The effect of central noradrenergic system lesion on dopamine (DA) and serotonin (5-HT) synthesis rate following administration of 5-HT3 receptor ligands in chosen parts of the rat brain
Abstract
Introduction: Since little has been known about the effect of the central noradrenergic system on the reactivity of serotonin 5-HT3 receptors, the aim of the current study was to find out whether this reactivity could be altered by chemical damage to the system in adult rats in early developmental stage. Materials and methods: Adult male Wistar rats with central noradrenergic lesion induced by DSP-4 on day 1 and 3 of life were injected with analgesic model substance – morphine, serotoninergic 5-HT3 receptor agonist (1-phenylbiguanide, PBG), 5-HT3 receptor antagonist (ondansetron) or both compounds jointly followed by decarboxylase inhibitor of aromatic amino acids (NSD-1050). After 30 min following NSD-1050 injection, the animals were decapitated using a guillotine. Chosen cerebral structures were dissected, and the contents of 5-hydroxytryptofan (5-HTP) and L-dihydroxyphenyla- lanine (L-DOPA) were determined using high-pressure liquid chromatography with electrochemical detection (HPLC/ED).
Results: Neither PBG nor morphine affected L-DOPA contents in the hippocampus in control rats; however, DSP-4 lesion caused a significant decrease in the synthesis rate of DA in this structure. Hippocampal contents of 5-HTP increased after morphine or PBG administration, and central noradrenergic lesion attenuated this effect. Morphine or PBG decreased cerebellar DA synthesis rate in control rats and DSP-4 lesion did not modify it. Cerebellar levels of 5-HTP increased after morphine or PBG challenge in control rats. DSP-4 lesion intensified the effect of morphine and attenuated that of PBG. Ondansetron abolished the effects mediated by PBG. We did not observe any impact of PBG or ondansetron on DA and 5-HT synthesis in the striatum.
Conclusion: Damage to the central noradrenergic system in rat newborns, through altered reactivity of central 5-HT3 receptors, results in permanent disorders in serotoninergic transmission in hippocampus and cerebellum as well as dopaminergic transmission in hippocampus, which may attenuate the activity of the descending pathways that derive from these structures.
Introduction
Functional regulation of one neurotransmission system in the brain may affect the action of other systems of the CNS. The serotonergic and noradrenergic systems and their connections with other neurotransmission systems are currently an object of numerous studies.
The central noradrenergic system mediates pain sensation inhibition, especially chronic pain [1]. Many literature data also indicate its involvement in the mechanism of anxiety and depression which may be accompanied by pain [2–6]. It has been shown that the peripheral 5-HT3 receptors take part in the generation of pain induced by inflammatory processes, but not by mechanical or thermal stimuli. The 5-HT released in tissues, by affecting the peripheral 5-HT3 receptors sensitizes neurons that are responsible for pain sensation to the action of bradykinin (pro- nociceptive effect) [7,8].
On the other hand, centrally released 5-HT exerts an anti- nociceptive effect, by stimulating e.g. 5-HT3 receptors. This effect can be blocked by a central 5-HT3 receptor antagonist [9].As it has been reported, the serotonergic and noradrenergic systems cooperate in pain modulation at the level of the spinal cord, although the central interactions of these neurotransmitters are poorly known [1].The analgesic action of tricyclic antidepressants (inhibiting the reuptake of NA and 5-HT synaptic cleft) as well as serotonin- norepinephrine reuptake inhibitors and to the less extent – selective serotonin reuptake inhibitors in some types of chronic pain needs to be explained.
The 5-HT3 receptors are known to affect the release of certain neurotransmitters or modulatory substances such as dopamine (DA) in the limbic system. Agonists of the 5-HT3 receptor enhance the release of endogenous 5-HT and CCK, but inhibit the secretion of endogenous NA and acetylcholine in the cerebral cortex [10,11]. Data concerning the effect of 5-HT3 receptors on the release of neurotransmitters still remain quite scarce and the knowledge of mutual interactions of the respective types of receptors is extremely important to elucidate the functioning of the given system.
Behavioral studies conducted in the last decade have shown that the administration of 5-HT3 ligands to animals does not basically alter their behavior, yet it affects the action of other substances in the animal models of anxiety, psychoses and drug addictions [12,13].
We used a model of central noradrenergic lesion induced by DSP-4, designed in the Department of Pharmacology in Zabrze. DSP-4 is a neurotoxin which easily permeates through the blood– brain barrier and can be administered peripherally (sc, ip). The neurotoxin causes permanent inhibition of NA reuptake in the CNS pathways and a decrease in the content of endogenous NA in the central and peripheral nervous system in rats. The reduction in NA content in the peripheral noradrenergic system is temporary and approximately 80% of neurons regain the ability to synthesize NA 4–6 weeks after DSP-4 injection. This substance does not affect the content of catecholamines in the adrenal medulla [14]. In turn, dysfunction of the adrenergic neurons is permanent and the action of DSP-4 on nerve cells of the dorsal pathway is stronger than in the ventral one [15].
The application of the central noradrenergic system lesion model in the developing brain allows for compensatory changes from other neurotransmission systems. Previously, using the same model and 5-HT3 receptor agonist (1-phenylbiguanide, PBG) and antagonist (ondansetron) we have demonstrated the effect of lesion of the central noradrenergic system on analgesic effects mediated by serotoninergic 5-HT3 receptors in behavioral experiments as well as during the assessment of DA and 5-HT synthesis rate in chosen parts of rat brain. We have found that in animals with central noradrenergic system lesion, the reactivity of 5-HT3 receptors is diminished in the main structuresinvolved in nociception: in frontal cortex and thalamus [16–18]. In the current study, we decided to examine other subcortical structures with a potent ability to modify the perception of pain stimuli giving their projections to the thalamus, where 5-HT3 receptor expression can be found: cerebellum, striatum and hippocampus [19]. As we were particu- larly interested in the elucidation of the involvement of serotonin- ergic–noradrenergic central interactions in the context of analgesia we used morphine as a model analgesic reference substance.
Materials and methods
The study used male Wistar rats, newborns and adults aged 8–10 weeks. The animals were kept in a room at a constant temperature of approximately 22 8C and 12 h/12 h day/night artificial light cycle (light from 7:00 to 19:00). Throughout the experiment, the animals had free access to water and standard diet. Rat newborns were injected (sc) with the neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromo-bensylamine] on day 1 and 3 of life at a dose of 50 mg/kg × 2 to induce permanent damage to the central noradrenergic system. The control animals received 0.9% NaCl solution (1.0 ml/kg sc). Previously, we demonstrated, that such procedure reduced noradrenaline content in frontal cortex and hippocampus by 96.6% and 95.5%, respectively as compared to the control rats. Conversely, in the cerebellum it increased by 66% suggestive of reactive neuronal sprouting [15]. It has been shown that other neurotransmitter systems are not influenced in this model [20].
Adult male rats were injected with morphine (7.5 mg/kg b.w. sc), 5-HT3 receptor antagonist (ondansetron; 1.0 mg/kg b.w. ip), 5-HT3 receptor agonist (1-phenylbiguanide, PBG; 7.5 mg/kg b.w. ip) and jointly ondansetron and PBG in the above mentioned doses. Next, after 30 min decarboxylase inhibitor of aromatic amino acids (NSD- 1050) was injected ip in a dose of 100 mg/kg b.w. After another 30 min following NSD-1050 injection the animals were decapitated with a guillotine. Then, the skin and cranial vault bones were removed to take out the brain, which was placed on a glass plate with ice at 0 8C. Next, striatum, hippocampus and cerebellum were dissected and frozen on solidified CO2. On the day when amino acids were determined, the tissue was homogenized at 0 8C (in water bath) in a solution of 0.1 M HClO4 (Fluka, Germany) with addition of 25 mg/l of ascorbic acid for approximately 10 s. Then, the homogenate was centrifuged at 4 8C at 15,000 rotations/min for 20 min. After centrifugation, the supernatant was centrifuged again, this time using a cellulose filter (pore diameter of 0.2 mm) for 10 min at 4 8C at 10,000 rotations per minute, and the supernatant was frozen at —18 8C for 24 h. Next, the sample was defrosted and subjected to chromatography with the use of high-pressure chromatography with electrochemical detection (Gilson, France). The intensity of DA and 5-HT synthesis was measured by an indirect method, determining chromatographically the intensity of accu- mulation of L-DOPA (DA precursor) and 5-HTP (5-HT precursor). The respective groups consisted of 10 animals.The current study was approved by the local Bioethics Committee of the Silesian Medical University (consent no 66 of 11 December, 2007).
Statistical analysis
Licensed version of the computer software STATISTICA 6.0 (StatSoft, Tulsa OK, USA) was used for the analysis.After the analysis of normally distributed data (Kolmogorov– Smirnov test), the analysis of variance (ANOVA univariate and bivariate) and Newman–Keuls post hoc test were applied. The Kruskal–Wallis test was used for nonparametric data. The significance criterion were the values of p < 0.05. Results We showed that in the hippocampus neither morphine (7.5 mg/ kg b.w. sc) nor 5-HT3 receptor agonist – PBG (7.5 mg/kg b.w. ip) affected dopamine synthesis rate in control rats. However, in DSP- 4 lesioned rats a significant decrease in dopamine synthesis was observed after morphine as well as PBG injection in comparison to control rats injected with these substances and DSP-4 lesioned animals which received 0.9% NaCl. The effect of PBG was attenuated by concomitant application of 5-HT3 receptor antago- nist, ondansetron (1.0 mg/kg b.w. ip) (Fig. 1). When investigating the content of 5-HTP in the hippocampus we found that the 5-HT synthesis rate in this structure in rats with DSP-4 lesion was statistically significantly lower in animals which received 0.9% NaCl (1.0 ml/kg b.w. ip), morphine (7.5 mg/kg b.w. sc) or 5-HT3 receptor agonist, PBG (7.5 mg/kg b.w. ip), as compared to the control group. In turn, in the control rats, the hippocampus content of 5-HTP after administration of morphine (7.5 mg/kg b.w. sc) or PBG (7.5 mg/kg b.w. ip) was statistically significantly higher as compared to the control animals receiving saline. The effect of PBG was abrogated by concomitant administration of 5-HT3 receptor antagonist, ondansetron (1.0 mg/kg b.w. ip) (Fig. 2). In the cerebellum of control rats, morphine (7.5 mg/kg b.w. sc) as well as 5-HT3 receptor agonist, PBG, decreased L-DOPA contents in this structure in comparison to saline. The effect of PBG was attenuated by concomitant administration of 5-HT3 receptor antagonist, ondansetron (1.0 mg/kg b.w. ip). However, in rats after DSP-4 lesion DA synthesis increased only after morphine administration as compared to control animals (Fig. 3). The cerebellar content of 5-HTP exhibited statistically signifi- cant differences after administration of morphine (7.5 mg/kg b.w. sc), 5-HT3 receptor agonist, PBG (7.5 mg/kg b.w. ip) and 5-HT3 receptor antagonist, ondansetron (1.0 mg/kg b.w. ip). In control rats, both morphine and PBG increased the 5-HT synthesis rate in this structure in comparison to those treated with saline. The 5-HT synthesis rate in the cerebellum of animals with DSP-4 induced lesion was significantly increased after morphine administration, but was decreased by PBG as compared to control animals. Ondansetron abolished the effect of PBG when applied concomi- tantly (Fig. 4). DA synthesis rate in the striatum after administration of morphine (7.5 mg/kg b.w. sc), PBG (7.5 mg/kg b.w. ip) or ondansetron (1.0 mg/kg b.w. ip) separately or jointly was similar in the two groups of study animals (Fig. 5). We found no effect of DSP-4 lesion on the 5-HT synthesis rate in rat striatum (Fig. 6). Fig. 5. The effect of DSP-4 lesion on the content of L-DOPA in the striatum following administration of morphine (7.5 mg/kg b.w. sc), PBG (7.5 mg/kg b.w. ip) or ondansetron (1.0 mg/kg b.w. ip) in rats (x¯ SEM; n = 10). Fig. 6. The effect of DSP-4 lesion on the content of 5-HTP in the striatum following administration of morphine (7.5 mg/kg b.w. sc), PBG (7.5 mg/kg b.w. ip) or ondansetron (1.0 mg/kg b.w. ip) in rats (x¯ SEM; n = 10). Discussion The molecular mechanism of DSP-4 action consists in the inhibition of NA storage capacity in the synaptic granules both in the central and peripheral noradrenergic neurons. DSP-4 in noradrenergic neurons is hydrolyzed to aziridinium ion, which then reacts with amino acid and thiol residues of the subcellular structures that bind and store NA, thus leading to their dysfunction. This in consequence results in the decreased content of NA in neurons, which impairs neurotransmission [21]. The effect of DSP-4 has two phases. The first, called ‘‘the acute phase’’ is characterized by rapid loss of neurotransmitter (NA). The second one, known as ‘‘neurodegenerative’’, is associated with biochemi- cal and morphological disorders and functional inhibition of DBH, the DA to NA converting enzyme [22]. DSP-4 has also the ability to inhibit MAO activity [23]. The inhibition of NA reuptake in the peripheral and central neurons occurs rapidly (already an hour after neurotoxin injection) and is dose-dependent. The effect of DSP-4 on NA content appears already after administration of 20 mg/kg b.w. ip, whereas the maximum effect can be obtained when a dose of 100 mg/kg b.w. ip is applied. It should be added that when prior to DSP-4 administration the animals are injected with a NA reuptake inhibitor, e.g. desipramine, the action of the neurotoxin is ineffective [24]. Following administration of DSP-4 (50 mg/kg b.w. ip), the activity of DBH in the animal brain and heart decreases rapidly and this effect is also inhibited by earlier administration of desipra- mine. The activity of the enzyme on the periphery after a single dose of DSP-4 returns to the initial state after approximately 2 weeks. However, in the CNS, the activity of DBH is reduced for the period of 8–10 months after administration of the neurotoxin. It has been also found that the biological activity of DSP-4 differs among rat strains used for investigations, e.g. Long–Evans rats are less vulnerable to the action of DSP-4 as compared to the Sprague– Dawley ones [25]. It has been reported that the application of the DSP-4 induced lesion model leads to permanent damage to the noradrenergic system in the early extra-fetal life period of animals, which causes an increase in the reactivity of the central dopamine system (D2 and D3 receptors). It also affects the activity of the central serotonergic system, inducing desensitization of 5-HT1A auto- receptors, and results in GABA-ergic system dysfunction in adult animals [20,26]. Previously, our behavioral studies revealed that damage to the central noradrenergic system in the early period of extra-fetal life attenuates the anti-nociceptive effects mediated by the serotoner- gic 5-HT3 receptor, integrated at the level of higher structures of the central nervous system, but remains ineffective in the processes integrated in the spinal cord [8,10]. Animals with DSP-4 lesion also showed DA and 5-HT synthesis disturbances in the cerebral cortex and stem structures after administration of central 5-HT3 receptor ligands [17]. The above observations indicate permanent alterations in the reactivity of this receptor caused by damage to the noradrenergic system. The hypothesis is also confirmed by our current findings. Currently, we found that the synthesis rate of DA in the hippocampus was not affected by morphine or 5-HT3 receptor agonist – PBG in control rats, but was significantly reduced in animals after DSP-4 lesion. The rate of 5-HT synthesis in this structure was increased by morphine and PBG, although this effect was attenuated by central noradrenergic lesion. In the above- mentioned experiments the effect of PBG was abolished by concomitant administration of ondansetron. In the cerebellum the effect of morphine and PBG on L-DOPA content was observed only in control rats. The effect of PBG was blocked by concomitant administration of 5-HT3 receptor antago- nist – ondansetron. Neonatal DSP-4 lesion did not affect DA synthesis rate in this structure after 5-HT3 receptor ligands challenge. Therefore. it is unlikely that dopaminergic transmission in the cerebellum is involved in the effects mediated by 5HT3 receptors in these animals. Also the level of 5-HTP in this structure in control rats was significantly elevated by morphine as well as 5-HT3 receptor agonist – PBG. Interestingly, in animals with neonatal noradrener- gic lesion the effect of morphine on 5-HT synthesis rate was intensified, but decreased by PBG as compared to control group. In the striatum, the synthesis rate of DA and 5-HT after administration of morphine, PBG or ondansetron jointly or separately was similar in both study groups of animals. No reports have been available worldwide concerning the effect of agonists and antagonists of 5-HT3 serotonin receptor on the synthesis of monoamines in the CNS of animals with injured central noradrenergic system, which hinders interpretation of the results. Johnson et al. [27] have found that 60 min after administration, morphine increases the synthesis of 5-HT measured by determi- nation of 5-HTP accumulation on the pre-optic and suprachias- matic areas, arcuate nuclei of the thalamus and striatum. In turn, Curade et al. [28], who used paracetamol (200–400 mg/kg b.w.), i.e. another analgesic with a central action, found no effect on the accumulation of 5-HTP in the posterior cortex, in the hypothala- mus, striatum, hippocampus or brain stem. Sastre-Coll et al. [29] demonstrated that the acute treatment with morphine (3 mg/kg) in rats had no effect on DA synthesis rate in the hippocampus, cortex, hypothalamus and striatum, but higher doses (10–100 mg/ kg) decreased the accumulation of L-DOPA (synthesis of DA) in the cerebral cortex and hippocampus of intact rats what is in line with our findings. They suggest that this effect probably results from the activation of inhibitory m-receptors (heteroreceptors) located on noradrenergic terminals, in agreement with the reported decrease in the release of NA induced by morphine [30]. In contrast, the above cited authors [29] showed that acute morphine treatment of intact rats increased the synthesis of 5-HT and DA in various brain regions (what is generally in agreement with our results), the most probably through an indirect effect mediated by GABA. Thus, the activation of inhibitory m-heteroreceptors located on GABAergic interneurons would relieve the inhibitory effect mediated by GABA on 5-HT [31,32] and DA [33] neuronal firing and neurotransmitter release. In our study, we also found that morphine and PBG injected to rats lesioned with DSP-4 as neonates reduced DA and 5-HT synthesis rate in the hippocampus in comparison to control (intact) animals. Other researchers have found a well-established interaction between noradrenergic, serotoninergic and GABAergic systems, whereby the noradrenergic and serotoninergic systems are involved in the regulation of basal GABA release, while GABAergic neurons simultaneously exert tonic inhibitory regula- tion of locus ceruleus norepinephrine and raphe nuclei serotonin neurons. Previously, we have demonstrated that neonatal DSP-4 treatment modifies (1) GABAergic and serotoninergic neurotrans- mission in the brain [25,34] and simultaneously (2) the sensitivity of GABAA and 5-HT1A receptors to their agonists [22,35]. The opposite effect of morphine and PBG on DA synthesis in cerebellum in comparison to hippocampus may be explained by the fact that L- DOPA accumulation is not a sole indicator of DA synthesis rate, but also depends on NA production. Therefore, in brain structures densely innervated by NA neurons with sparse DA innervation (e.g. cerebellum), L-DOPA accumulation is a more reliable indicator of NA synthesis. This is in line with our previous results showing that neonatal DSP-4 treatment genuinely caused noradrenergic hyper- innervation of brain stem and cerebellum [35]. Summing up, the effect of morphine on DA and 5-HT brain synthesis rate is sometimes problematic to define since it seems to be a dose and region specific. Mongeau et al. [36] have shown that the 5-HT3 receptor agonist (2-metyl-5-HT) increases NA release in the frontal cortex; however, damage to serotonergic system induced by neurotoxin – 5,7-dihydroxytriptamine (5,7-DHT) has no effect. Thus, their observations indicate that the increased NA release under the impact of 2-methyl-5-HT is not an effect of 5-HT3 receptor stimulation on serotoninergic endings [36]. Other authors have stated that the stimulation of 5-HT3 receptors enhances the release of DA and 5-HT [37,38], and it is well known that the substances increasing the release of monoamines (e.g. amphetamine) increase their synthesis rate [39]. On the basis of the cited reports it can be assumed that the 5-HT3 agonist mimics, to some extent, the effect of morphine on the release of neurotransmitters, and it was therefore chosen for this study. Conclusion Our findings seem to indirectly explain previous behavioral observations showing a decrease in the analgesic effect of morphine and 5-HT3 receptor agonist – PBG in acute and chronic pain models by the involvement of supraspinal perception mechanisms [8,10]. Damage to the central noradrenergic system in rat newborns via altered reactivity of central 5-HT3 receptors leads to permanent disorders in serotoninergic transmission in hippocampus and cerebellum as well as dopaminergic transmis- sion in hippocampus, which may attenuate the activity of the descending anti-nociceptive UNC8153 serotonergic pathways that arise from these structures.