1992. N-methyl-D-aspartate increases acetylcholine release from rat striatum and cor-
275
tex: its effect is augmented by choline. J. Pharmacy Exp. Ther. 261, 1122 – 1128. Vezzani, A., Ruiz, R., Monno, A., Rizzi, M., Lindefors, N., Samanin, R., Brodin, E., 1993. Extracellular somatostatin measured by microdialysis in the hippocampus of freely moving rats: evidence for neuronal release. J. Neurochem. 60, 671 – 677. Vincent, S., Johansson, O., 1983. Striatal neurons containing both somatostatin- and avianpancreatic polypeptide (APP)-like immunoreactivities and NADPH-diaphorase activity: a light and electronic microscopiic study. J. Comp. Neurol. 217, 264 –270. Vincent, S.R., Staines, W.A., Fibiger, H.C., 1983. Histochemical demonstration of separate populations of somatostatin and cholinergic neurons in the rat striatum. Neurosci. Lett. 35, 111 – 114. Vizi, E.S., Mike, A., Tarnava, I., 1996. 2,3-Benzodiazepines (GYKI 52466 and analogs): Negative allosteric modulators of AMPA receptors. CNS Drug Reviews, Branford, Connecticut: Neva Press, 2(1), 91 – 126. Vuillet, J., Dimova, R., Nieoullon, A., Kerkerian-Le Golf, L., 1992. Ultrastructural relationships between choine acetyltransferaseand neuropeptide Y-containing neurons in the rat striatum. Neuroscience 46, 351 – 360. Vuillet, J., Kerkerion, L., Kachidian, P., Boster, O., Nieullon, A., 1989. Ultrastructural correlates of functional relationships bethween nigral dopaminergic or cortical afferent fibers and neuropeptide-Y-containing neurons in the rat striatum. Neurosci. Lett. 100, 99 – 104. Wilson, C.J., Chang, H.T., Kitai, S.T., 1990. Firing patterns and synaptic potentials of identified giant aspiny interneurons in the rat neostriatum. J. Neurosci. 10, 508 – 519. Wisden, W., Seeberg, P.H., 1993. A complex mosaic of the affinity kainate receptors in rat brain. J. Neurosci. 13 (8), 3582 – 3598.