Neonatal DSP-4 Treatment Modifies Antinociceptive Effects of the CB1 Receptor Agonist Methanandamide in Adult Rats
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To study the influence of the central noradrenergic system on antinociceptive effects mediated by the CB1-receptor agonist methanandamide, intact rats were contrasted with rats in which noradrenergic nerves were largely destroyed shortly after birth with the neurotoxin DSP-4 [N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (50 mg/kg sc × 2, P1 and P3); zimelidine (10 mg/kg sc, 30 min pretreatment, selective serotonin reuptake inhibitor). When rats attained 10 weeks of age, monoamine and their metabolite concentrations were determined in the frontal cortex, thalamus, and spinal cord by an HPLC/ED method. Antinociceptive effects after methanandamide (10 mg/kg ip) apply were evaluated by a battery of tests. In addition, immunohistochemistry and densitometric analysis of the cannabinoid CB1 receptor in the rat brain was performed. DSP-4 lesioning was associated with a reduction in norepinephrine content of the frontal cortex (>90 %) and spinal cord (>80 %) with no changes in the thalamus. Neonatal DSP-4 treatment produced a significant reduction in the antinociceptive effect of methanandamide in the tail-immersion test, hot-plate test and writhing tests. In the paw pressure and formalin hind paw tests results were ambiguous. These findings indicate that the noradrenergic system exerts a prominent influence on analgesia acting via the cannabinoid system in brain, without directly altering CB1 receptor density in the brain.
KeywordsNoradrenergic Lesion CB1 receptor Antinociception Rats
Cannabinoids exert palliative effects in cancer patients by stimulating appetite and by abating nausea, vomiting, and pain. To date, cannabinoids have been licensed for clinical use as palliative treatment of chemotherapy, but increased evidence indicates a direct antiproliferative action of these drugs on several tumor cell lines, both in vitro and in animal models (Walsh et al. 2003). Also, recent evidence suggests that the endocannabinoid system mediates stress responses and regulates emotional homeostasis, in part, by targeting noradrenergic circuits. Simultaneously, the midbrain locus coeruleus (LC), that contains most noradrenergic neurons and projects to multiple cortical, limbic and autonomic-related brain structures, regulates arousal, attention, vigilance, stress, and pain (Berridge and Waterhouse 2003; Dunn et al. 2004). Alteration of norepinephrine (NE) exocytosis in the thalamus, brain stem and other nuclei alters output of nociceptive information to higher brain centers from projection neurons. Also, LC stimulation, which increases NE release in spinal cord, inhibits nociceptive transmission in the dorsal horn via α2-adrenergic receptors (Cenci et al. 1992; Delaney et al. 2007). Clinical evidence indicates a link between NE with pain modulation and opioid withdrawal syndrome. Abrupt cessation of opioid intake precipitates opioid withdrawal, which produces several aversive responses and symptoms, i.e., an abnormal increase in pain sensitivity (hyperalgesia) (Van Bockstaele et al. 2008).
LC activity is tightly controlled by presynaptic α2 autoreceptors (Lichtman and Martin 1991; Pudovkina and Westerink 2005) and by afferent pathways from several brain areas (Lee et al. 2005). Among these, serotonin (5-HT) and γ-aminobutyric acid (GABA)-containing neurons appear to play a major role (Aston-Jones et al. 1991). LC neurons possess a high density of postsynaptic mu-opioid receptors (Van Bockstaele and Commons 2001); and cannabinoids modulate noradrenergic neuronal activity. Scavone et al. (2010) provided evidence for a heterogeneous distribution of CB1 receptors in the LC and demonstrated that this receptor and mu-opioid receptors co-exist in cellular profiles in this region. Others have shown an interaction between the cannabinoid system and the NE system in areas such as the prefrontal cortex (Oropeza et al. 2007), nucleus accumbens (Carvalho et al. 2010) and the nucleus of the solitary tract (Jelsing et al. 2009).
Previously, we showed that N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 50 mg/kg sc per day), administered on the 1st and 3rd days of postnatal life, alters noradrenergic input to the hippocampus and prefrontal cortex (endogenous NE content was reduced by 98.5 and 95.0 %, respectively), without impairing dopaminergic and serotoninergic input to these regions. An elevated NE level was found in brainstem and cerebellum of these DSP-4-treated rats, suggestive of reactive neuronal sprouting (Brus et al. 2004; Nowak et al. 2006; Dąbrowska et al. 2007). In addition, we reported that neonatal DSP-4 treatment modifies convulsant effects of bicuculine and pentetrazole, as well as sensitivity to the anxiolytic-like effect of benzodiazepines, and the sedative-hypnotic effect of phenobarbital and ethanol in adult rats (Bortel et al. 2007, 2008a, b). And we established that the GABA transaminase inhibitor vigabatrin causes a twofold increase in the extracellular GABA concentration in DSP-4-lesioned rat brain (Bortel et al. 2008c). In the present study, a lesion of noradrenergic neurons was made in early postnatal ontogeny, in order to assess how subsequent postnatal compensatory processes influence cannabinoid-induced antinociception. This phenomenon may be of importance, because adrenergic receptor dysfunction is suspect in patients with clinical depression, anxiety disorder and other neuropsychiatric disorders (Ressler and Nemeroff 1999; Anand and Charney 2000) and neurological disease (Parkinson’s and Alzheimer’s disease) (Narabayashi 1999; Haglund et al. 2006). Furthermore, the difficulties of relieving painful symptoms associated with some pathological entities, i.e., bone cancer pain, justify experimental efforts to define new analgesic targets. Cannabinoid CB1 receptor agonists represent a class with this desired analgesic potential (Farquhar-Smith 2009).
Materials and Methods
Animals and Treatment
Wistar rats (University Animal Department, Katowice, Poland) were housed under controlled environmental conditions, in a well-ventilated room, at 22 ± 2 °C and under a 12 h light:12 h dark cycle (lights on from 7:00 a.m. to 7:00 p.m.). Animals received food and water ad libitum. Litters remained with dams until the 21st day after birth and then were placed in individual cages according to sex. Experiments were carried out in the morning in only male rats, handled in accordance with the principles and guidelines described in the NIH Guide for the Care and Use of Laboratory Animals. All procedures were reviewed and approved by the Local Bioethical Committee for Animal Care at the Medical University of Silesia (decision no. 49/2009 issued on 17.06.2009).
The central noradrenergic system of newborn rats was lesioned with DSP-4 (Sigma, St. Louis, MO, USA). Rats were injected on the 1st and 3rd day of postnatal life with either DSP-4 (50 mg/kg sc) or 0.9 % NaCl (1.0 ml/kg sc). DSP-4 was dissolved in distilled water immediately before injection, and preceded 30 min beforehand by treatment with the selective serotonin reuptake inhibitor zimelidine (10 mg/kg ip) (Sigma, St. Louis, MO, USA)—in order to prevent serotoninergic effects of DSP-4. The dose and the days of treatment were selected on the basis of the reports by Jonsson et al. (1982), Brus et al. (2004) and Dąbrowska et al. (2007). Rats continued to be housed as above until 10 weeks, for further experimentation.
Assessment of Biogenic Amine and Metabolite Content
At 10 weeks after birth control and DSP-4-treated rats were injected with saline (1.0 ml/kg ip) or methanandamide (10 mg/kg ip) (Tocris Bioscience, Ellisville, MO, USA), and were terminated by decapitation 60 min later. The frontal cortex, thalamus, and spinal cord were rapidly dissected and placed on dry ice, weighed and stored at −70 °C, pending assay. Samples were homogenized for 15–20 s in ice-cold trichloracetic acid (0.1 M), containing 0.05 mM ascorbic acid. After centrifugation (5,000×g, 5 min), supernatants were filtered through 0.2 μm cellulose membranes (Titan MSF Microspin filters, Scientific Resources Inc., Eatontown GB) and filtrates were injected onto the HPLC/ED column. Levels of NE, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were assayed by HPLC/ED (Nowak et al. 2003). The composition of the mobile phase was: 75 mM NaH2PO4, 1.7 mM 1-octanesulfonic acid, 5 μM EDTA (Avocado, Research Chemical Ltd., Morecambe, GB), 100 μl triethylamine (Sigma, St. Louis, USA), 9.5 % acetonitrile (J.T. Baker, Deventer, Holland), pH 3 adjusted with phosphoric acid (Fluka, Steinheim, Switzerland). The flow rate was maintained at 0.7 ml/min, at a temperature of 22 °C, and the oxidation potential was fixed at +700 mV, 10 nA/V sensitivity. Peaks were automatically integrated by universal chromatographic interface UCI-100 (Dionex Softron Gmbh, Germering, Germany). The instrumentation included an electrochemical detector (Gilson, Villiers-le-Bel, France) model 141 with flow cell, piston pump model 302 with head 5SC (Gilson, Villiers-le-Bel, France), manometric module model 802 (Gilson, Villiers-le-Bel, France), thermostat for STH 595 column (Dionex Softron Gmbh, Germering, Germany), precolumn Hypersil BDS C18, 10 × 4 mm, 3 μm (ThermoQuest, Waltham, GB), and chromatographic column Hypersil BDS C18, 250 × 4.6 mm, 3 μm (ThermoQuest, Waltham, GB). The data were quantified using the area under the peaks and external standards, using Chromeleon software (Dionex, Germany).
Immunohistochemistry and Densitometric Analysis
Immunostaining for CB1 receptor expression was carried out according to Kuter et al. (2011). At 10 weeks after birth control and DSP-4-treated rats were decapitated. The brains were rapidly removed, postfixed in cold 4 % paraformaldehyde for 7 days and cryoprotected in 20 % sucrose solution in phosphate-buffered saline (PBS). The brains were cut on a freezing microtome into 30 μm frontal sections (AP = 2.52 mm from bregma according to Paxinos and Watson 2007). Free-floating sections were incubated for 48 h at 4 °C in a primary antibodies (anti-CB1 receptor, 1: 1,000; Sigma, Germany), rinsed in PBS, then incubated for 30 min in secondary antibodies (anti rabbit biotinylated, 1:200, Vector Laboratories, UK) and processed using an ABC-peroxidase kit (Vector Laboratories, UK) and 3,3′-diaminobenzidine as a chromogen. The stained sections were mounted on slides, dried, dehydrated, cleared in xylene and cover-slipped in a Permount medium (Fisher Scientific, USA).
Two sections stained immunohistochemically for CB1 receptor were analyzed densitometrically in the region of striatum and frontal cortex. The sections were scanned all together, digitalized, adjusted for brightness, and regions of interest outlined using Multi Gauge programme (FUJIFILM). The optical density and area (OD) were counted. Background signal was subtracted from each section separately, from the region of corpus callosum. The results are presented as mean of each OD/area2 value minus backgrounds.
Inflammatory pain and analgesia were determined using the formalin test in the rat (Acton et al. 1992). Rats were placed in a clear plastic chamber (30 × 30 × 30 cm) for 30 min to allow them to accommodate to their surroundings with a mirror placed at a 45° angle beneath the floor to allow an unobstructed view of the paws. Then, 30 min after methanandamide (10 mg/kg ip) administration, 50 μl of 5 % formalin solution was injected subcutaneously, 30-gauge needle, into the right hind paw plantar surface. Rats were then returned to the chambers, and nociceptive behavior was observed immediately after formalin injection. Nociceptive behavior was quantified using the scale 0–3 points. Formalin-induced pain is biphasic. The initial acute phase (0–10 min) is followed by a relatively short quiescent period, which is then followed by a prolonged tonic response (15–60 min). A reduction of formalin-induced behavior observed after administration of a given drug is interpreted as an analgesic response.
Group differences in monoamines were assessed by an analysis of variance (ANOVA) and the post-ANOVA test of Newman–Keuls. Group differences in behavioral studies were analyzed by Student’s t test. A P value <0.05 was taken as the level of significant difference.
Effect of DSP-4 Treatment on Monoamine Concentration in the Frontal Cortex, Thalamus, and Spinal Cord
In the thalamus methanandamide treatment significantly reduced NE, 5-HT and 5-HIAA concentration only in control rats, while there was no consisted change in DA and its metabolite DOPAC (Fig. 1b).
Immunohistochemistry and Densitometric Analysis for CB1 Receptor Expression
The major finding of the present study is that neonatal DSP-4 treatment—resulting in permanent destruction of noradrenergic inputs to spinal cord and cortical areas—diminishes adulthood CB1-receptor agonist mediated antinoception, in the absence of change in CB1 receptor density in brain.
The antinociceptive effects of endocannabinoids have been well described in animal models of acute and chronic pain (Walker and Huang 2002). Cannabinoid analgesia involves effects at the supraspinal, spinal and peripheral levels (Agarwal et al. 2007; Hohmann and Suplita 2006). Furthermore, there is a large body of evidence associating the cannabinoid system with regulation of noradrenergic activity. Systemic administration of the synthetic cannabinoid agonist WIN 55,212-2 enhances NE release in prefrontal cortex and increases c-fos expression in the LC. In addition, there is a significant alteration in adrenergic receptor and NE transporter expression in the frontal cortex (Oropeza et al. 2005). McLaughlin et al. (2009) found that the acute administration of exogenous cannabinoid ligands activates the hypothalamic–pituitary–adrenal axis through an increase in serotoninergic and noradrenergic neurotransmission. All of the above may underlie the reported changes in attention, cognition, anxiety and pain threshold commonly observed after cannabinoid exposure (Reyes et al. 2009). Yet, there are no data regarding such an association in NE-denervated animals. Neonatal treatment with neurotoxins, such as 6-hydroxydopamine (6-OHDA) or DSP-4 produces marked impairment in development of the central noradrenergic system, i.e., permanent and robust NE-denervation of the frontal cortex and spinal cord (Fig. 1a, c), accompanied by NE hyperinnervation of brainstem and cerebellum (Jaim-Etcheverry and Zieher 1980; Medina and Novas 1983).
In the present study, destruction of noradrenergic neurons by DSP-4 significantly decreased the antinociceptive effect of methanandamide (10 mg/kg ip) in the tail-immersion test, hot-plate test, and writhing test (Figs. 3, 4, 5). Ambiguous results were obtained in the paw pressure and formalin tests (Figs. 6, 7). Despite marked antinociceptive effects of methanandamide, there was no change in CB1 receptor density in rat brain (Fig. 2). Gutierrez et al. (2003) demonstrated that intrathecal administration of 6-OHDA, resulting in selective 85 % NE depletion in rat lumbar spinal cord, attenuated the antinociceptive effect of the cannabinoid agonist WIN55,212-2 (5 or 10 mg/kg, ip) as assessed in the tail-flick and formalin tests. They also found that WIN55,212-2 suppressed formalin-evoked fos protein expression, a marker of neuronal activity, in the lumbar dorsal horn of sham-operated rats, while no suppression was observed in lesioned rats. Thus, cannabinoids produce antinociception, in part, by modulating descending noradrenergic systems. To the best of our knowledge there are no data relating to the analgesic effect of CB1 receptor agonists in neonatally DSP-4 treated rats. Kushikata et al. (2011) showed that DSP-4 (50 mg/kg ip in adult rats) significantly reduced ketamine analgesia in the hot-plate test. Others (Zhong et al. 1985) found that in rats pretreated intrathecally with DSP-4, the analgesic effect of morphine, given either icv or ip and assessed in a tail-flick test, was significantly attenuated.
Previous reports (Oropeza et al. 2005; Page et al. 2008) indicate that CB1 receptor agonists activate the noradrenergic pathway. Martin et al. (1999) reported that WIN55,212-2 elevated tail-flick latencies when injected into the noradrenergic A5 region. Yoon and Choi (2003) demonstrated synergistic interaction after intrathecal delivery of WIN 55,212-2 and clonidine in the formalin test. And, Tham et al. (2005) showed that an α2-adrenoceptor agonist (dexmedetomidine) when combined with a cannabinoid receptor agonist (CP55,940) resulted in a synergistic antinociceptive effect in the hot-plate test. Summing up, the above reports, plus results of the current study, indicate that a reduction in methanandamide induced analgesia may be attributable to destruction of noradrenergic neurons.
Nevertheless, we cannot definitely exclude participation of the 5-HT system in the obtained results, particularly considering the fact that Mendiguren and Pineda (2009) showed that anandamide inhibited 5-HT neuronal firing in rat brain slices of the dorsal raphe nucleus. Also, pain sensation is known to be modified by the serotoninergic system, another monoaminergic pathway. As already noted, DSP-4-induced NE depletion attenuated 5-HT ligand mediated analgesia (Minor et al. 1986; Archer et al. 1987). And in our study we found that in the thalamus methanandamide reduced 5-HT and 5-HIAA concentrations, although only in control rats. Therefore, it is quite possible that the serotoninergic system in particular and other phenotypic systems in general, may be influenced by DSP-4 lesioning of noradrenergic innervation and its effect on antinociception.
In conclusion, the present study demonstrates a prominent effect of noradrenergic neurons in regulating the antinociceptive effects of methanandamide—the CB1 receptor agonist—without altering CB1 receptor density in brain. The associated pathways involved in this effect remain to be resolved.
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