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Effects of repeated injections of the neurotensin analog NT69L on dopamine release and uptake in rat striatum in vitro
Brain Research 1025 (2004) 21 – 28 www.elsevier.com/locate/brainres
Research report
Effects of repeated injections of the neurotensin analog NT69L on dopamine release and uptake in rat striatum in vitro Rui Wang, Mona Boules, William Tiner, Elliott Richelson* Neuropsychopharmacology Laboratory, Mayo Foundation for Medical Education and Research, and Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA Accepted 9 July 2004 Available online 11 September 2004
Abstract The effect of five daily intraperitoneal (i.p.) injections of NT69L on in vitro dopamine release, uptake, and [3H]NT binding in rat striatal tissue was investigated. NT69L perfusion increased K+-evoked and electrically evoked [3H]DA release. NT receptor-1 antagonist SR48692 inhibited the stimulatory effect of NT69L on K+-evoked [3H]DA release, but not on electrical depolarization. Pretreatment with NT69L, in vivo, daily for 5 days, did not cause significant change in K+ evoked [3H]DA release, but reduced electrically evoked [3H]DA release induced by NT69L perfusion. Repeated perfusion with NT69L in vitro caused marked reduction on K+-evoked [3H]DA release and no change in electrically evoked [3H]DA release. [3H]NT binding was not significantly changed by one injection but was decreased after five injections of NT69L. Desensitization to the effects of NT69L in vitro was different depending upon whether tissue was preexposed to the compound in vivo or in vitro. These results provide further proof for the involvement of different NT receptor subtypes in mediating the effect of NT69L on dopamine release evoked by K+ or electrical depolarization. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Receptor modulation, up- and down-regulation Keywords: Dopamine release; Tolerance; Neurotensin receptor; Striatum; Rat
1. Introduction Neurotensin (NT) was isolated from bovine hypothalamus [7] and was subsequently shown to be widely and heterogeneously distributed in the central nervous system (CNS) [14] and in the digestive tract [39] of mammals. NT exerts a wide range of pharmacological effects in laboratory animals. The interaction between NT, via its receptors, and central dopaminergic systems has been well documented. NT functionally antagonizes dopamine in the mesolimbic system, while increasing dopamine transmission in the nigrostriatal system. It acts as a neuromodulator in the brain and, in particular, as a modulator of dopamine transmission in the nigrostriatal and mesocorticolimbic systems [1,36]. * Corresponding author. Tel.: +1 904 953 2439; fax: +1 904 935 2482. E-mail address: [email protected] (E. Richelson). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.07.069
Many preclinical studies have shown that injection of NT into brain has remarkable similarities to systemically administered antipsychotic drugs. In fact, centrally administered NT behaves in certain paradigms like an atypical neuroleptic drug, such as clozapine [24,25]. That is, it induces antipsychotic-like effects without causing motor disturbances. Such findings, together with studies showing that administration of antipsychotic drugs increases NT content in discrete brain nuclei [20,33,43], have led researchers to suggest that at least some of the beneficial clinical effects of antipsychotic treatment are mediated via enhanced NT neurotransmission [35,28,32]. NT mediates its effects through its receptors [49,31]. Therefore, direct activation of NT receptors in brain might have therapeutic effects in certain neuropsychiatric diseases and in abuse of psychostimulants. Roles for the three molecularly cloned NT receptors in brain are far from being
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fully elucidated. However, it is very likely that in rats and in mice, NTR-1 activation mediates antinociception, hypothermia, hypotension, and sedation [46,38]. NTR-2 may be associated with long-term metabolic effects, rather than with rapid, synaptic actions [41]. However, NT does not cross the blood–brain barrier (BBB), thus, it must be directly injected into the CNS to cause its effects. Over the years, many groups, including ours, have been developing NT receptor agonists that could be delivered systemically and cross the highly selective BBB. One of these compounds, developed by our group, is a brain-penetrating analog of the 8–13 fragment of NT (NT69L). In binding studies, NT69L has high affinity for human neurotensin receptor NTR-1 [9,47] and for human NTR-2 (unpublished observations). CNS effects of NT69L in rats after intraperitoneal (i.p.) injection include hypothermia, antinociception [9,48], and reduced food intake [5]. Additionally, pretreatment of rats with NT69L blocks the catalepsy caused by haloperidol [9], the climbing behavior caused by apomorphine [9], the turning behavior caused by d-amphetamine and apomorphine in rats with supersensitive dopamine receptors after unilateral lesioning of the nigrostriatal pathway [3], and blocks the locomotor stimulatory effects of the psychostimulants cocaine, d-amphetamine [4], and nicotine [16]. Interestingly, also in rats, we have shown that pretreatment with NT69L 30 min before nicotine once per week for 5 weeks blocks the behavioral sensitization seen with nicotine [17]. Our in vivo studies [6] have shown that tolerance develops rapidly (after a single dose) to some effects of NT69L (e.g., hypothermia and antinociception) and very slowly if at all (no tolerance after five daily doses) to other effects (e.g., blockade of the hyperactivity caused by psychostimulants) of this peptide. Here, we present the results of acute versus repeated treatment of animals with NT69L on dopamine release and uptake in striatal slices and synaptosomes in vitro, respectively, as well as, binding sites for [3H]NT in striatal slices by autoradiographic techniques.
2. Materials and methods 2.1. Animals, chemicals, and experimental treatment Male Sprague–Dawley rats weighing 150–250 g were divided into two groups of 4–8 rats and housed under 12-h light/dark conditions and allowed free access to food and water. All procedures were approved by the Mayo Foundation Institutional Animal Use and Care Committee. The rats were injected intraperitoneally with either NT69L (2 mg/kg) or an equal volume of saline once or daily for 5 days. Tissue from untreated rats (3–8) was used for dose–response studies, the double-stimulation of NT69L and for the antagonistic effect of SR48692. SR48692 {2-[(1-(7-chloro4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo (3.3.1.1.3.7)decan-2-carboxylic acid} was synthesized and generously provided by Sanofi
Synthe´labo (Toulouse, France). NT69L, synthesized by the Mayo Protein Core Facility as previously described [9], was dissolved in saline (0.9% NaCl) for in vivo intraperitoneal administration or in Kreb’s medium for in vitro perfusion. SR48692 was solubilized in dimethyl sulfoxide (DMSO). All other chemicals were from commercial sources. 2.2. Dopamine release Striatal slices were prepared as previously described [10]. Briefly, rats were killed by decapitation 1 h after their last injection. The brains were quickly removed and immersed for about 30 s in chilled (4 8C) Kreb’s media (in mM: NaCl 125, KCl 5, CaCl2 1.2, MgCl2 1.1, NaHCO3 20, NaH2PO4 1.2, glucose 10, pH 7.4). All buffers were equilibrated with atmospheric oxygen throughout the experiments. Subsequently the striata were dissected free of surrounding tissue and rapidly placed on the moistened circular stage of McIIwain chopper (Bachofer, Germany). 0.4 mm thick coronal slices of the striatum were prepared and immediately transferred to a preincubation chamber containing Kreb’s buffer at room temperature (22–24 8C) to allow recovery from the slicing procedure. After 60 min preincubation, slices were suspended in 1 ml of Kreb’s medium containing 1 AM pargyline, 1 mM ascorbic acid, 0.1 AM desipramine, saturated with 5% CO2 and 95% O2. [3H]DA (120 nM, 20 Ci/mmol; Moravek Biochemicals, Brea, CA) was added and the incubation was carried out at 37 8C for 30 min under a continuous flow of 5% CO2 and 95% O2. After rinsing with Kreb’s medium, three slices were transferred into a chamber (World Precision Instruments, Sarasota, FL) connected to two electrodes and perfused at 35 8C with Kreb’s solution (0.5 ml/min) for 60 min after which 3-min serial fractions (1.5 ml) were collected. The first nine fractions were used to establish basal release. The neurotensin receptor antagonist SR142948A (10 7 M) or the same dilution of the vehicle, DMSO, was added 18 min before depolarization. NT69L (10 11 to 10 7 M) was perfused 9 min before depolarization. To compare the effects of successive treatments of NT69L in vitro to 5 days pretreatment of NT69L in vivo on K+-evoked and electrically evoked [3H]DA release, two successive homologous depolarizations (K+ followed by K+ or electric followed by electric) were performed during the perfusion on slices from untreated animals. There was a 45 min washout period between the two depolarizations. With potassium, depolarization was performed using a 6 min application of a buffer containing 20 mM KCl. For electrical depolarization, electric field stimulation was applied in 5 trains of seven monophasic, rectangular pulses (1 ms duration, 22 mA intensity, 7 Hz frequency; Grass-Telefactor S48 Stimulator, W. Warwick, RI). At the end of the experiment, the slices were removed from the chamber and solubilized in Soluene350 (Perkin–Elmer Life Sciences, Boston, MA) for measurement of total radioactivity. The radioactivity was measured in 0.5 ml of each fractional perfusate with a
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Beckman LS6000TA liquid scintillation counter (Fullerton, CA). The fractional efflux of [3H]DA was expressed as percentage of the amount of radioactivity in the perfusate fraction, relative to the initial total amount of radioactivity in the slices. The initial amount of radioactivity was estimated from the sum of radioactivity measured in the solubilized slices at the end of the perfusion and the total radioactivity collected in the medium samples throughout the experiment. To determine stimulated release, we subtracted the basal efflux of radioactivity in the fraction prior to stimulation, from each of the subsequent three KCl fractions or electrical depolarization fractions. These values were then summed, averaged, and expressed as meansFS.E.M. of three to eight independent determinations. 2.3. Measurement of [3H]DA uptake into synaptosomes The synaptosomal uptake assay was conducted as described previously [40]. The assay was performed in Krebs’–Ringer’s buffer containing 0.64 mM ascorbic acid, 0.8 mM pargyline, and 0.1 AM [3H]DA (20 Ci/mmol). Various concentrations of nonradioactively labeled DA were added to give the final concentrations in the range of 6.310 6 to 110 2 M. The uptake assay was initiated by the addition of aliquots (50–100 Ag) of the synaptosomal fraction followed by incubation for 10 min at 37 8C. The assay was terminated by placing the samples on ice and adding 5 ml of ice-cold Krebs’–Ringer’s buffer. The synaptosomes were filtered through Whatman glass microfiber filters (GF/B), which had been presoaked in 0.1% polyethylenimine to reduce nonspecific binding, using a Brandel cell-harvester filtration apparatus. The synaptosomes, trapped on the filters, were washed twice with 5 ml of ice-cold Krebs’–Ringer’s buffer. The radioactivity of filters was determined by liquid scintillation spectrometry as described before. Protein concentration was measured using the Bio-Rad assay (Hercules, CA). 2.4. Determination of the levels of NT receptors in the striatum by autoradiography Adult male Sprague–Dawley rats 200–250 g were injected with NT69L or saline, once or daily for 5 days. The rats were sacrificed 1 h after the last injection by decapitation and their brains were rapidly frozen on dry ice and then stored at 80 8C until used. Coronal sections (20 Am) were cut on a cryostat at 16 8C, mounted on superfrost plus slides (VWR), and stored at 20 8C. Binding on sections was done as described by others [29], except for the use of a different radioligand. Briefly, neurotensin binding was performed by incubating the sections with 10 nM tritium labeled neurotensin {[3,11tyrosyl-3,5-3H(N)]NT}(91 Ci/mmol) in 50 mM Tris–HCl buffer containing 5 mM MgCl2, 0.2% bovine serum albumin and 50 AM bacitracin, pH 7.6, at 4 8C for 60 min. Additional sections were incubated in the presence of 1 AM nonradioactively labeled NT for determinations of nonspecific
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binding. The slides were washed in 40 mM Tris–HCl, pH 7.4, at 4 8C in two consecutive baths for 10 s. Slides were then dried by cool air. Film radiographs were processed by apposition of the radiolabeled section to Kodak MR film with 3H standards (Amersham Biosciences), exposed for 4 weeks and developed for 2 min in Kodak developer, fixer, and was then rinsed in water. The film was scanned and the optical density (OD) of the autoradiogram quantitated using the MCID-M5+ (version 5.1) software (Image Research, Ontario, Canada). The optical densities of nonspecific binding were subtracted from total binding to obtain specific binding. Measurements were done bilaterally on 4 brain sections. The values were expressed as nCi/mg wet tissue weight by using autoradiographic radiolabeled microscales (Amersham) that were exposed together with the tissue sections. The binding in different brain structures were identified using the rat brain atlas [37]. 2.5. Statistical analysis Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s test for multiple comparisons and t-test, using SigmaStat 2.0 software (SPSS, Chicago, IL), with Pb0.05 being considered significant.
3. Results 3.1. Effects of NT receptor agonist NT69L and antagonist SR48692 on K+-evoked and electrically evoked [3H]DA release In striatal slices prelabeled with [3H]DA, 9-min perfusion with the NT receptor agonist NT69L or 18-min perfusion with antagonist SR48692 (up to 10 7 M) had no effect on spontaneous, basal [3H]DA release (data not shown). However, NT69L increased K+-evoked and electrically evoked [3H]DA release in a concentration-dependent manner (Fig. 1A) reaching a plateau around 10 8 M. Concentration above 10 8 M caused desensitization for both types of stimulation. The EC50 and E max were 0.15 nM (95% confidence interval: 0.02 to 1 nM) and 200% over control (95% confidence interval: 100 to 300%), and 0.3 nM (95% confidence interval: 0.002 to 20 nM) and 400% of control (95% confidence interval: 100 to 800%), for the effect of NT69L on K+-evoked and electrically evoked [3H]DA release, respectively. The NT receptor antagonist, SR48692, blocked the stimulatory effect of NT69L on K+-evoked [3H]DA release ( Pb0.05), but not on electrically evoked [3H]DA release ( PN0.05; Fig. 1B). 3.2. Effect of NT69L on K+-evoked and electrically evoked [3H]DA release after five daily injections of NT69L A 6-min 20 mM K+ pulse and electrical depolarization caused an average of 0.04F0.02% and 0.05F0.05 %
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Fig. 1. Effect of NTR agonist (NT69L) and antagonist (SR48692) on [3H]DA release evoked by potassium and electrical depolarization in rat striatal slices. NT69L was added 9 min before K+ (20 mM, 6 min) or electrical depolarization (1 ms, 22 mA, 7 Hz, 40 s) and remained until the end of the stimulations. SR48692 was added 9 min before NT69L perfusion and were maintained during NT69L perfusion and depolarization. Each value represents meanFS.E.M. of four to eight independent determinations in triplicate. (A) Dose–response curve for the effect of NT69L on [3H]DA release evoked by potassium (E) and electrical (n) depolarization in rat striatal slices. [3H]DA release was expressed as percentage of stimulation of K+ or electrical depolarization above the baseline with Kreb’s medium plus K+- or electrically-evoked [3H]DA release as control. (B) Inhibition of NT69L-induced enhancement of K+or electrical-evoked [3H]DA release by SR48692. NT69L plus the vehicle of antagonist were used as control. Student’s t-test was used to determine the difference between control and antagonist treatment in each depolarization. *Pb0.05 vs. control.
increase, respectively, in the fractional rate of release over unstimulated release in the slices pretreated with saline for 5 days in vivo, Fig. 2A–B. Superfusion of the slices (pretreated with saline for 5 days in vivo) with 10 nM NT69L caused a significant increase in K+-evoked and electrically evoked [3H]DA release. Although the average enhancement evoked by electrical depolarization was greater than that with potassium-evoked release, there was no significant difference between the two stimulations. NT69L perfusion of slices from animals treated with five daily injections of NT69L in vivo caused an 80% reduction in electrically evoked [3H]DA release as
Fig. 2. Effect of chronic administration of NT69L in vivo on [3H]DA release evoked by K+-induced and electrically induced depolarization in striatal slices. Rats were treated with NT69L (2 mg/kg, i.p.) or saline for 5 days in vivo. Rats were sacrificed 1 h after the last injection and striatal slices were prepared as detailed in materials and methods. NT69L (10 8 M) or Kreb’s media were added 9 min before K+ (20 mM, 6 min) or electrical depolarization (1 ms, 22 mA, 7 Hz, 40 s) and remained until the end of the stimulations. Radioactivity in 3-min fractions is expressed as fractional release (percent of total tissue radioactivity at the start of the collection period). K+-evoked (A) or electrically evoked (B) fractional release of [3H]DA in different treatments. (C) Expressed as increased fractional release of [3H]DA over basal release. About four to eight independent determinations in triplicate were carried out in each group. Data is expressed as meansFS.E.M. Student’s t-test was used to determine statistical significance. *Pb0.05, **Pb0.01 vs. saline+Kreb’s control; # Pb0.05 vs. saline+NT69L treatment.
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compared to five daily injections of saline ( P=0.009; Fig. 2C). However, perfusion of NT69L in vitro still caused significant increase of [3H]DA release evoked by high potassium stimulation after five daily treatment of NT69L in vivo (Fig. 2C). 3.3. Effect of two successive applications of NT69L on K+evoked and electrically evoked [3H]DA release To compare the difference between five daily injections of NT69L in vivo and repeated applications of NT69L in vitro, two successive K+ or electrical depolarizations were performed. Compared to control slices (NT69L was replaced by Kreb’s medium), the first exposure to NT69L (10 8 M) induced a significant increase in both K+-evoked and electrically evoked [3H]DA release. However, with the second application of NT69L (10 8 M), slices lost responsiveness to NT69L on K+, but not the electrically evoked [3H]DA release which was significantly increased over control (160F10%, P=0.01; Table 1). 3.4. Effect of NT69L after five daily injections of NT69L on [3H]DA uptake into synaptosomes Five daily injections of NT69L had no effect on either the K t (concentration at one-half maximal transport) or the V max for transport of [3H]DA. The best-fit K t values (F S.D.) of [3H]DA uptake in synaptosomes from animals treated for 5 days with NT69L or with saline were identical at 2.0 F 0.510 4 M. Their V max values (F S.D.) were not significantly different at 5.9 F 0.510 9 and 4.8 F 0.410 9.
Fig. 3. Effect of NT69L on [3H]NT binding to striatal slices by autoradiographic techniques. Four 20-Am sections in each group were incubated in 10 nM [3H]NT for 1 h, washed, and processed as described in Materials and methods. The films were scanned and the optical density (OD) of the autoradiogram was quantitated using the MCID-M5+ (version 5.1) software. Sections from 4 rats in each treatment were analyzed. Shown are meanFS.E.M. *Pb0.05 vs. saline control and #Pb0.05 vs. one injection of NT69L.
3.5. Effect of five daily administrations of N69L on NT receptor binding in striatum There was no significant difference between one injection and five daily injections of saline on [3H]NT binding (data not shown). Five daily treatments of NT69L significantly decreased [3H]NT binding in striatum compared to saline control and one treatment of NT69L, respectively ( pb0.05). The reduction of one injection of NT69L was not significantly different from control (Figs. 3 and 4). The
Table 1 Effects of two successive applications of NT69L in vitro on [3H]DA fractional release under potassium or electrical depolarization [3H]DA fractional release (%) First stimulation, S1
Second stimulation, S2
Prestimulation
Poststimulation
Prestimulation
Poststimulation
Potassium Kreb’s control +NT69L (10 8 M) %Kreb’s control
0.55F0.04 0.50F0.03 91F5%
0.56F0.05 0.70F0.04* 125F7%
0.28F0.03 0.26F0.04 93F14%
0.30F0.02 0.28F0.04 90F10%
Electrical Kreb’s control +NT69L (10 8 M) %Kreb’s control
0.52F0.05 0.64F0.10 123F15%
0.56F0.07 0.96F0.15* 170F30%
0.26F0.03 0.24F0.03 92F12%
0.27F0.03 0.43F0.04** 160F10%
Slices of nontreated rat striatum labeled with [3H]DA were stimulated twice with two successive K+ (20 mM, 6 min) depolarizations or with two successive electrical depolarizations (1 ms, 22 mA, 7 Hz, 40 s). In each case, a 45-min washout period was allowed between depolarizations. The medium with NT69L (10 8 M) was switched into chambers 9 min before each depolarization and remained during the duration of depolarization. Shown are meansFS.E.M. Values are expressed as the sum of the amount of [3H]DA in the superfusion medium collected from three fractions after stimulation as a percentage of the stored [3H]DA in the tissue (see Materials and methods for more information on the determination of fractional release). +NT69L (10 8 M) represents NT69L perfusion before and during depolarization of slices instead of Kreb’s media. Each value represents the mean of three independent determinations in triplicate. T-test was used for testing the difference between Kreb’s control and NT69L treatment in each depolarization. * Pb0.05 vs. Kreb’s control. ** Pb0.01 vs. Kreb’s control.
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Fig. 4. Representative autoradiograms of brain sections of rat showing (A) saline control (five daily injections); (B) one injection of NT69L and (C) five daily injections of NT69L.
results suggest that NT receptors were downregulated after five daily injections of NT69L.
4. Discussion High potassium and electrical stimulation are two approaches used by most researchers to evoke the release of neurotransmitters in vivo and in vitro. Most studies in which potassium was used to evaluate DA release, involved in vivo or in vitro infusion of striatal tissue with high potassium solution for a short period [11,18,19,30,42]. High potassium produces a continuous depolarization of neurons, leading to sustained elevations in intracellular calcium.
Electrical fields and currents applied to the nervous systems generally cause excitation and generate synchronized activity. Electrical stimulation has traditionally been used in the nervous system to excite axons and neurons for studying basic mechanisms or to replace a function lost following injury. Numerous studies have shown that relative low-stimulation frequencies are required to cause dopamine release from the mesotelencephalic dopamine populations. For instance, activation of dopamine neurons in the rat striatum is evoked at 5.0–15 Hz [13,34], in the rabbit striatum at 10 Hz [8], and in the cat caudate at 30 Hz [12]. Although it has been proven that the dopamine release evoked both by potassium and electrical stimulation is calcium independent [12], different mechanisms might be involved in either case. Much evidence has accumulated implicating different neurotensin receptors in dopamine release [2,15,22,23]. However, the precise mechanism is still unclear. Similar to neurotensin (NT), the present study showed that NT69L, a novel neurotensin receptor agonist, stimulated both K+evoked and electrically evoked [3H]DA release in dosedependent manner. These in vitro results are similar to those reported for NT by others [22,15]. Additionally, our data support the hypothesis that distinct NT receptors are involved in the control of [3H]DA release, depending on the type of depolarization used, electrical versus chemical. The fact that SR48692, a putative high-affinity receptor (NTR-1) antagonist, blocked the effect of NT69L on K+ but not on electrical depolarization, further indicates that distinct neurotensin receptors are involved in the effect of NT69L on either type of depolarization. The second important, and perhaps more conclusive, observation is related to the differential profile for the effects of NT69L on K+-evoked and electrically evoked [3H]DA release after five daily injections of NT69L in vivo. There was no development of tolerance to K+-evoked [3H]DA release, but electrically evoked [3H]DA release was significantly reduced. These results strongly suggest that different neurotensin receptors are involved in the facilitation of NT69L on [3H]DA release evoked by K+ and electrical stimulation. Rapid development of tolerance to some of NT69L effects, namely hypothermia, antinociception, and blockade of catalepsy, but slowly, if at all, to other effects such as apomorphine-induced climbing and amphetamine-induced activity, has been previously reported in rats [6]. The evidence above strongly supports that different neurotensin receptors are involved in the differential profile of NT69Linduced tolerance. The slow-to-develop tolerance to some effects induced by NT69L, such as apomorphine-induced climbing behavior and blockade of d-amphetamine and cocaine-induced hyperactivity, may be mediated through NTR-1. Effects to which tolerance develops rapidly, such as antinociception and hypothermia, may be mediated by other NTRs, such as NTR-2. However, our previous antisense and antigene studies [44,45], as well as studies with mice
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lacking NTR-1 [38], suggest that NTR-1 receptors mediate the hypothermic and antinociceptive effects of NT agonists. In addition to testing the effect of repeated in vivo injections or in vitro applications of NT69L on the [3H] DA release from rat striatal slices, the effect of repeated injections of NT69L on [3H]NT binding and uptake was studied. Five daily injections of NT69L significantly reduced striatal [3H]NT binding, suggesting that NT receptors were downregulated by the treatment of five daily injections of NT69L and that there was no significant difference in dopamine uptake between chronic administration of NT69L and saline controls. From our results and those in the literature: (1)
(2)
(3)
(4)
(5)
Chronic administration of NT69L resulted in development of tolerance to electrically evoked but not to K+evoked [3H]DA release; SR48692, a putative high-affinity receptor (NTR-1) antagonist, blocked the effect of NT69L on K+-evoked [3H]DA release, but had no effect on electrically evoked [3H]DA release; SR48692 inhibits most of the in vitro and in vivo effects of NT and confirms the modulatory role of NT in the central nervous system, in particular, in regulating the activity of DA neurons, and its inability to antagonize hypothermia and analgesia induced by neurotensin. However, SR142948A, a nonselective neurotensin receptor antagonist, is able to reduce markedly NT-induced hypothermia and analgesia [21]. Five daily injections of NT69L significantly reduced striatal [3H]NT binding, suggesting that NT receptors were downregulated by the treatment of five daily injections of NT69L. Furthermore, there was no significant difference in dopamine uptake between chronic administration of NT69L and saline controls, implying that dopamine uptake is not involved in the development of tolerance to the effect of NT69L on dopamine release.
In addition to the involvement of various receptor subtypes, the difference in tolerance between repeated exposure to NT69L in vivo and in vitro might be due to the different effects of NT69L when applied locally versus systemically. Injection of NT69L systemically will cause global activation of NT receptors in the brain and can affect other neurotransmitter systems influencing dopamine release, while localized application would only affect localized NT receptors. As reported by others [26,27], the effect of direct injection of NT into discrete areas of the brain such as the VTA causes motor stimulation and release of dopamine that are augmented after repeated injections of NT. In contrast, injecting the NT analog, NT69L systemically did not cause stimulation of activity either acutely or after repeated injections (unpublished observations). These results further support our previous findings related to the presence to multiple NT receptor subtypes and to differential tolerance.
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Acknowledgments This work was funded by grant MH 27692 from the National Institute of Mental Health, the Forrest C. Lattner Foundation, Inc. and by the Mayo Clinic School of Medicine.
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[34] L.C. Murrin, R.H. Toth, Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis, Mol. Pharmacol. 12 (1976) 463 – 475. [35] C.B. Nemeroff, Neurotensin: perchance an endogenous neuroleptic? Biol. Psychiatry 15 (1980) 283 – 302. [36] C.B. Nemeroff, The interaction of neurotensin with dopaminergic pathways in the central nervous system: basic neurobiology and implications for the pathogenesis and treatment of schizophrenia, Psychoneuroendocrinology 11 (1986) 15 – 37. [37] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, third ed., Academic Press, San Francisco, 1997. [38] D.J. Pettibone, J.F. Hess, P.J. Hey, M.A. Jacobson, M. Leviten, E.V. Lis, P.J. Mallorga, D.M. Pascarella, M.A. Snyder, J.B. Williams, Z. Zeng, The effects of deleting the mouse neurotensin receptor NTR1 on central and peripheral responses to neurotensin, J. Pharmacol. Exp. Ther. 300 (2002) 305 – 313. [39] C. Pothoulakis, I. Castagliuolo, S.E. Leeman, Neuroimmune mechanisms of intestinal responses to stress. Role of corticotropin-releasing factor and neurotensin, Ann. N. Y. Acad. Sci. 840 (1998) 635 – 648. [40] E. Richelson, M. Pfenning, Blockade by antidepressants and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake, Eur. J. Pharmacol. 104 (1984) 277 – 286. [41] P. Sarret, L. Gendron, P. Kilian, H.M. Nguyen, N. Gallo-Payet, M.D. Payet, A. Beaudet, Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells, J. Biol. Chem. 277 (2002) 36233 – 36243. [42] D.W. Schulz, G.A. Kuchel, R.E. Zigmond, Decline in response to nicotine in aged rat striatum: correlation with a decrease in a subpopulation of nicotinic receptors, J. Neurochem. 61 (1993) 2225 – 2232. [43] R.E. See, A.M. Lynch, M. Aravagiri, C.B. Nemeroff, M.J. Owens, Chronic haloperidol-induced changes in regional dopamine release and metabolism and neurotensin content in rats, Brain Res. 704 (1995) 202 – 209. [44] B.M. Tyler, D.J. McCormick, C.V. Hoshall, C.L. Douglas, K. Jansen, B.W. Lacy, B. Cusack, E. Richelson, Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo, FEBS Lett. 421 (1998) 280 – 284. [45] B.M. Tyler, K. Jansen, D.J. McCormick, C.L. Douglas, M. Boules, J.A. Stewart, L. Zhao, B. Lacy, B. Cusack, A. Fauq, E. Richelson, Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood–brain barrier and specifically reduce gene expression, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7053 – 7058. [46] B.M. Tyler, C.L. Douglas, A. Fauq, Y.P. Pang, J.A. Stewart, B. Cusack, D.J. McCormick, E. Richelson, In vitro binding and CNS effects of novel neurotensin agonists that cross the blood–brain barrier, Neuropharmacology 38 (1999) 1027 – 1034. [47] B.M. Tyler-McMahon, M. Boules, E. Richelson, Neurotensin: peptide for the next millennium, Regul. Pept. 93 (2000) 125 – 136. [48] B.M. Tyler-McMahon, J.A. Stewart, F. Farinas, D.J. McCormick, E. Richelson, Highly potent neurotensin analog that causes hypothermia and antinociception, Eur. J. Pharmacol. 390 (2000) 107 – 111. [49] J.P. Vincent, J. Mazella, P. Kitabgi, Neurotensin and neurotensin receptors, Trends Pharmacol. Sci. 20 (1999) 302 – 309.
Research report
Effects of repeated injections of the neurotensin analog NT69L on dopamine release and uptake in rat striatum in vitro Rui Wang, Mona Boules, William Tiner, Elliott Richelson* Neuropsychopharmacology Laboratory, Mayo Foundation for Medical Education and Research, and Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA Accepted 9 July 2004 Available online 11 September 2004
Abstract The effect of five daily intraperitoneal (i.p.) injections of NT69L on in vitro dopamine release, uptake, and [3H]NT binding in rat striatal tissue was investigated. NT69L perfusion increased K+-evoked and electrically evoked [3H]DA release. NT receptor-1 antagonist SR48692 inhibited the stimulatory effect of NT69L on K+-evoked [3H]DA release, but not on electrical depolarization. Pretreatment with NT69L, in vivo, daily for 5 days, did not cause significant change in K+ evoked [3H]DA release, but reduced electrically evoked [3H]DA release induced by NT69L perfusion. Repeated perfusion with NT69L in vitro caused marked reduction on K+-evoked [3H]DA release and no change in electrically evoked [3H]DA release. [3H]NT binding was not significantly changed by one injection but was decreased after five injections of NT69L. Desensitization to the effects of NT69L in vitro was different depending upon whether tissue was preexposed to the compound in vivo or in vitro. These results provide further proof for the involvement of different NT receptor subtypes in mediating the effect of NT69L on dopamine release evoked by K+ or electrical depolarization. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Receptor modulation, up- and down-regulation Keywords: Dopamine release; Tolerance; Neurotensin receptor; Striatum; Rat
1. Introduction Neurotensin (NT) was isolated from bovine hypothalamus [7] and was subsequently shown to be widely and heterogeneously distributed in the central nervous system (CNS) [14] and in the digestive tract [39] of mammals. NT exerts a wide range of pharmacological effects in laboratory animals. The interaction between NT, via its receptors, and central dopaminergic systems has been well documented. NT functionally antagonizes dopamine in the mesolimbic system, while increasing dopamine transmission in the nigrostriatal system. It acts as a neuromodulator in the brain and, in particular, as a modulator of dopamine transmission in the nigrostriatal and mesocorticolimbic systems [1,36]. * Corresponding author. Tel.: +1 904 953 2439; fax: +1 904 935 2482. E-mail address: [email protected] (E. Richelson). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.07.069
Many preclinical studies have shown that injection of NT into brain has remarkable similarities to systemically administered antipsychotic drugs. In fact, centrally administered NT behaves in certain paradigms like an atypical neuroleptic drug, such as clozapine [24,25]. That is, it induces antipsychotic-like effects without causing motor disturbances. Such findings, together with studies showing that administration of antipsychotic drugs increases NT content in discrete brain nuclei [20,33,43], have led researchers to suggest that at least some of the beneficial clinical effects of antipsychotic treatment are mediated via enhanced NT neurotransmission [35,28,32]. NT mediates its effects through its receptors [49,31]. Therefore, direct activation of NT receptors in brain might have therapeutic effects in certain neuropsychiatric diseases and in abuse of psychostimulants. Roles for the three molecularly cloned NT receptors in brain are far from being
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fully elucidated. However, it is very likely that in rats and in mice, NTR-1 activation mediates antinociception, hypothermia, hypotension, and sedation [46,38]. NTR-2 may be associated with long-term metabolic effects, rather than with rapid, synaptic actions [41]. However, NT does not cross the blood–brain barrier (BBB), thus, it must be directly injected into the CNS to cause its effects. Over the years, many groups, including ours, have been developing NT receptor agonists that could be delivered systemically and cross the highly selective BBB. One of these compounds, developed by our group, is a brain-penetrating analog of the 8–13 fragment of NT (NT69L). In binding studies, NT69L has high affinity for human neurotensin receptor NTR-1 [9,47] and for human NTR-2 (unpublished observations). CNS effects of NT69L in rats after intraperitoneal (i.p.) injection include hypothermia, antinociception [9,48], and reduced food intake [5]. Additionally, pretreatment of rats with NT69L blocks the catalepsy caused by haloperidol [9], the climbing behavior caused by apomorphine [9], the turning behavior caused by d-amphetamine and apomorphine in rats with supersensitive dopamine receptors after unilateral lesioning of the nigrostriatal pathway [3], and blocks the locomotor stimulatory effects of the psychostimulants cocaine, d-amphetamine [4], and nicotine [16]. Interestingly, also in rats, we have shown that pretreatment with NT69L 30 min before nicotine once per week for 5 weeks blocks the behavioral sensitization seen with nicotine [17]. Our in vivo studies [6] have shown that tolerance develops rapidly (after a single dose) to some effects of NT69L (e.g., hypothermia and antinociception) and very slowly if at all (no tolerance after five daily doses) to other effects (e.g., blockade of the hyperactivity caused by psychostimulants) of this peptide. Here, we present the results of acute versus repeated treatment of animals with NT69L on dopamine release and uptake in striatal slices and synaptosomes in vitro, respectively, as well as, binding sites for [3H]NT in striatal slices by autoradiographic techniques.
2. Materials and methods 2.1. Animals, chemicals, and experimental treatment Male Sprague–Dawley rats weighing 150–250 g were divided into two groups of 4–8 rats and housed under 12-h light/dark conditions and allowed free access to food and water. All procedures were approved by the Mayo Foundation Institutional Animal Use and Care Committee. The rats were injected intraperitoneally with either NT69L (2 mg/kg) or an equal volume of saline once or daily for 5 days. Tissue from untreated rats (3–8) was used for dose–response studies, the double-stimulation of NT69L and for the antagonistic effect of SR48692. SR48692 {2-[(1-(7-chloro4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo (3.3.1.1.3.7)decan-2-carboxylic acid} was synthesized and generously provided by Sanofi
Synthe´labo (Toulouse, France). NT69L, synthesized by the Mayo Protein Core Facility as previously described [9], was dissolved in saline (0.9% NaCl) for in vivo intraperitoneal administration or in Kreb’s medium for in vitro perfusion. SR48692 was solubilized in dimethyl sulfoxide (DMSO). All other chemicals were from commercial sources. 2.2. Dopamine release Striatal slices were prepared as previously described [10]. Briefly, rats were killed by decapitation 1 h after their last injection. The brains were quickly removed and immersed for about 30 s in chilled (4 8C) Kreb’s media (in mM: NaCl 125, KCl 5, CaCl2 1.2, MgCl2 1.1, NaHCO3 20, NaH2PO4 1.2, glucose 10, pH 7.4). All buffers were equilibrated with atmospheric oxygen throughout the experiments. Subsequently the striata were dissected free of surrounding tissue and rapidly placed on the moistened circular stage of McIIwain chopper (Bachofer, Germany). 0.4 mm thick coronal slices of the striatum were prepared and immediately transferred to a preincubation chamber containing Kreb’s buffer at room temperature (22–24 8C) to allow recovery from the slicing procedure. After 60 min preincubation, slices were suspended in 1 ml of Kreb’s medium containing 1 AM pargyline, 1 mM ascorbic acid, 0.1 AM desipramine, saturated with 5% CO2 and 95% O2. [3H]DA (120 nM, 20 Ci/mmol; Moravek Biochemicals, Brea, CA) was added and the incubation was carried out at 37 8C for 30 min under a continuous flow of 5% CO2 and 95% O2. After rinsing with Kreb’s medium, three slices were transferred into a chamber (World Precision Instruments, Sarasota, FL) connected to two electrodes and perfused at 35 8C with Kreb’s solution (0.5 ml/min) for 60 min after which 3-min serial fractions (1.5 ml) were collected. The first nine fractions were used to establish basal release. The neurotensin receptor antagonist SR142948A (10 7 M) or the same dilution of the vehicle, DMSO, was added 18 min before depolarization. NT69L (10 11 to 10 7 M) was perfused 9 min before depolarization. To compare the effects of successive treatments of NT69L in vitro to 5 days pretreatment of NT69L in vivo on K+-evoked and electrically evoked [3H]DA release, two successive homologous depolarizations (K+ followed by K+ or electric followed by electric) were performed during the perfusion on slices from untreated animals. There was a 45 min washout period between the two depolarizations. With potassium, depolarization was performed using a 6 min application of a buffer containing 20 mM KCl. For electrical depolarization, electric field stimulation was applied in 5 trains of seven monophasic, rectangular pulses (1 ms duration, 22 mA intensity, 7 Hz frequency; Grass-Telefactor S48 Stimulator, W. Warwick, RI). At the end of the experiment, the slices were removed from the chamber and solubilized in Soluene350 (Perkin–Elmer Life Sciences, Boston, MA) for measurement of total radioactivity. The radioactivity was measured in 0.5 ml of each fractional perfusate with a
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Beckman LS6000TA liquid scintillation counter (Fullerton, CA). The fractional efflux of [3H]DA was expressed as percentage of the amount of radioactivity in the perfusate fraction, relative to the initial total amount of radioactivity in the slices. The initial amount of radioactivity was estimated from the sum of radioactivity measured in the solubilized slices at the end of the perfusion and the total radioactivity collected in the medium samples throughout the experiment. To determine stimulated release, we subtracted the basal efflux of radioactivity in the fraction prior to stimulation, from each of the subsequent three KCl fractions or electrical depolarization fractions. These values were then summed, averaged, and expressed as meansFS.E.M. of three to eight independent determinations. 2.3. Measurement of [3H]DA uptake into synaptosomes The synaptosomal uptake assay was conducted as described previously [40]. The assay was performed in Krebs’–Ringer’s buffer containing 0.64 mM ascorbic acid, 0.8 mM pargyline, and 0.1 AM [3H]DA (20 Ci/mmol). Various concentrations of nonradioactively labeled DA were added to give the final concentrations in the range of 6.310 6 to 110 2 M. The uptake assay was initiated by the addition of aliquots (50–100 Ag) of the synaptosomal fraction followed by incubation for 10 min at 37 8C. The assay was terminated by placing the samples on ice and adding 5 ml of ice-cold Krebs’–Ringer’s buffer. The synaptosomes were filtered through Whatman glass microfiber filters (GF/B), which had been presoaked in 0.1% polyethylenimine to reduce nonspecific binding, using a Brandel cell-harvester filtration apparatus. The synaptosomes, trapped on the filters, were washed twice with 5 ml of ice-cold Krebs’–Ringer’s buffer. The radioactivity of filters was determined by liquid scintillation spectrometry as described before. Protein concentration was measured using the Bio-Rad assay (Hercules, CA). 2.4. Determination of the levels of NT receptors in the striatum by autoradiography Adult male Sprague–Dawley rats 200–250 g were injected with NT69L or saline, once or daily for 5 days. The rats were sacrificed 1 h after the last injection by decapitation and their brains were rapidly frozen on dry ice and then stored at 80 8C until used. Coronal sections (20 Am) were cut on a cryostat at 16 8C, mounted on superfrost plus slides (VWR), and stored at 20 8C. Binding on sections was done as described by others [29], except for the use of a different radioligand. Briefly, neurotensin binding was performed by incubating the sections with 10 nM tritium labeled neurotensin {[3,11tyrosyl-3,5-3H(N)]NT}(91 Ci/mmol) in 50 mM Tris–HCl buffer containing 5 mM MgCl2, 0.2% bovine serum albumin and 50 AM bacitracin, pH 7.6, at 4 8C for 60 min. Additional sections were incubated in the presence of 1 AM nonradioactively labeled NT for determinations of nonspecific
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binding. The slides were washed in 40 mM Tris–HCl, pH 7.4, at 4 8C in two consecutive baths for 10 s. Slides were then dried by cool air. Film radiographs were processed by apposition of the radiolabeled section to Kodak MR film with 3H standards (Amersham Biosciences), exposed for 4 weeks and developed for 2 min in Kodak developer, fixer, and was then rinsed in water. The film was scanned and the optical density (OD) of the autoradiogram quantitated using the MCID-M5+ (version 5.1) software (Image Research, Ontario, Canada). The optical densities of nonspecific binding were subtracted from total binding to obtain specific binding. Measurements were done bilaterally on 4 brain sections. The values were expressed as nCi/mg wet tissue weight by using autoradiographic radiolabeled microscales (Amersham) that were exposed together with the tissue sections. The binding in different brain structures were identified using the rat brain atlas [37]. 2.5. Statistical analysis Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s test for multiple comparisons and t-test, using SigmaStat 2.0 software (SPSS, Chicago, IL), with Pb0.05 being considered significant.
3. Results 3.1. Effects of NT receptor agonist NT69L and antagonist SR48692 on K+-evoked and electrically evoked [3H]DA release In striatal slices prelabeled with [3H]DA, 9-min perfusion with the NT receptor agonist NT69L or 18-min perfusion with antagonist SR48692 (up to 10 7 M) had no effect on spontaneous, basal [3H]DA release (data not shown). However, NT69L increased K+-evoked and electrically evoked [3H]DA release in a concentration-dependent manner (Fig. 1A) reaching a plateau around 10 8 M. Concentration above 10 8 M caused desensitization for both types of stimulation. The EC50 and E max were 0.15 nM (95% confidence interval: 0.02 to 1 nM) and 200% over control (95% confidence interval: 100 to 300%), and 0.3 nM (95% confidence interval: 0.002 to 20 nM) and 400% of control (95% confidence interval: 100 to 800%), for the effect of NT69L on K+-evoked and electrically evoked [3H]DA release, respectively. The NT receptor antagonist, SR48692, blocked the stimulatory effect of NT69L on K+-evoked [3H]DA release ( Pb0.05), but not on electrically evoked [3H]DA release ( PN0.05; Fig. 1B). 3.2. Effect of NT69L on K+-evoked and electrically evoked [3H]DA release after five daily injections of NT69L A 6-min 20 mM K+ pulse and electrical depolarization caused an average of 0.04F0.02% and 0.05F0.05 %
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Fig. 1. Effect of NTR agonist (NT69L) and antagonist (SR48692) on [3H]DA release evoked by potassium and electrical depolarization in rat striatal slices. NT69L was added 9 min before K+ (20 mM, 6 min) or electrical depolarization (1 ms, 22 mA, 7 Hz, 40 s) and remained until the end of the stimulations. SR48692 was added 9 min before NT69L perfusion and were maintained during NT69L perfusion and depolarization. Each value represents meanFS.E.M. of four to eight independent determinations in triplicate. (A) Dose–response curve for the effect of NT69L on [3H]DA release evoked by potassium (E) and electrical (n) depolarization in rat striatal slices. [3H]DA release was expressed as percentage of stimulation of K+ or electrical depolarization above the baseline with Kreb’s medium plus K+- or electrically-evoked [3H]DA release as control. (B) Inhibition of NT69L-induced enhancement of K+or electrical-evoked [3H]DA release by SR48692. NT69L plus the vehicle of antagonist were used as control. Student’s t-test was used to determine the difference between control and antagonist treatment in each depolarization. *Pb0.05 vs. control.
increase, respectively, in the fractional rate of release over unstimulated release in the slices pretreated with saline for 5 days in vivo, Fig. 2A–B. Superfusion of the slices (pretreated with saline for 5 days in vivo) with 10 nM NT69L caused a significant increase in K+-evoked and electrically evoked [3H]DA release. Although the average enhancement evoked by electrical depolarization was greater than that with potassium-evoked release, there was no significant difference between the two stimulations. NT69L perfusion of slices from animals treated with five daily injections of NT69L in vivo caused an 80% reduction in electrically evoked [3H]DA release as
Fig. 2. Effect of chronic administration of NT69L in vivo on [3H]DA release evoked by K+-induced and electrically induced depolarization in striatal slices. Rats were treated with NT69L (2 mg/kg, i.p.) or saline for 5 days in vivo. Rats were sacrificed 1 h after the last injection and striatal slices were prepared as detailed in materials and methods. NT69L (10 8 M) or Kreb’s media were added 9 min before K+ (20 mM, 6 min) or electrical depolarization (1 ms, 22 mA, 7 Hz, 40 s) and remained until the end of the stimulations. Radioactivity in 3-min fractions is expressed as fractional release (percent of total tissue radioactivity at the start of the collection period). K+-evoked (A) or electrically evoked (B) fractional release of [3H]DA in different treatments. (C) Expressed as increased fractional release of [3H]DA over basal release. About four to eight independent determinations in triplicate were carried out in each group. Data is expressed as meansFS.E.M. Student’s t-test was used to determine statistical significance. *Pb0.05, **Pb0.01 vs. saline+Kreb’s control; # Pb0.05 vs. saline+NT69L treatment.
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compared to five daily injections of saline ( P=0.009; Fig. 2C). However, perfusion of NT69L in vitro still caused significant increase of [3H]DA release evoked by high potassium stimulation after five daily treatment of NT69L in vivo (Fig. 2C). 3.3. Effect of two successive applications of NT69L on K+evoked and electrically evoked [3H]DA release To compare the difference between five daily injections of NT69L in vivo and repeated applications of NT69L in vitro, two successive K+ or electrical depolarizations were performed. Compared to control slices (NT69L was replaced by Kreb’s medium), the first exposure to NT69L (10 8 M) induced a significant increase in both K+-evoked and electrically evoked [3H]DA release. However, with the second application of NT69L (10 8 M), slices lost responsiveness to NT69L on K+, but not the electrically evoked [3H]DA release which was significantly increased over control (160F10%, P=0.01; Table 1). 3.4. Effect of NT69L after five daily injections of NT69L on [3H]DA uptake into synaptosomes Five daily injections of NT69L had no effect on either the K t (concentration at one-half maximal transport) or the V max for transport of [3H]DA. The best-fit K t values (F S.D.) of [3H]DA uptake in synaptosomes from animals treated for 5 days with NT69L or with saline were identical at 2.0 F 0.510 4 M. Their V max values (F S.D.) were not significantly different at 5.9 F 0.510 9 and 4.8 F 0.410 9.
Fig. 3. Effect of NT69L on [3H]NT binding to striatal slices by autoradiographic techniques. Four 20-Am sections in each group were incubated in 10 nM [3H]NT for 1 h, washed, and processed as described in Materials and methods. The films were scanned and the optical density (OD) of the autoradiogram was quantitated using the MCID-M5+ (version 5.1) software. Sections from 4 rats in each treatment were analyzed. Shown are meanFS.E.M. *Pb0.05 vs. saline control and #Pb0.05 vs. one injection of NT69L.
3.5. Effect of five daily administrations of N69L on NT receptor binding in striatum There was no significant difference between one injection and five daily injections of saline on [3H]NT binding (data not shown). Five daily treatments of NT69L significantly decreased [3H]NT binding in striatum compared to saline control and one treatment of NT69L, respectively ( pb0.05). The reduction of one injection of NT69L was not significantly different from control (Figs. 3 and 4). The
Table 1 Effects of two successive applications of NT69L in vitro on [3H]DA fractional release under potassium or electrical depolarization [3H]DA fractional release (%) First stimulation, S1
Second stimulation, S2
Prestimulation
Poststimulation
Prestimulation
Poststimulation
Potassium Kreb’s control +NT69L (10 8 M) %Kreb’s control
0.55F0.04 0.50F0.03 91F5%
0.56F0.05 0.70F0.04* 125F7%
0.28F0.03 0.26F0.04 93F14%
0.30F0.02 0.28F0.04 90F10%
Electrical Kreb’s control +NT69L (10 8 M) %Kreb’s control
0.52F0.05 0.64F0.10 123F15%
0.56F0.07 0.96F0.15* 170F30%
0.26F0.03 0.24F0.03 92F12%
0.27F0.03 0.43F0.04** 160F10%
Slices of nontreated rat striatum labeled with [3H]DA were stimulated twice with two successive K+ (20 mM, 6 min) depolarizations or with two successive electrical depolarizations (1 ms, 22 mA, 7 Hz, 40 s). In each case, a 45-min washout period was allowed between depolarizations. The medium with NT69L (10 8 M) was switched into chambers 9 min before each depolarization and remained during the duration of depolarization. Shown are meansFS.E.M. Values are expressed as the sum of the amount of [3H]DA in the superfusion medium collected from three fractions after stimulation as a percentage of the stored [3H]DA in the tissue (see Materials and methods for more information on the determination of fractional release). +NT69L (10 8 M) represents NT69L perfusion before and during depolarization of slices instead of Kreb’s media. Each value represents the mean of three independent determinations in triplicate. T-test was used for testing the difference between Kreb’s control and NT69L treatment in each depolarization. * Pb0.05 vs. Kreb’s control. ** Pb0.01 vs. Kreb’s control.
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Fig. 4. Representative autoradiograms of brain sections of rat showing (A) saline control (five daily injections); (B) one injection of NT69L and (C) five daily injections of NT69L.
results suggest that NT receptors were downregulated after five daily injections of NT69L.
4. Discussion High potassium and electrical stimulation are two approaches used by most researchers to evoke the release of neurotransmitters in vivo and in vitro. Most studies in which potassium was used to evaluate DA release, involved in vivo or in vitro infusion of striatal tissue with high potassium solution for a short period [11,18,19,30,42]. High potassium produces a continuous depolarization of neurons, leading to sustained elevations in intracellular calcium.
Electrical fields and currents applied to the nervous systems generally cause excitation and generate synchronized activity. Electrical stimulation has traditionally been used in the nervous system to excite axons and neurons for studying basic mechanisms or to replace a function lost following injury. Numerous studies have shown that relative low-stimulation frequencies are required to cause dopamine release from the mesotelencephalic dopamine populations. For instance, activation of dopamine neurons in the rat striatum is evoked at 5.0–15 Hz [13,34], in the rabbit striatum at 10 Hz [8], and in the cat caudate at 30 Hz [12]. Although it has been proven that the dopamine release evoked both by potassium and electrical stimulation is calcium independent [12], different mechanisms might be involved in either case. Much evidence has accumulated implicating different neurotensin receptors in dopamine release [2,15,22,23]. However, the precise mechanism is still unclear. Similar to neurotensin (NT), the present study showed that NT69L, a novel neurotensin receptor agonist, stimulated both K+evoked and electrically evoked [3H]DA release in dosedependent manner. These in vitro results are similar to those reported for NT by others [22,15]. Additionally, our data support the hypothesis that distinct NT receptors are involved in the control of [3H]DA release, depending on the type of depolarization used, electrical versus chemical. The fact that SR48692, a putative high-affinity receptor (NTR-1) antagonist, blocked the effect of NT69L on K+ but not on electrical depolarization, further indicates that distinct neurotensin receptors are involved in the effect of NT69L on either type of depolarization. The second important, and perhaps more conclusive, observation is related to the differential profile for the effects of NT69L on K+-evoked and electrically evoked [3H]DA release after five daily injections of NT69L in vivo. There was no development of tolerance to K+-evoked [3H]DA release, but electrically evoked [3H]DA release was significantly reduced. These results strongly suggest that different neurotensin receptors are involved in the facilitation of NT69L on [3H]DA release evoked by K+ and electrical stimulation. Rapid development of tolerance to some of NT69L effects, namely hypothermia, antinociception, and blockade of catalepsy, but slowly, if at all, to other effects such as apomorphine-induced climbing and amphetamine-induced activity, has been previously reported in rats [6]. The evidence above strongly supports that different neurotensin receptors are involved in the differential profile of NT69Linduced tolerance. The slow-to-develop tolerance to some effects induced by NT69L, such as apomorphine-induced climbing behavior and blockade of d-amphetamine and cocaine-induced hyperactivity, may be mediated through NTR-1. Effects to which tolerance develops rapidly, such as antinociception and hypothermia, may be mediated by other NTRs, such as NTR-2. However, our previous antisense and antigene studies [44,45], as well as studies with mice
R. Wang et al. / Brain Research 1025 (2004) 21–28
lacking NTR-1 [38], suggest that NTR-1 receptors mediate the hypothermic and antinociceptive effects of NT agonists. In addition to testing the effect of repeated in vivo injections or in vitro applications of NT69L on the [3H] DA release from rat striatal slices, the effect of repeated injections of NT69L on [3H]NT binding and uptake was studied. Five daily injections of NT69L significantly reduced striatal [3H]NT binding, suggesting that NT receptors were downregulated by the treatment of five daily injections of NT69L and that there was no significant difference in dopamine uptake between chronic administration of NT69L and saline controls. From our results and those in the literature: (1)
(2)
(3)
(4)
(5)
Chronic administration of NT69L resulted in development of tolerance to electrically evoked but not to K+evoked [3H]DA release; SR48692, a putative high-affinity receptor (NTR-1) antagonist, blocked the effect of NT69L on K+-evoked [3H]DA release, but had no effect on electrically evoked [3H]DA release; SR48692 inhibits most of the in vitro and in vivo effects of NT and confirms the modulatory role of NT in the central nervous system, in particular, in regulating the activity of DA neurons, and its inability to antagonize hypothermia and analgesia induced by neurotensin. However, SR142948A, a nonselective neurotensin receptor antagonist, is able to reduce markedly NT-induced hypothermia and analgesia [21]. Five daily injections of NT69L significantly reduced striatal [3H]NT binding, suggesting that NT receptors were downregulated by the treatment of five daily injections of NT69L. Furthermore, there was no significant difference in dopamine uptake between chronic administration of NT69L and saline controls, implying that dopamine uptake is not involved in the development of tolerance to the effect of NT69L on dopamine release.
In addition to the involvement of various receptor subtypes, the difference in tolerance between repeated exposure to NT69L in vivo and in vitro might be due to the different effects of NT69L when applied locally versus systemically. Injection of NT69L systemically will cause global activation of NT receptors in the brain and can affect other neurotransmitter systems influencing dopamine release, while localized application would only affect localized NT receptors. As reported by others [26,27], the effect of direct injection of NT into discrete areas of the brain such as the VTA causes motor stimulation and release of dopamine that are augmented after repeated injections of NT. In contrast, injecting the NT analog, NT69L systemically did not cause stimulation of activity either acutely or after repeated injections (unpublished observations). These results further support our previous findings related to the presence to multiple NT receptor subtypes and to differential tolerance.
27
Acknowledgments This work was funded by grant MH 27692 from the National Institute of Mental Health, the Forrest C. Lattner Foundation, Inc. and by the Mayo Clinic School of Medicine.
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