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Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats
Author’s Accepted Manuscript Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats Pourzitaki Chryssa, Tsaousi Georgia, Papazisis Georgios, Kyrgidis Athanassios, Zacharis Constantinos, Kritis Aristeidis, Malliou Faye, Kouvelas Dimitrios
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S0014-2999(18)30397-2 https://doi.org/10.1016/j.ejphar.2018.07.029 EJP71894
To appear in: European Journal of Pharmacology Received date: 29 January 2018 Revised date: 12 July 2018 Accepted date: 18 July 2018 Cite this article as: Pourzitaki Chryssa, Tsaousi Georgia, Papazisis Georgios, Kyrgidis Athanassios, Zacharis Constantinos, Kritis Aristeidis, Malliou Faye and Kouvelas Dimitrios, Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats
Pourzitaki Chryssa1*, Tsaousi Georgia2, Papazisis Georgios1, Kyrgidis Athanassios1, Zacharis Constantinos3, Kritis Aristeidis4, Malliou Faye1 Kouvelas Dimitrios1
1
Department of Clinical Pharmacology, Faculty of Medicine, School of Health
Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 2
Clinic of Anesthesiology and Intensive Care, AHEPA University Hospital, Faculty of
Medicine, School of Health Sciences, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece 3
Analytical Development Laboratory, R&D API Operations, Pharmathen SA,
Thessaloniki, Greece 4
Department of Experimental Physiology, Faculty of Medicine, School of Health
Sciences, Aristotle University of Thessaloniki
*
Corresponding author. Pourzitaki Chryssa, MD, MSc, MHA, PhD Assistant
Professor of Pharmacology and Clinical Pharmacology Department of Clinical Pharmacology, Faculty of Medicine, School of Health Sciences, Aristotle University of Thessaloniki University Campus, 54124, Thessaloniki, Greece Tel: +302310999025, Mobile: +306945492971; fax: +302310999312. [email protected]
ABSTRACT Fentanyl, a μ-opioid receptor agonist, has been studied for its neuro/psychopharmacological effects since its first clinical use; however, its effect on the release rate of the Central Nervous System (CNS) neurotransmitters has not been yet elucidated. In the present study the influence of fentanyl on the release rates of glutamate and GABA is investigated. Specifically, we examined the effects of
intravenous (10μg/kg) as well as intrahypothalamic (0.1nmol/min) fentanyl administration on the release rates of GABA and glutamate in the superfusate of anterior hypothalamus, under tail pinch manipulation. The release rate of the neurotransmitters was monitored by the push–pull superfusion technique. To investigate the role of fentanyl the opioid antagonist, naloxone 0.1mg/kg was administered intravenously, or 50nmol/min intrahypothalamicaly. The amino acids were determined by High Performance Liquid Chromatography (HPLC) and fluorimetric detection after NBD-Cl derivatisation. After intravenous fentanyl administration a significant decrease of glutamate and increase of GABA release rates were observed. However during the pain manipulations, the release rate of glutamate was increased. Intravenous naloxone did not affect significantly the release rates of both amino acids, while intrahypothalamic antagonist administration reversed the alterations in both neurotransmitters release rates. Our results demonstrate that there is an opioid-glutamatergic transmission pathway, located in hypothalamus and that opioids can activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect.
KEYWORDS: fentanyl, naloxone, hypothalamus, tail pinch, push pull superfusion, high performance liquid chromatography, GABA, glutamate
1. INTRODUCTION Glutamatergic neurotransmission plays a crucial role in both noxious stimuli creation and pain perception (Fundytus 2001). Pain stimuli from spinal cord end up to the hypothalamus, with its final endings being the ventromedial, periventricular, suprachiasmatic and paraventricular nuclei (Bester et al. 1997). The ventromedial hypothalamic nucleus corresponds to the center of aversion and has also been described as an important center for anger and aggressive behavior. The connections of the hypothalamus with the grey matter of aqueduct regulate the
homeostasis through metabolism, adding another regulatory mechanism of the body's response to the pain (Malick et al. 2000). Similar properties have been addressed to the arcuate nucleus of hypothalamus (Porro et al. 1998). Animal experiments involving the paraventricular nucleus of the hypothalamus have shown that the removal of pituitary gland did not inhibit the analgesic effect of i.c.v. administration of L-glutamate. These results seem to contradict the aspect that the activation of hypothalamus in the analgesia process is elicited by the hypothalamicpituitary tract. Thus, it is assumed that the hypothalamic control is due to its connections with other nuclei (Yang et al. 2006a; 2006b). A similar mechanism of analgesia has been assigned to the paraventricular hypothalamic nucleus suggesting its possible involvement in the endogenous opiate peptide system in spinal cord independently (Yang, et al. 2008; 2009). The anterior hypothalamus includes the paraventricular (PV), the supraoptic (SO) and the suprachiasmatic (SC) nuclei, all of which seem to be involved in the perception of pain. Apart from the pain perception, hypothalamus is also involved in learning, memory and response to stress in rats (Kohsaka et al. 1999). In addition, the hypothalamus receives nerve stimuli from baro- and chemo-receptors and interacts with the solitary tract and the motor nucleus of vagus. Furthermore, evidence suggests that peripheral nervous signaling mechanisms control excitation at central terminals, consisting major sites of signal integration of peripheral and central inputs particularly, from the hypothalamus (Andresen et al. 2012). Experimental studies have shown that the co-administration of glutamate and opioids intravenously exerts a synergistic analgesic effect. Moreover, opioids enhance the presynaptic inhibitory effects mediated by GABA-A receptors, through inhibition of glutamate re-uptake and thus its concentration in the synapsis is increased. This fact further promotes the analgesic effect of opioids. However, it is known that glutamate release is involved in both creation and perception of pain stimuli. (Popik et al. 2000; Popik and Kozela 1999).
The purpose of the present research was to investigate the processes taking place in the hypothalamus at the influence of noxious stimuli and administration of intravenous opioids. These processes are probably expressed as alterations in the release rate of neurotransmitters from hypothalamic nuclei over time. For this purpose, the tail pinch test was carried out on adult, male rats. The release of GABA and glutamate in the hypothalamus was assessed using the push–pull superfusion technique, while fentanyl and naloxone as opioid agonist and antagonist respectively were applied both intravenously (i.v.) and intracerebroventricularly (i.c.v.).
2. MATERIALS AND METHODS The study was approved by the Research Council of School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki and has been conducted in a manner that does not inflict unnecessary pain or discomfort upon the animals. All animal experiments were in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). 2.1 Experimental setting Eighty (80) male Wistar rats (aged 10 -12 weeks old, weighting 230 – 300 g) were used in this experimental setting. All animals were single housed, maintained in a climate-controlled room on a 12-hour light-dark cycle and allowed food and water ad libitum. All animals were provided from the Veterinary School, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. Rats were anaesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (40mg/kg) and ketamine (50mg/kg). Push pull cannulas used were custom made according to the literature. (Philippu, 2017) For intravenous (i.v.) infusions of drugs a PE 50 tubing was inserted into the jugular vein (Kouvelas et al. 2006). Push pull perfusion technique was applied on each animal. In detail, the head was fixed in a stereotaxic frame and a guide cannula (outer diameter 1.25mm, inner diameter 0.90 mm) was stereotaxically inserted according to the Paxinos and Watson atlas (Paxinos and Watson 1996), until its tip was 2mm above the left anterior hypothalamus (AP -1.5mm, V 8.2mm, L 0.5mm). The guide cannula was fixed with screws and dental cement. At least two days after surgery, the stylet of the guide cannula was replaced by a push-pull cannula of the following diameters (mm): outer needle; outer diameter 0.82, inner diameter 0.50, inner needle; outer diameter 0.20, inner diameter 0.10. Protrusion of the inner needle was 0.20mm. The push-pull
cannula was 2mm longer than the guide cannula thus reaching the anterior hypothalamus. According to the literature 48h after the placement of the cannula under pentobarbital and ketamine anesthesia both drugs were eliminated, while no effects of sensitization nor tolerance are described in the low doses used (Lin et al, 1973;Kosten and Bombace, 2001;Popik et al, 2008,Dodelet-Devillers et al, 2016) The hypothalamus of the conscious, freely moving rat was superfused at a rate of 20μl/min with artificial cerebrospinal fluid (aCSF) of the following composition (mmol/l): NaCl 140, KCl 3.0, CaCl2 2.5, MgCl2 1.0, Na2HPO4 1.0, NaH2PO4 1.0, glucose 3.0, pH 7.2 (Prast et al. 1996). After an equilibration period of 80min, superfusates were collected in time periods of 5min into tubes kept at −50oC. The samples were stored at−80oC until biochemical analysis was carried out. During the experiment, animals were deprived of food and water. 2.2 Study outline The drugs tested in our experiment study protocol were fentanyl [N-(1phenethyl-4-piperidyl)-N-phenyl-propanamide] from Jansen Cilag (Fentanyl) and naloxone [(5a)-4,5-Epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one] from Vianex (Narcan) in commercial forms. Both fentanyl and naloxone were diluted in 0.9% NaCl solution for i.v. (intravenous) infusion, while for i.c.v. (intracerebroventricular) infusion into the hypothalamus drugs were dissolved in aCSF. For fentanyl and naloxone i.v. and i.c.v. administration the doses used were in accordance with previously published studies. (Yoshida et al, 1999;Verbogh et al, 1999;Genco et al, 2003;Cao et al, 2004) According to the type of drug regimen applied, the animals were randomly assigned into 10 groups (of 8 animals each) using an internet-based computer- generated random number table (www.randomizer.gr). In detail the animal groups were treated with: 1) normal saline 0,9% i.v., 2) fentanyl 10μg/kg (i.v), 3) naloxone 0.1mg/kg (i.v.), 4) vehicle (aCSF) (i.c.v), 5) 0.1nmol/min fentanyl (i.c.v), 6) 50nmol/min (i.c.v.), of
naloxone 7) normal saline 0,9% (i.v.) premedicated with naloxone 0.1mg/kg (i.v), 8) fentanyl 10μg/kg (i.v) premedicated with naloxone 0.1mg/kg (i.v), 9) vehicle (aCSF) (i.c.v.) premedicated with naloxone 50nmol/min (i.c.v.) and 10) fentanyl 0.1nmol/min (i.c.v.) premedicated with naloxone 50nmol/min (i.c.v.) (Table 1). Push pull superfusion was conducted in normal conditions of temperature and humidity, between 09.00 a.m. and 12.00 p.m. Fifteen min (15) after initiation of superfusate collection, we administered intravenously to the rats in Groups 1-3 either saline 0,9%, fentanyl, or naloxone i.v., at a rate of 2μl/min (Bruins Slot et al. 2002;Yoshida et al. 1999). In Groups 4 to 6, the animals received i.c.v., either aCSF as vehicle, or fentanyl or naloxone with infusion rate 2μl/min. Forty-five min after initiation of superfusate collection a tail pinch test was applied to all animals of Groups 1 to 6. Tail pinch (not traumatic) was applied continuously for 3min by an appropriate clamp (force: 3.5 N) placed approximately 2cm from the tip of the tail (Singewald et al. 1995). According to the literature the area is very sensitive to pain and the stimulus causes great stress (Park et al. 2015). Sampling stopped 30min after tail pinch. In Groups 7 and 8, fifteen (15) min after initiation of sampling the animals were premedicated with i.v. administration of naloxone. Forty-five (45) min after initiation the animals received intravenously either saline 0.9% (Group 7) or fentanyl (Group 8) (Cao et al., 2004). In Groups 9 and 10, fifteen (15) min after initiation of sampling the animals were premedicated with naloxone i.c.v. with infusion rate 2μl/min. Naloxone i.c.v. infusion lasted 2min. Forty-five (45) min after starting the experiment the animals received i.c.v. either vehicle (aCSF) (Group 9) or fentanyl (Group 10) with infusion rate 2μl/min. Fentanyl i.c.v. infusion lasted 5min. Seventyfive (75) min after initiation of superfusate collection a tail pinch test was applied to all animals of Groups 7 to 10. When the i.c.v. infusion was taking place, sampling was omitted (1 sample skipped) while it was completely stopped 30min after tail pinch.
At the end of the superfusion, the brain was removed and the position of the cannula was verified in 50mm histologic slices stained with cresyl violet and luxol fast blue (data non shown) (Kluver and Barrera, 1953;Singewald et al, 1995;Prast et al, 1996;Kouvelas et al, 2006). Superfusates from experiments, in which the cannula was found to be localized outside of the anterior hypothalamus, were discarded. 2.3 Determination of GABA and glutamate All reagents used throughout this study were of analytical grade. Double distilled water was used throughout this study. Standard stock solutions of amino acids and the internal standard (methylamine) were prepared in water and stored at 4oC. Working standards solutions were prepared daily by appropriate dilutions of aliquots obtained by the stock solutions in water. NBD-Cl was dissolved in ethanol to prepare standard stock solution at 6 mM. This solution was stored at 4oC and protected from the light. The mobile phase A consisted of 10 mM NaH2PO4 and 10 mM Na2HPO4 (pH 7.0) and was prepared in daily basis. This solution was filtered through 0.22 μm PTFE membrane filter (Millipore) prior to use. The derivatization protocol of amino acids (AA) was adopted by Yang and his colleagues with some modifications (Yang et al. 2007). Briefly, 150 μl of standard AA solution or sample was mixed with 50μl of internal standard (120μM methylamine) and 50μl of borate buffer (100mM, pH 8.2). Then 50μl of 4-chloro-7-nitrobenzo-2oxa-1,3-diazole (NBD-Cl) solution (6mM) was added and the resulted mixture was vortexed and left to react at 60oC for 1h in water bath. An aliquot of 600μl of cold water was finally added in the mixture while 10μl was used for HPLC analysis. Blank solutions without AA were processed as above. The HPLC setup comprised the following parts: a quaternary gradient solvent system (LC-10ADvp, Shimadzu), a fluorescence detector (RF-10AXL, Shimadzu) and an auto-sampler (SIL-20AC – Prominence, Shimadzu). The analytical column was a reversed-phase Lichrospher 100-5, C18 (250 x 4.0mm, 5μm, MZ Analysentechnik) and the data acquisition was carried out via Clarity® software
(DataApex). The mobile phase A (A) consisted of a 20 mM phosphate buffer (pH 7.0):CH3OH, 99:1 % v/v while the mobile phase B (B) was a mixture of mobile phase A:CH3OH, 30:70 %, v/v. The flow rate was 1 ml/min throughout the analysis and all separations were performed at ambient temperature. The initial concentration of eluent B was 10 %. The gradient elution was as follows: eluent B 10-35% (0-20min), 35-100% (20-30min). At time of 30.1min the gradient elution was returned to its initial composition and stated for a period 15min to maintain a stable and reproducible separation. The analyte derivatives were detected at 470/540nm (λext / λem). The retention time of glutamate and γ-aminobutyric acid (GABA) was 7.2 and 23.9 min respectively while the methylamine was eluted at 31.1 min. The compounds of interest were quantified using external calibration curves. 2.4 Statistical analysis The first 3 superfusate samples, taken until fentanyl or naloxone were administered, were used as controls. Normality of data was assessed by ShapiroWilk test. Neurotransmitter release rates were analyzed by Mann Whitney U and Friedman’s test followed by Wilcoxon’s rank test for paired data. Analyses of variance (ANOVA) for multifactor repeated measures with Greenhouse-Geisser correction were conducted to analyze continuous variables over time. If variance/covariance matrices were not equal across all groups and if sphericity of the combined variance/covariance matrix (overall sphericity) and sphericity for each group was violated, then we used contrasts for between/within tests and Bonferroni adjustment. Comparisons between different groups were performed using one-way ANOVA. Two-tailed levels of significance were used in all statistical calculations. Values are presented as mean and standard error (SE) unless otherwise stated. Statistical significance level for group differences was set at P≤0.05. Statistical Package for Social Sciences (SPSS, version 22.0; SPSS Inc., Chicago, IL, USA) was used for all calculations and analysis.
3. RESULTS Mean basal outputs of neurotransmitters in the anterior hypothalamus of the conscious adult rat were 652.6±89.2 fmol/min for glutamate (n=80) and 274.3±78.7 fmol/min for GABA (n = 80). The basal release rates in the first three samples preceding infusions were taken as 1. 3.1 Effects of intravenous fentanyl and naloxone on glutamate and GABA release rate in the anterior hypothalamus Administration of fentanyl 10μg/kg (i.v.) decreased the glutamate release rate from the anterior hypothalamus. This effect peaked approximately 10min after fentanyl reaching 32±13% of the control value (P<0.001) (Fig. 1A). Injection of naloxone, 0.1mg/kg (i.v.) induced a subtle increase of the basal rate of glutamate release, while saline 0,9% (i.v.) was ineffective (Fig. 1A). Immediately after the tail pinch test glutamate release in the anterior hypothalamus from rats receiving saline and naloxone i.v. increased up to 295±25% and 282±18% of the control value respectively and returned to baseline values after about 20min (P<0.001). In contrast, in the group of fentanyl i.v. administration a delayed (15min after the tail pinch), and progressive increase in glutamate release rate up to 191±21% (P=0.002) was recorded. To the former effect, administration of fentanyl 10 μg/kg i.v. enhanced the GABA release rate from the anterior hypothalamus. The peak effect occurred approximately 15 min after fentanyl injection being 253±22% of the control value (P<0.001) (Fig. 1B). The tail pinch test decreased the GABA release rate in the groups of rats receiving saline i.v. or naloxone i.v, approximating 74±15% and 43±24% of the control value, respectively (P=0.046) and 25min later they reversed to baseline values. Whereas, in the group of animals receiving fentanyl i.v. a notable augmentation of GABA release rate up to 182±17% (P<0.001) was recorded. (Fig. 1B)
3.2 Effects of fentanyl and naloxone i.c.v. infusion into the hypothalamus on glutamate and GABA release rate in the anterior hypothalamus Infusion of fentanyl 0.1nmol/min i.c.v reduced the glutamate release rate, immediately after injection, which peaked 5min post-infusion up to 34±18% of the control value (P<0.001) (Fig. 2A). Infusion of naloxone 50 nmol/min i.c.v. increased basal release rate of glutamate in the anterior hypothalamus up to 214±24%, while aCSF as vehicle i.c.v (n=8) was ineffective (Fig. 2A). Immediately after the tail pinch test glutamate release in the anterior hypothalamus from rats receiving saline and naloxone i.c.v. increased up to 280±23% and 261±16% of the control value respectively and returned to baseline values within 20min post its peak (P<0.001). Infusion of fentanyl 0.1nmol/min i.c.v increased the GABA release rate, up to 192±24% of the control value (P<0.001) (Fig. 2B). Immediately after the tail pinch test GABA release in the group of rats receiving saline and naloxone i.c.v. presented a significant (P=0.002) deterioration (53±16% and 68±24% of the baseline, respectively) and returned to control values 15min later. However, the rats with fentanyl i.c.v. administration presented an abrupt increase in GABA release rate up to 163±19% of the control value that lasted approximately 30min (P=0.004). (Fig. 2B) 3.3 Effects of intravenous fentanyl after intravenous premedication with naloxone on glutamate and GABA release rate in the anterior hypothalamus Premedication with naloxone 0.1 mg/kg i.v. ameliorated fentanyl effects on glutamate release rates (Fig. 3A). The tail pinch test caused a statistically significant increase in glutamate release in the anterior hypothalamus of rats receiving saline which peaked after 15min reaching 278±14% of the control value and reversed to baseline values after approximately 30min (P<0.001). Moreover, the rats with fentanyl i.v. administration showed a slight increase in glutamate release rate up to 142±16% (P=0.04).
Premedication with naloxone 0.1mg/kg i.v. abolished the effect of fentanyl i.v. administration on GABA release rate. (Fig. 3B). The tail pinch test decreased the GABA release rate in the group of rats receiving saline i.v. which reached its lowest value 15 min later (62±16% of the baseline; P=0.05) and returned to baseline values after 30 min, while the rats that received fentanyl i.v. presented a marginally statistically significant increase in GABA release rate up to 144±16% (P=0.05) (Fig. 3B). 3.4 Effects of fentanyl infusion into the hypothalamus after i.c.v. premedication with naloxone on glutamate and GABA release rate in the anterior hypothalamus Premedication with naloxone 50 nmol/min i.c.v. induced an abrupt and notable (P<0.001) increase in glutamate release rate from anterior hypothalamus which peaked 10 min after naloxone infusion (235±27% of baseline), which during the next 30min gradually deteriorated but without reversing to baseline (Fig. 4A). Application of tail pinch test in the group of rats receiving aCSF as vehicle i.c.v resulted in an instant and notable glutamate release (276±29% of the baseline; P<0.001) which lasted about 10min and reversed almost to control values after 30min. However, in the group of rats with fentanyl i.v. administration a delayed increase in glutamate release rate was recorded but its peak (174±28% of baseline; P=0.026) was lower compared to aCSF i.c.v group. Injection of fentanyl 0.1nmol/min i.c.v increased the GABA release rate, up to 166±15% of the control value (P=0.003), while in the group of aCSF as vehicle i.c.v. GABA release rate remained constant (Fig. 4B). After the tail pinch test the GABA release rate in the group of rats receiving vehicle i.c.v. remained close to control values, while in the group of rats with fentanyl i.c.v. administration a marked increase in GABA release rate up to 168±19% of the baseline (P=0.007) was recorded. (Fig. 4B)
4. DISCUSSION Pain involves sensory-discriminative, cognitive-evaluative, and affectiveemotional components. Nociceptive transmission is modulated by several pathways including brainstem and hypothalamus which play a key role in pain stimuli processing (Denuelle et al. 2007). Pain stimuli information can reach to the hypothalamus via multi-synaptic pathways through the brainstem, thalamus and cortex. Opioid signaling in the central nervous system is critical for controlling cellular excitability. The present study examined the modulation of anterior hypothalamus response to a painful stimulus by the administration of both a strong μ-opioid receptor agonist and an opioid antagonist (fentanyl, and naloxone, respectively), on an experimental setting of freely moving rats. For the determination of GABA and glutamate release rates, push pull perfusion was combined with a new derivatization protocol for HPLC analysis, developed according to the literature with some modifications (Klinker and Bowser 2007;Yang et al, 2009;Wu et al. 2014). According to our results, application of fentanyl promoted a notable reduction of glutamate release rate from the anterior hypothalamus, irrespectively of the route of administration (i.v. or i.c.v). According to Iremonger and Bains, endogenous opioids are released from dendritic vesicles in response to postsynaptic activity and act in a retrograde manner to inhibit excitatory synaptic transmission (Iremonger and Bains 2009). This opioid inhibition involves a downstream entry of calcium ions (Ca+2) and is modulated presynaptically. It appears that neurons have the ability to selfregulate their excitability through the dendritic release of opioids to inhibit excitatory synaptic transmission (Iremonger and Bains 2009). Nevertheless, the pattern of glutamate release in the two routes of fentanyl administration after the tail pinch was not alike. Intravenous infusion of fentanyl promoted a delayed but notable augmentation of glutamate release, while i.c.v administration of fentanyl induced only a subtle enhancement of glutamate release
rate. The role of glutamate in the process of central regulation of pain is not fully elucidated yet. The activation of glutamatergic receptors may have an either analgesic or the opposite effect. In the past the inhibition of glutamatergic transmission by the administration of NMDA antagonists (ketamine, MK801dizocilpine, memantine, dextromethorphan, glycine, LY274614, LY235959 and CGP 39551) also resulted in an inhibition of the development of the opioid addiction phenomenon (Bilsky et al. 1996;Elliott et al. 1994a;Elliott et al. 1994b;Gonzalez et al. 1997;Popik et al. 2000;Popik & Kozela 1999). Furthermore, the administration of LY354740 without reversing the analgesic effect of morphine revealed the involvement of glutamatergic metabotropic type II receptors in pain perception (Bhat et al. 1998). The decline of glutamate release rate after fentanyl infusion and its significant augmentation after the tail pinch test could be explained by the existence of an opioid-NO-glutamatergic transmission pathway, located in hypothalamus, which is responsible for neuro-endocrinological regulatory response after noxious stimulus (Bhat et al. 1998). There are data showing that remifentanyl administration allosterically stimulates different types of NMDA receptors NR1A / 2A and NR1A / 2B, an effect unrelated to its concentration (Hahnenkamp et al. 2004). Furthermore, the withdrawal response, known as acute dependence, seems to reflect the beginning of an adaptive change in the rats. In rat models, it is suggested that a single dose of morphine modifies the expression of the NMDA receptor subunit NR1, NR2A and NR2B mRNAs in the hippocampus and hypothalamus which probably leads to a late chronic opiate dependence (Le Greves et al. 1998). It has been reported that μ-opioid receptor activation selectively inhibits excitatory activity in SON hypothalamic neurons via a presynaptic mechanism (Liu et al. 1999). In the present study although intravenous infusion of naloxone had no impact on the release rate of glutamate in the anterior hypothalamus, when it was given i.c.v. it increased glutamate release in an important manner. Fentanyl i.v.
administration delayed increase in glutamate release rates may indicate that naloxone prevented the increase in glutamate after fentanyl treatment. Moreover, the administration of naloxone from either route as premedication abolished the effect of fentanyl infusion on glutamate release rate. This is in accordance to the reversed effects of D-Ala(2), N-CH(3)-Phe(4), Gly(5)-ol-enkephalin (DAGO) on glutamatergic transmission in supraoptic nucleus by addition of naloxone (Liu et al. 1999). Previous reports suggested that when morphine was given in striatum and limbic forebrain, it promoted a decline in extracellular glutamate levels; when it was infused in substantia nigra it failed to increase dopamine release and decreased glutamate (Desole et al. 1996;Enrico et al. 1998;Miele et al. 1994). In the same experimental settings, intravenous administration of naloxone within one hour after systemic morphine, presented an antagonizing effect on morphine-induced changes, without affecting the depletion of glutamate dialysate concentrations. The above results combined with the results from the present study suggest that an inhibitory μopioid receptor mediated-mechanism controls certain brain sites coupled with neuronal glutamate uptake (Desole et al. 1996;Enrico et al. 1998;Miele et al. 1994). Concerning the GABA release rate in our setting, this was significantly enhanced by the application of fentanyl, regardless of the route of its administration (i.v. or i.c.v), while naloxone did not affect GABA concentrations. Unlike our findings it has been shown that the push-pull perfusion of morphine within the hypothalamus affected the activity of GABA at only 3 out of 17 hypothalamic sites at which the opioid agonist was perfused (Noto and Myers 1984). However, Yoneda and colleagues have demonstrated that morphine administered acutely exerts little effect on the content or distribution of GABA within the whole brain or its anatomical constituents including the hypothalamus (Yoneda et al. 1977). This inconsistency between the results of the present study and previous ones could probably be attributed to divergent pharmacokinetics and selectivity for μ-opioid receptors
between morphine and fentanyl. Furthermore, morphine is a partial agonist at the opioid μ-receptors, while fentanyl may have higher efficacy (Popik et al. 2000). This discrepancy underlines the importance of the direct determination of amino acids release by the push-pull technique under in vivo conditions when modulatory mechanisms are investigated. Noxious stimulus in the rats receiving fentanyl i.v. promoted GABA release, while in those receiving saline or naloxone, the inhibitory amino acid basal release rates remained unaffected. It should be noted that when the tail pinch test was performed in fentanyl i.v. group GABA release rates had not fully reversed to baseline values, so it is not clear if the certain enhancement is due to the tail pinch test or the effect caused by the infusion of an opioid. Premedication with naloxone i.v. had no significant impact on GABA release rate and fully antagonized the effect of fentanyl i.v. administration. The tail pinch test decreased the GABA release rate in the rats receiving saline i.v. while, in those receiving fentanyl i.v., a marginal increase in GABA release rate was presented. When premedication with naloxone was administered i.c.v. the administration of fentanyl by the same route increased the GABA release rate, which was maintained almost constant after the tail pinch test (Fig. 4B). This is in accordance with previous results showing that morphine increased GABAergic tone in the hypothalamic paraventricular nucleus and reduced the release of glutamate and the activation of oxytocinergic neurons mediating penile erection (Succu et al. 2006). The limitation of our study is the lack of cardiovascular monitoring during the specific experimental procedure; however this would require an arterial catherization, leading to significant perioperative risk and adding stressful manipulations during the push pull superfusion in freely moving rats. Moreover, opioid and especially fentanyl effects on cardiovascular parameters have already been studied in rats thoroughly (Albrecht et al. 2014;Baechtold et al. 2001).
To the authors knowledge this is the first attempt to relate the administration of fentanyl and naloxone with pain test and release rates of glutamate and GABA from anterior hypothalamus of freely moving rats. It can be speculated that opioids activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect. It is probable that in addition to down-regulation of opioid receptor function, opioid tolerance may involve upregulation of NMDA receptor activity and that opioid tolerance may be due to a combination of synaptic glutamate accumulation and postsynaptic NMDA receptors activation (Wen et al. 2004). Our findings support the hypothesis that the NMDA receptor is involved both in short-term and long-term neuronal changes subsequent to the initial behavior activating effects of pain stimuli and opioids (Le Greves et al. 1998). They also underline the need of conducting push pull perfusion in freely moving rats under certain conditions as trigeminal pain or surgical pain.
AKNOWLEDGEMENTS The authors are grateful to Professor Emeritus Athineos Philippu for his donation in order to obtain the HPLC equipment. This work was supported by the Research Committee of Aristotle University of Thessaloniki, Greece and by the Foundation Tsagka-Despotidou in Athens, Greece.
CONFLICT OF INTEREST We declare that there is no conflict of interest
FIG. 1. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Effects of i.v. injection of saline in Group 1, fentanyl in Group 2 or naloxone in Group 3 at 15min. The tail pinch test was performed at 45min. Basal release rate in the three samples preceding the i.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 2. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Effects of i.c.v. injection of aCSF in Group 4, fentanyl in Group 5 or naloxone in Group 6 at 15min. The tail pinch test was performed at 45min. Basal release rate in the three samples preceding the i.c.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 3. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Premedication with i.v. naloxone at 15min. Effects of i.v. injection of saline in Group 7 or fentanyl in Group 8 at 45min. The tail pinch test was performed at 75min. Basal release rate in the three samples preceding the i.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 4. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Premedication with i.c.v. naloxone at 15min. Effects of i.c.v. injection of aCSF in Group 9 or fentanyl in Group 10 at 45min. The tail pinch test was performed at 75min. Basal release rate in the three samples preceding the i.c.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate.
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Table 1. Description of drug administration and tail pinch manipulation during push pull superfusion per group (n= number of rats included in each group)
Tim Grou ps
Administrat Premedicat ion route
ion
Time of Premedicat ion
Treatm ent
Time of
e of
Treatm
Tail
ent
Pinc h
Grou p1
i.v.
None
-
Saline
15min
i.v.
None
-
Fentanyl
15min
i.v.
None
-
i.c.v.
None
-
aCSF
15min
i.c.v.
None
-
Fentanyl
15min
i.c.v.
None
-
i.v.
Naloxone
15min
Saline
45min
i.v.
Naloxone
15min
Fentanyl
45min
n=8 Grou p2 n=8 Grou p3 n=8
Naloxon e
15min
Grou p4 n=8 Grou p5 n=8 Grou p6 n=8
Naloxon e
15min
Grou p7 n=8 Grou p8 n=8
45m in
45m in
45m in
45m in
45m in
45m in
75m in
75m in
Grou p9
i.c.v.
Naloxone
15min
aCSF
45min
i.c.v.
Naloxone
15min
Fentanyl
45min
n=8 Grou p 10 n=8
75m in
75m in
PII: DOI: Reference:
www.elsevier.com/locate/ejphar
S0014-2999(18)30397-2 https://doi.org/10.1016/j.ejphar.2018.07.029 EJP71894
To appear in: European Journal of Pharmacology Received date: 29 January 2018 Revised date: 12 July 2018 Accepted date: 18 July 2018 Cite this article as: Pourzitaki Chryssa, Tsaousi Georgia, Papazisis Georgios, Kyrgidis Athanassios, Zacharis Constantinos, Kritis Aristeidis, Malliou Faye and Kouvelas Dimitrios, Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats
Pourzitaki Chryssa1*, Tsaousi Georgia2, Papazisis Georgios1, Kyrgidis Athanassios1, Zacharis Constantinos3, Kritis Aristeidis4, Malliou Faye1 Kouvelas Dimitrios1
1
Department of Clinical Pharmacology, Faculty of Medicine, School of Health
Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 2
Clinic of Anesthesiology and Intensive Care, AHEPA University Hospital, Faculty of
Medicine, School of Health Sciences, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece 3
Analytical Development Laboratory, R&D API Operations, Pharmathen SA,
Thessaloniki, Greece 4
Department of Experimental Physiology, Faculty of Medicine, School of Health
Sciences, Aristotle University of Thessaloniki
*
Corresponding author. Pourzitaki Chryssa, MD, MSc, MHA, PhD Assistant
Professor of Pharmacology and Clinical Pharmacology Department of Clinical Pharmacology, Faculty of Medicine, School of Health Sciences, Aristotle University of Thessaloniki University Campus, 54124, Thessaloniki, Greece Tel: +302310999025, Mobile: +306945492971; fax: +302310999312. [email protected]
ABSTRACT Fentanyl, a μ-opioid receptor agonist, has been studied for its neuro/psychopharmacological effects since its first clinical use; however, its effect on the release rate of the Central Nervous System (CNS) neurotransmitters has not been yet elucidated. In the present study the influence of fentanyl on the release rates of glutamate and GABA is investigated. Specifically, we examined the effects of
intravenous (10μg/kg) as well as intrahypothalamic (0.1nmol/min) fentanyl administration on the release rates of GABA and glutamate in the superfusate of anterior hypothalamus, under tail pinch manipulation. The release rate of the neurotransmitters was monitored by the push–pull superfusion technique. To investigate the role of fentanyl the opioid antagonist, naloxone 0.1mg/kg was administered intravenously, or 50nmol/min intrahypothalamicaly. The amino acids were determined by High Performance Liquid Chromatography (HPLC) and fluorimetric detection after NBD-Cl derivatisation. After intravenous fentanyl administration a significant decrease of glutamate and increase of GABA release rates were observed. However during the pain manipulations, the release rate of glutamate was increased. Intravenous naloxone did not affect significantly the release rates of both amino acids, while intrahypothalamic antagonist administration reversed the alterations in both neurotransmitters release rates. Our results demonstrate that there is an opioid-glutamatergic transmission pathway, located in hypothalamus and that opioids can activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect.
KEYWORDS: fentanyl, naloxone, hypothalamus, tail pinch, push pull superfusion, high performance liquid chromatography, GABA, glutamate
1. INTRODUCTION Glutamatergic neurotransmission plays a crucial role in both noxious stimuli creation and pain perception (Fundytus 2001). Pain stimuli from spinal cord end up to the hypothalamus, with its final endings being the ventromedial, periventricular, suprachiasmatic and paraventricular nuclei (Bester et al. 1997). The ventromedial hypothalamic nucleus corresponds to the center of aversion and has also been described as an important center for anger and aggressive behavior. The connections of the hypothalamus with the grey matter of aqueduct regulate the
homeostasis through metabolism, adding another regulatory mechanism of the body's response to the pain (Malick et al. 2000). Similar properties have been addressed to the arcuate nucleus of hypothalamus (Porro et al. 1998). Animal experiments involving the paraventricular nucleus of the hypothalamus have shown that the removal of pituitary gland did not inhibit the analgesic effect of i.c.v. administration of L-glutamate. These results seem to contradict the aspect that the activation of hypothalamus in the analgesia process is elicited by the hypothalamicpituitary tract. Thus, it is assumed that the hypothalamic control is due to its connections with other nuclei (Yang et al. 2006a; 2006b). A similar mechanism of analgesia has been assigned to the paraventricular hypothalamic nucleus suggesting its possible involvement in the endogenous opiate peptide system in spinal cord independently (Yang, et al. 2008; 2009). The anterior hypothalamus includes the paraventricular (PV), the supraoptic (SO) and the suprachiasmatic (SC) nuclei, all of which seem to be involved in the perception of pain. Apart from the pain perception, hypothalamus is also involved in learning, memory and response to stress in rats (Kohsaka et al. 1999). In addition, the hypothalamus receives nerve stimuli from baro- and chemo-receptors and interacts with the solitary tract and the motor nucleus of vagus. Furthermore, evidence suggests that peripheral nervous signaling mechanisms control excitation at central terminals, consisting major sites of signal integration of peripheral and central inputs particularly, from the hypothalamus (Andresen et al. 2012). Experimental studies have shown that the co-administration of glutamate and opioids intravenously exerts a synergistic analgesic effect. Moreover, opioids enhance the presynaptic inhibitory effects mediated by GABA-A receptors, through inhibition of glutamate re-uptake and thus its concentration in the synapsis is increased. This fact further promotes the analgesic effect of opioids. However, it is known that glutamate release is involved in both creation and perception of pain stimuli. (Popik et al. 2000; Popik and Kozela 1999).
The purpose of the present research was to investigate the processes taking place in the hypothalamus at the influence of noxious stimuli and administration of intravenous opioids. These processes are probably expressed as alterations in the release rate of neurotransmitters from hypothalamic nuclei over time. For this purpose, the tail pinch test was carried out on adult, male rats. The release of GABA and glutamate in the hypothalamus was assessed using the push–pull superfusion technique, while fentanyl and naloxone as opioid agonist and antagonist respectively were applied both intravenously (i.v.) and intracerebroventricularly (i.c.v.).
2. MATERIALS AND METHODS The study was approved by the Research Council of School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki and has been conducted in a manner that does not inflict unnecessary pain or discomfort upon the animals. All animal experiments were in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). 2.1 Experimental setting Eighty (80) male Wistar rats (aged 10 -12 weeks old, weighting 230 – 300 g) were used in this experimental setting. All animals were single housed, maintained in a climate-controlled room on a 12-hour light-dark cycle and allowed food and water ad libitum. All animals were provided from the Veterinary School, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. Rats were anaesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (40mg/kg) and ketamine (50mg/kg). Push pull cannulas used were custom made according to the literature. (Philippu, 2017) For intravenous (i.v.) infusions of drugs a PE 50 tubing was inserted into the jugular vein (Kouvelas et al. 2006). Push pull perfusion technique was applied on each animal. In detail, the head was fixed in a stereotaxic frame and a guide cannula (outer diameter 1.25mm, inner diameter 0.90 mm) was stereotaxically inserted according to the Paxinos and Watson atlas (Paxinos and Watson 1996), until its tip was 2mm above the left anterior hypothalamus (AP -1.5mm, V 8.2mm, L 0.5mm). The guide cannula was fixed with screws and dental cement. At least two days after surgery, the stylet of the guide cannula was replaced by a push-pull cannula of the following diameters (mm): outer needle; outer diameter 0.82, inner diameter 0.50, inner needle; outer diameter 0.20, inner diameter 0.10. Protrusion of the inner needle was 0.20mm. The push-pull
cannula was 2mm longer than the guide cannula thus reaching the anterior hypothalamus. According to the literature 48h after the placement of the cannula under pentobarbital and ketamine anesthesia both drugs were eliminated, while no effects of sensitization nor tolerance are described in the low doses used (Lin et al, 1973;Kosten and Bombace, 2001;Popik et al, 2008,Dodelet-Devillers et al, 2016) The hypothalamus of the conscious, freely moving rat was superfused at a rate of 20μl/min with artificial cerebrospinal fluid (aCSF) of the following composition (mmol/l): NaCl 140, KCl 3.0, CaCl2 2.5, MgCl2 1.0, Na2HPO4 1.0, NaH2PO4 1.0, glucose 3.0, pH 7.2 (Prast et al. 1996). After an equilibration period of 80min, superfusates were collected in time periods of 5min into tubes kept at −50oC. The samples were stored at−80oC until biochemical analysis was carried out. During the experiment, animals were deprived of food and water. 2.2 Study outline The drugs tested in our experiment study protocol were fentanyl [N-(1phenethyl-4-piperidyl)-N-phenyl-propanamide] from Jansen Cilag (Fentanyl) and naloxone [(5a)-4,5-Epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one] from Vianex (Narcan) in commercial forms. Both fentanyl and naloxone were diluted in 0.9% NaCl solution for i.v. (intravenous) infusion, while for i.c.v. (intracerebroventricular) infusion into the hypothalamus drugs were dissolved in aCSF. For fentanyl and naloxone i.v. and i.c.v. administration the doses used were in accordance with previously published studies. (Yoshida et al, 1999;Verbogh et al, 1999;Genco et al, 2003;Cao et al, 2004) According to the type of drug regimen applied, the animals were randomly assigned into 10 groups (of 8 animals each) using an internet-based computer- generated random number table (www.randomizer.gr). In detail the animal groups were treated with: 1) normal saline 0,9% i.v., 2) fentanyl 10μg/kg (i.v), 3) naloxone 0.1mg/kg (i.v.), 4) vehicle (aCSF) (i.c.v), 5) 0.1nmol/min fentanyl (i.c.v), 6) 50nmol/min (i.c.v.), of
naloxone 7) normal saline 0,9% (i.v.) premedicated with naloxone 0.1mg/kg (i.v), 8) fentanyl 10μg/kg (i.v) premedicated with naloxone 0.1mg/kg (i.v), 9) vehicle (aCSF) (i.c.v.) premedicated with naloxone 50nmol/min (i.c.v.) and 10) fentanyl 0.1nmol/min (i.c.v.) premedicated with naloxone 50nmol/min (i.c.v.) (Table 1). Push pull superfusion was conducted in normal conditions of temperature and humidity, between 09.00 a.m. and 12.00 p.m. Fifteen min (15) after initiation of superfusate collection, we administered intravenously to the rats in Groups 1-3 either saline 0,9%, fentanyl, or naloxone i.v., at a rate of 2μl/min (Bruins Slot et al. 2002;Yoshida et al. 1999). In Groups 4 to 6, the animals received i.c.v., either aCSF as vehicle, or fentanyl or naloxone with infusion rate 2μl/min. Forty-five min after initiation of superfusate collection a tail pinch test was applied to all animals of Groups 1 to 6. Tail pinch (not traumatic) was applied continuously for 3min by an appropriate clamp (force: 3.5 N) placed approximately 2cm from the tip of the tail (Singewald et al. 1995). According to the literature the area is very sensitive to pain and the stimulus causes great stress (Park et al. 2015). Sampling stopped 30min after tail pinch. In Groups 7 and 8, fifteen (15) min after initiation of sampling the animals were premedicated with i.v. administration of naloxone. Forty-five (45) min after initiation the animals received intravenously either saline 0.9% (Group 7) or fentanyl (Group 8) (Cao et al., 2004). In Groups 9 and 10, fifteen (15) min after initiation of sampling the animals were premedicated with naloxone i.c.v. with infusion rate 2μl/min. Naloxone i.c.v. infusion lasted 2min. Forty-five (45) min after starting the experiment the animals received i.c.v. either vehicle (aCSF) (Group 9) or fentanyl (Group 10) with infusion rate 2μl/min. Fentanyl i.c.v. infusion lasted 5min. Seventyfive (75) min after initiation of superfusate collection a tail pinch test was applied to all animals of Groups 7 to 10. When the i.c.v. infusion was taking place, sampling was omitted (1 sample skipped) while it was completely stopped 30min after tail pinch.
At the end of the superfusion, the brain was removed and the position of the cannula was verified in 50mm histologic slices stained with cresyl violet and luxol fast blue (data non shown) (Kluver and Barrera, 1953;Singewald et al, 1995;Prast et al, 1996;Kouvelas et al, 2006). Superfusates from experiments, in which the cannula was found to be localized outside of the anterior hypothalamus, were discarded. 2.3 Determination of GABA and glutamate All reagents used throughout this study were of analytical grade. Double distilled water was used throughout this study. Standard stock solutions of amino acids and the internal standard (methylamine) were prepared in water and stored at 4oC. Working standards solutions were prepared daily by appropriate dilutions of aliquots obtained by the stock solutions in water. NBD-Cl was dissolved in ethanol to prepare standard stock solution at 6 mM. This solution was stored at 4oC and protected from the light. The mobile phase A consisted of 10 mM NaH2PO4 and 10 mM Na2HPO4 (pH 7.0) and was prepared in daily basis. This solution was filtered through 0.22 μm PTFE membrane filter (Millipore) prior to use. The derivatization protocol of amino acids (AA) was adopted by Yang and his colleagues with some modifications (Yang et al. 2007). Briefly, 150 μl of standard AA solution or sample was mixed with 50μl of internal standard (120μM methylamine) and 50μl of borate buffer (100mM, pH 8.2). Then 50μl of 4-chloro-7-nitrobenzo-2oxa-1,3-diazole (NBD-Cl) solution (6mM) was added and the resulted mixture was vortexed and left to react at 60oC for 1h in water bath. An aliquot of 600μl of cold water was finally added in the mixture while 10μl was used for HPLC analysis. Blank solutions without AA were processed as above. The HPLC setup comprised the following parts: a quaternary gradient solvent system (LC-10ADvp, Shimadzu), a fluorescence detector (RF-10AXL, Shimadzu) and an auto-sampler (SIL-20AC – Prominence, Shimadzu). The analytical column was a reversed-phase Lichrospher 100-5, C18 (250 x 4.0mm, 5μm, MZ Analysentechnik) and the data acquisition was carried out via Clarity® software
(DataApex). The mobile phase A (A) consisted of a 20 mM phosphate buffer (pH 7.0):CH3OH, 99:1 % v/v while the mobile phase B (B) was a mixture of mobile phase A:CH3OH, 30:70 %, v/v. The flow rate was 1 ml/min throughout the analysis and all separations were performed at ambient temperature. The initial concentration of eluent B was 10 %. The gradient elution was as follows: eluent B 10-35% (0-20min), 35-100% (20-30min). At time of 30.1min the gradient elution was returned to its initial composition and stated for a period 15min to maintain a stable and reproducible separation. The analyte derivatives were detected at 470/540nm (λext / λem). The retention time of glutamate and γ-aminobutyric acid (GABA) was 7.2 and 23.9 min respectively while the methylamine was eluted at 31.1 min. The compounds of interest were quantified using external calibration curves. 2.4 Statistical analysis The first 3 superfusate samples, taken until fentanyl or naloxone were administered, were used as controls. Normality of data was assessed by ShapiroWilk test. Neurotransmitter release rates were analyzed by Mann Whitney U and Friedman’s test followed by Wilcoxon’s rank test for paired data. Analyses of variance (ANOVA) for multifactor repeated measures with Greenhouse-Geisser correction were conducted to analyze continuous variables over time. If variance/covariance matrices were not equal across all groups and if sphericity of the combined variance/covariance matrix (overall sphericity) and sphericity for each group was violated, then we used contrasts for between/within tests and Bonferroni adjustment. Comparisons between different groups were performed using one-way ANOVA. Two-tailed levels of significance were used in all statistical calculations. Values are presented as mean and standard error (SE) unless otherwise stated. Statistical significance level for group differences was set at P≤0.05. Statistical Package for Social Sciences (SPSS, version 22.0; SPSS Inc., Chicago, IL, USA) was used for all calculations and analysis.
3. RESULTS Mean basal outputs of neurotransmitters in the anterior hypothalamus of the conscious adult rat were 652.6±89.2 fmol/min for glutamate (n=80) and 274.3±78.7 fmol/min for GABA (n = 80). The basal release rates in the first three samples preceding infusions were taken as 1. 3.1 Effects of intravenous fentanyl and naloxone on glutamate and GABA release rate in the anterior hypothalamus Administration of fentanyl 10μg/kg (i.v.) decreased the glutamate release rate from the anterior hypothalamus. This effect peaked approximately 10min after fentanyl reaching 32±13% of the control value (P<0.001) (Fig. 1A). Injection of naloxone, 0.1mg/kg (i.v.) induced a subtle increase of the basal rate of glutamate release, while saline 0,9% (i.v.) was ineffective (Fig. 1A). Immediately after the tail pinch test glutamate release in the anterior hypothalamus from rats receiving saline and naloxone i.v. increased up to 295±25% and 282±18% of the control value respectively and returned to baseline values after about 20min (P<0.001). In contrast, in the group of fentanyl i.v. administration a delayed (15min after the tail pinch), and progressive increase in glutamate release rate up to 191±21% (P=0.002) was recorded. To the former effect, administration of fentanyl 10 μg/kg i.v. enhanced the GABA release rate from the anterior hypothalamus. The peak effect occurred approximately 15 min after fentanyl injection being 253±22% of the control value (P<0.001) (Fig. 1B). The tail pinch test decreased the GABA release rate in the groups of rats receiving saline i.v. or naloxone i.v, approximating 74±15% and 43±24% of the control value, respectively (P=0.046) and 25min later they reversed to baseline values. Whereas, in the group of animals receiving fentanyl i.v. a notable augmentation of GABA release rate up to 182±17% (P<0.001) was recorded. (Fig. 1B)
3.2 Effects of fentanyl and naloxone i.c.v. infusion into the hypothalamus on glutamate and GABA release rate in the anterior hypothalamus Infusion of fentanyl 0.1nmol/min i.c.v reduced the glutamate release rate, immediately after injection, which peaked 5min post-infusion up to 34±18% of the control value (P<0.001) (Fig. 2A). Infusion of naloxone 50 nmol/min i.c.v. increased basal release rate of glutamate in the anterior hypothalamus up to 214±24%, while aCSF as vehicle i.c.v (n=8) was ineffective (Fig. 2A). Immediately after the tail pinch test glutamate release in the anterior hypothalamus from rats receiving saline and naloxone i.c.v. increased up to 280±23% and 261±16% of the control value respectively and returned to baseline values within 20min post its peak (P<0.001). Infusion of fentanyl 0.1nmol/min i.c.v increased the GABA release rate, up to 192±24% of the control value (P<0.001) (Fig. 2B). Immediately after the tail pinch test GABA release in the group of rats receiving saline and naloxone i.c.v. presented a significant (P=0.002) deterioration (53±16% and 68±24% of the baseline, respectively) and returned to control values 15min later. However, the rats with fentanyl i.c.v. administration presented an abrupt increase in GABA release rate up to 163±19% of the control value that lasted approximately 30min (P=0.004). (Fig. 2B) 3.3 Effects of intravenous fentanyl after intravenous premedication with naloxone on glutamate and GABA release rate in the anterior hypothalamus Premedication with naloxone 0.1 mg/kg i.v. ameliorated fentanyl effects on glutamate release rates (Fig. 3A). The tail pinch test caused a statistically significant increase in glutamate release in the anterior hypothalamus of rats receiving saline which peaked after 15min reaching 278±14% of the control value and reversed to baseline values after approximately 30min (P<0.001). Moreover, the rats with fentanyl i.v. administration showed a slight increase in glutamate release rate up to 142±16% (P=0.04).
Premedication with naloxone 0.1mg/kg i.v. abolished the effect of fentanyl i.v. administration on GABA release rate. (Fig. 3B). The tail pinch test decreased the GABA release rate in the group of rats receiving saline i.v. which reached its lowest value 15 min later (62±16% of the baseline; P=0.05) and returned to baseline values after 30 min, while the rats that received fentanyl i.v. presented a marginally statistically significant increase in GABA release rate up to 144±16% (P=0.05) (Fig. 3B). 3.4 Effects of fentanyl infusion into the hypothalamus after i.c.v. premedication with naloxone on glutamate and GABA release rate in the anterior hypothalamus Premedication with naloxone 50 nmol/min i.c.v. induced an abrupt and notable (P<0.001) increase in glutamate release rate from anterior hypothalamus which peaked 10 min after naloxone infusion (235±27% of baseline), which during the next 30min gradually deteriorated but without reversing to baseline (Fig. 4A). Application of tail pinch test in the group of rats receiving aCSF as vehicle i.c.v resulted in an instant and notable glutamate release (276±29% of the baseline; P<0.001) which lasted about 10min and reversed almost to control values after 30min. However, in the group of rats with fentanyl i.v. administration a delayed increase in glutamate release rate was recorded but its peak (174±28% of baseline; P=0.026) was lower compared to aCSF i.c.v group. Injection of fentanyl 0.1nmol/min i.c.v increased the GABA release rate, up to 166±15% of the control value (P=0.003), while in the group of aCSF as vehicle i.c.v. GABA release rate remained constant (Fig. 4B). After the tail pinch test the GABA release rate in the group of rats receiving vehicle i.c.v. remained close to control values, while in the group of rats with fentanyl i.c.v. administration a marked increase in GABA release rate up to 168±19% of the baseline (P=0.007) was recorded. (Fig. 4B)
4. DISCUSSION Pain involves sensory-discriminative, cognitive-evaluative, and affectiveemotional components. Nociceptive transmission is modulated by several pathways including brainstem and hypothalamus which play a key role in pain stimuli processing (Denuelle et al. 2007). Pain stimuli information can reach to the hypothalamus via multi-synaptic pathways through the brainstem, thalamus and cortex. Opioid signaling in the central nervous system is critical for controlling cellular excitability. The present study examined the modulation of anterior hypothalamus response to a painful stimulus by the administration of both a strong μ-opioid receptor agonist and an opioid antagonist (fentanyl, and naloxone, respectively), on an experimental setting of freely moving rats. For the determination of GABA and glutamate release rates, push pull perfusion was combined with a new derivatization protocol for HPLC analysis, developed according to the literature with some modifications (Klinker and Bowser 2007;Yang et al, 2009;Wu et al. 2014). According to our results, application of fentanyl promoted a notable reduction of glutamate release rate from the anterior hypothalamus, irrespectively of the route of administration (i.v. or i.c.v). According to Iremonger and Bains, endogenous opioids are released from dendritic vesicles in response to postsynaptic activity and act in a retrograde manner to inhibit excitatory synaptic transmission (Iremonger and Bains 2009). This opioid inhibition involves a downstream entry of calcium ions (Ca+2) and is modulated presynaptically. It appears that neurons have the ability to selfregulate their excitability through the dendritic release of opioids to inhibit excitatory synaptic transmission (Iremonger and Bains 2009). Nevertheless, the pattern of glutamate release in the two routes of fentanyl administration after the tail pinch was not alike. Intravenous infusion of fentanyl promoted a delayed but notable augmentation of glutamate release, while i.c.v administration of fentanyl induced only a subtle enhancement of glutamate release
rate. The role of glutamate in the process of central regulation of pain is not fully elucidated yet. The activation of glutamatergic receptors may have an either analgesic or the opposite effect. In the past the inhibition of glutamatergic transmission by the administration of NMDA antagonists (ketamine, MK801dizocilpine, memantine, dextromethorphan, glycine, LY274614, LY235959 and CGP 39551) also resulted in an inhibition of the development of the opioid addiction phenomenon (Bilsky et al. 1996;Elliott et al. 1994a;Elliott et al. 1994b;Gonzalez et al. 1997;Popik et al. 2000;Popik & Kozela 1999). Furthermore, the administration of LY354740 without reversing the analgesic effect of morphine revealed the involvement of glutamatergic metabotropic type II receptors in pain perception (Bhat et al. 1998). The decline of glutamate release rate after fentanyl infusion and its significant augmentation after the tail pinch test could be explained by the existence of an opioid-NO-glutamatergic transmission pathway, located in hypothalamus, which is responsible for neuro-endocrinological regulatory response after noxious stimulus (Bhat et al. 1998). There are data showing that remifentanyl administration allosterically stimulates different types of NMDA receptors NR1A / 2A and NR1A / 2B, an effect unrelated to its concentration (Hahnenkamp et al. 2004). Furthermore, the withdrawal response, known as acute dependence, seems to reflect the beginning of an adaptive change in the rats. In rat models, it is suggested that a single dose of morphine modifies the expression of the NMDA receptor subunit NR1, NR2A and NR2B mRNAs in the hippocampus and hypothalamus which probably leads to a late chronic opiate dependence (Le Greves et al. 1998). It has been reported that μ-opioid receptor activation selectively inhibits excitatory activity in SON hypothalamic neurons via a presynaptic mechanism (Liu et al. 1999). In the present study although intravenous infusion of naloxone had no impact on the release rate of glutamate in the anterior hypothalamus, when it was given i.c.v. it increased glutamate release in an important manner. Fentanyl i.v.
administration delayed increase in glutamate release rates may indicate that naloxone prevented the increase in glutamate after fentanyl treatment. Moreover, the administration of naloxone from either route as premedication abolished the effect of fentanyl infusion on glutamate release rate. This is in accordance to the reversed effects of D-Ala(2), N-CH(3)-Phe(4), Gly(5)-ol-enkephalin (DAGO) on glutamatergic transmission in supraoptic nucleus by addition of naloxone (Liu et al. 1999). Previous reports suggested that when morphine was given in striatum and limbic forebrain, it promoted a decline in extracellular glutamate levels; when it was infused in substantia nigra it failed to increase dopamine release and decreased glutamate (Desole et al. 1996;Enrico et al. 1998;Miele et al. 1994). In the same experimental settings, intravenous administration of naloxone within one hour after systemic morphine, presented an antagonizing effect on morphine-induced changes, without affecting the depletion of glutamate dialysate concentrations. The above results combined with the results from the present study suggest that an inhibitory μopioid receptor mediated-mechanism controls certain brain sites coupled with neuronal glutamate uptake (Desole et al. 1996;Enrico et al. 1998;Miele et al. 1994). Concerning the GABA release rate in our setting, this was significantly enhanced by the application of fentanyl, regardless of the route of its administration (i.v. or i.c.v), while naloxone did not affect GABA concentrations. Unlike our findings it has been shown that the push-pull perfusion of morphine within the hypothalamus affected the activity of GABA at only 3 out of 17 hypothalamic sites at which the opioid agonist was perfused (Noto and Myers 1984). However, Yoneda and colleagues have demonstrated that morphine administered acutely exerts little effect on the content or distribution of GABA within the whole brain or its anatomical constituents including the hypothalamus (Yoneda et al. 1977). This inconsistency between the results of the present study and previous ones could probably be attributed to divergent pharmacokinetics and selectivity for μ-opioid receptors
between morphine and fentanyl. Furthermore, morphine is a partial agonist at the opioid μ-receptors, while fentanyl may have higher efficacy (Popik et al. 2000). This discrepancy underlines the importance of the direct determination of amino acids release by the push-pull technique under in vivo conditions when modulatory mechanisms are investigated. Noxious stimulus in the rats receiving fentanyl i.v. promoted GABA release, while in those receiving saline or naloxone, the inhibitory amino acid basal release rates remained unaffected. It should be noted that when the tail pinch test was performed in fentanyl i.v. group GABA release rates had not fully reversed to baseline values, so it is not clear if the certain enhancement is due to the tail pinch test or the effect caused by the infusion of an opioid. Premedication with naloxone i.v. had no significant impact on GABA release rate and fully antagonized the effect of fentanyl i.v. administration. The tail pinch test decreased the GABA release rate in the rats receiving saline i.v. while, in those receiving fentanyl i.v., a marginal increase in GABA release rate was presented. When premedication with naloxone was administered i.c.v. the administration of fentanyl by the same route increased the GABA release rate, which was maintained almost constant after the tail pinch test (Fig. 4B). This is in accordance with previous results showing that morphine increased GABAergic tone in the hypothalamic paraventricular nucleus and reduced the release of glutamate and the activation of oxytocinergic neurons mediating penile erection (Succu et al. 2006). The limitation of our study is the lack of cardiovascular monitoring during the specific experimental procedure; however this would require an arterial catherization, leading to significant perioperative risk and adding stressful manipulations during the push pull superfusion in freely moving rats. Moreover, opioid and especially fentanyl effects on cardiovascular parameters have already been studied in rats thoroughly (Albrecht et al. 2014;Baechtold et al. 2001).
To the authors knowledge this is the first attempt to relate the administration of fentanyl and naloxone with pain test and release rates of glutamate and GABA from anterior hypothalamus of freely moving rats. It can be speculated that opioids activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect. It is probable that in addition to down-regulation of opioid receptor function, opioid tolerance may involve upregulation of NMDA receptor activity and that opioid tolerance may be due to a combination of synaptic glutamate accumulation and postsynaptic NMDA receptors activation (Wen et al. 2004). Our findings support the hypothesis that the NMDA receptor is involved both in short-term and long-term neuronal changes subsequent to the initial behavior activating effects of pain stimuli and opioids (Le Greves et al. 1998). They also underline the need of conducting push pull perfusion in freely moving rats under certain conditions as trigeminal pain or surgical pain.
AKNOWLEDGEMENTS The authors are grateful to Professor Emeritus Athineos Philippu for his donation in order to obtain the HPLC equipment. This work was supported by the Research Committee of Aristotle University of Thessaloniki, Greece and by the Foundation Tsagka-Despotidou in Athens, Greece.
CONFLICT OF INTEREST We declare that there is no conflict of interest
FIG. 1. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Effects of i.v. injection of saline in Group 1, fentanyl in Group 2 or naloxone in Group 3 at 15min. The tail pinch test was performed at 45min. Basal release rate in the three samples preceding the i.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 2. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Effects of i.c.v. injection of aCSF in Group 4, fentanyl in Group 5 or naloxone in Group 6 at 15min. The tail pinch test was performed at 45min. Basal release rate in the three samples preceding the i.c.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 3. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Premedication with i.v. naloxone at 15min. Effects of i.v. injection of saline in Group 7 or fentanyl in Group 8 at 45min. The tail pinch test was performed at 75min. Basal release rate in the three samples preceding the i.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate. FIG. 4. Release rate of (A) glutamate and (B) GABA in the anterior hypothalamus during push pull perfusion. Premedication with i.c.v. naloxone at 15min. Effects of i.c.v. injection of aCSF in Group 9 or fentanyl in Group 10 at 45min. The tail pinch test was performed at 75min. Basal release rate in the three samples preceding the i.c.v. infusion was taken as 1. Mean values ± S.E.M. (n=8). *P < 0.05; **P < 0.01, ***P < 0.001 significantly different from basal release rate.
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Table 1. Description of drug administration and tail pinch manipulation during push pull superfusion per group (n= number of rats included in each group)
Tim Grou ps
Administrat Premedicat ion route
ion
Time of Premedicat ion
Treatm ent
Time of
e of
Treatm
Tail
ent
Pinc h
Grou p1
i.v.
None
-
Saline
15min
i.v.
None
-
Fentanyl
15min
i.v.
None
-
i.c.v.
None
-
aCSF
15min
i.c.v.
None
-
Fentanyl
15min
i.c.v.
None
-
i.v.
Naloxone
15min
Saline
45min
i.v.
Naloxone
15min
Fentanyl
45min
n=8 Grou p2 n=8 Grou p3 n=8
Naloxon e
15min
Grou p4 n=8 Grou p5 n=8 Grou p6 n=8
Naloxon e
15min
Grou p7 n=8 Grou p8 n=8
45m in
45m in
45m in
45m in
45m in
45m in
75m in
75m in
Grou p9
i.c.v.
Naloxone
15min
aCSF
45min
i.c.v.
Naloxone
15min
Fentanyl
45min
n=8 Grou p 10 n=8
75m in
75m in