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Endocrinology Vol. 139, No. 3 955-960
Copyright © 1998 by The Endocrine Society

ARTICLES

Opioid-Glutamate-Nitric Oxide Connection in the Regulation of Luteinizing Hormone Secretion in the Rat1

Ganapathy K. Bhat, Virendra B. Mahesh, Lin Ping, Lynn Chorich, Vernon T. Wiedmeier and Darrell W. Brann1

Neuroendocrine Research Laboratory, Department of Physiology and Endocrinology, School of Medicine, Medical College of Georgia, Augusta, Georgia 30912-3000

Address all correspondence and request for reprints to: Darrell W. Brann, Ph.D., Neuroendocrine Research Laboratory, Department of Physiology and Endocrinology, School of Medicine, Medical College of Georgia, Augusta, Georgia. E-mail: dbrann{at}mail.mcg.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Opioid neurons are recognized to be an important component of the inhibitory "brake" in the CNS that restrains LHRH secretion. Opioid inhibition could be exerted directly on LHRH neurons, or it could be achieved via indirect mechanisms involving restrainment of excitatory "accelerator" neurons that facilitate LHRH release. The purpose of the present study was to explore the second hypothesis by investigating whether removal of opioid inhibition by administering the opioid antagonist, naloxone leads to enhanced activation of glutamate and nitric oxide (NO) neurons, which are known to be important excitatory "accelerator" components for the control of LHRH secretion. Naloxone administration (2.5 mg/kg) to adult male rats induced a significant elevation of serum LH levels at 20 min post injection. NOS activity in preoptic area (POA) and medial basal hypothalamic (MBH) fragments was demonstrated to be significantly elevated 20 min post naloxone injection. Administration of a glutamate (NMDA) receptor antagonist (MK-801, 0.2 mg/kg) abolished the naloxone-induced increase in NOS activity in the POA and MBH, with a corresponding block of the naloxone-induced LH release. Glutamate appears to only be involved in LH surge generation and not to regulate basal LH levels, as MK-801 had no effect on basal LH release. Because previous work by our laboratory and others have provided evidence that NO is a mediator of glutamate effects in the hypothalamus, these findings are interpreted to mean that opioid inhibition is mediated on glutamate neurons that are upstream of NO neurons. In support of this contention, we found that NMDA treatment enhanced NOS activity in the male rat POA and MBH fragments in vitro, an effect that was specific as it was completely blocked by the NMDA receptor antagonist, MK-801. Additionally, in vivo microdialysis studies revealed that naloxone treatment significantly enhances glutamate release in the preoptic area (POA) at 15 min post injection in conscious, unanesthetized, freely moving male rats. Release rates of the control amino acid, serine did not change significantly following naloxone injection. Taken as a whole, these findings provide evidence for an opioid-glutamate-NO pathway in the control of LHRH secretion, and they demonstrate the importance of "brake-accelerator" interactions in the control of LHRH and LH secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DESPITE intense investigation, the mechanisms underlying control of LHRH secretion remains poorly understood. While steroids are the principal regulators of LHRH secretion, it is unlikely that they regulate LHRH secretion directly because several studies have demonstrated that LHRH neurons do not possess steroid receptors (1, 2, 3). Rather, it would appear that steroid control of LHRH secretion is indirect, involving mediation by other inhibitory and excitatory neurotransmitter neurons, which, in effect, relay steroid signals to the LHRH neuron. It has been proposed that the inhibitory and excitatory transmitter neurons constitute a "brake" and "accelerator" mechanism for the control of LHRH neurosecretion (see Refs. 4–6, for review). Opioid neurons appear to be the principal component of the inhibitory "brake", with gamma amino butyric acid and neuropeptide K neurons also participating (5, 7, 8, 9, 10, 11, 12, 13, 14, 15). Glutamate, norepinephrine, neuropeptide Y and nitric oxide (NO) neurons have been proposed to constitute the major components of the "accelerator" (4, 5, 6, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). The excitatory "accelerator" signals may be linked (i.e. sequential) in their control of LHRH secretion, or they could exist in parallel, where the LHRH surge would be the result of summation of their individual effects.

In support of the linkage or sequential model for "accelerator" signal control of LHRH release, glutamate receptors have been demonstrated on hypothalamic NO neurons (31) and glutamate has been demonstrated to be a potent stimulator of NO production in many parts of the brain, although reports on the hypothalamus are lacking (32, 33). Functional studies have further verified the importance of NO in mediating glutamate effects in the hypothalamus, as nitric oxide synthase (NOS) inhibitors have been shown to block the ability of glutamate to stimulate LHRH in vitro (34, 35) and LH secretion in vivo (36, 37, 38). Glutamate has also been demonstrated to enhance norepinephrine release in the hypothalamus, suggesting that glutamate may also help recruit catecholamine neurons into the signaling cascade (39, 40). Thus, glutamate is a pivotal and central transmitter in the control of LHRH and LH secretion.

An important unresolved question is whether the inhibitory opioid brake is exerted directly on the LHRH neuron or occurs via an indirect mechanism. Recent findings using dual label in situ hybridization studies have demonstrated that LHRH neurons do not synthesize mRNA for µ, k or {delta} opiate receptors (41), suggesting that opioid "braking" of LHRH neurons may occur via an indirect mechanism involving restrainment of one or more of the "accelerator" components. Along these lines, we hypothesized that opioids may restrain LHRH secretion by tonically restraining glutamate and/or NO neurons in the hypothalamus, which are a significant component of the accelerator mechanism controlling LHRH secretion. To test this hypothesis, the opioid antagonist, naloxone was used as a mechanism of releasing the opioid inhibitory brake in the male rat hypothalamus to determine whether glutamate and/or NO neurons are subsequently activated and mediate the LH surge, which results upon release of the opioid brake.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
All chemicals and reagents were from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Fifty-five-day-old adult male rats (Holtzman, Sprague-Dawley, Madison, WI) were obtained and housed in air conditioned rooms under a light regimen of 14 h of light each day (lights on from 0500 to 1900 h). Food and water were provided ad libitum. All animal studies carried out were approved by our Institutional Committee for the Care and Use of Animals in Research and Education (CAURE) in accordance with the guidelines of NIH and USDA.

Effect of naloxone upon POA and MBH nitric oxide synthase (NOS) activity
To determine whether release of the opioid brake by naloxone resulted in activation of NO neurons, the effect of naloxone upon NOS activity in the preoptic area (POA) and medial basal hypothalamus (MBH) was determined in male rats after treatment with naloxone. Adult male rats were injected sc with either vehicle (saline) or naloxone (2.5 mg/kg) and killed by decapitation 20 min later. The chosen dose of naloxone (2.5 mg/kg) has been previously demonstrated to effectively release the opioid brake in the hypothalamus leading to an LH surge within 20 min of injection (13). The MBH and POA were rapidly dissected after decapitation as described in detail previously by our laboratory (42) and assayed for NOS activity as described in detail below. Trunk blood was also collected for serum LH measurements.

To determine whether opioid neurons act on glutamate neurons that are up-stream of NO neurons, an additional group was included in which animals were injected sc with the glutamate (NMDA) receptor antagonist MK-801 ([+]5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclo-heptin-5,10-imine maleate) (0.2 mg/kg, Research Biochemical Inc, Natick, MA) 20 min before naloxone treatment, and the effect on NOS activity and LH levels was determined. The dose of MK-801 was chosen based on previous studies by our group demonstrating its effectiveness in antagonizing glutamate (NMDA) receptor mediated action (43). A second experiment was performed to confirm naloxone effects on NOS activity and LH secretion, as well as to determine whether the glutamate antagonist MK-801 affects basal LH and NOS activity levels.

NOS activity was determined by measuring the conversion of [3H] arginine to [3H] citrulline following the method of Bredt and Snyder (44) and as described previously by our laboratory (27). Briefly, the hypothalamic blocks (POA and MBH) were homogenized in 20 mM HEPES buffer (pH 7.2) containing 0.32 mM sucrose, 0.5 mM EDTA and 1 mM DTT and centrifuged for 15 min To 340 µl of the supernatant, a cocktail buffer containing 2 mM NADPH, 0.45 mM Ca2+ (1 µM free calcium) 20 µM arginine, [3H] arginine (1 µCi/ml), Amersham Life Science, Arlington Heights, IL) were added for a final reaction volume of 400 µl. The reaction was carried out at 37 C for 30 min and terminated by adding 2 ml of 20 mM ice-cold HEPES (pH 5.5) containing 20 mM EDTA. The reaction mixture was then passed through Dowex AG50 (Na2+ form) columns, and the material was eluted with 2 ml water. [3H]-citrulline was quantified by liquid scintillation counting (Beckman Scintillation Counter, model no. LS 5801, Fullerton, CA) of the eluent and the NOS activity was expressed as cpm of citrulline formed per milligram protein for 30 min. Protein estimation of the tissue sample was done by Lowry’s method (45). For determination of NOS activity after in vitro treatment of male rat POA and MBH tissue blocks with NMDA, a slightly modified method was adopted. In this instance, the POA or MBH tissue blocks were incubated with the test substance NMDA (50 mM) or vehicle (Krebs Ringer Bicarbonate buffer) for 30 min at 37 C. The NMDA receptor antagonist was also used to demonstrate the specificity of the observed effect of NMDA. MK-801 (100 µM) was preincubated with the hypothalamic fragments for 30 min followed by coincubation with NMDA for 30 min at 37 C. Following the incubation with test substances, the tissue was homogenized, followed by addition of the NOS assay cocktail and performance of a second incubation at 37 C for 30 min. The rest of the NOS assay procedure was performed as described previously above.

Effect of naloxone upon preoptic area (POA) release rates of glutamate and serine
To determine whether release of the opioid brake by naloxone treatment resulted in activation of glutamate neurons, a microdialysis probe was placed in the preoptic area (POA) of the adult male rat, and the effect of naloxone treatment (2.5 mg/kg) upon preoptic release rates of glutamate was determined. Serine release rates were also determined as a control. Cannulation and microdialysis were carried out as described previously by our laboratory (46). Briefly, at 58 days of age, the animals were anesthetized with ketamine-xyline (50 µg/kg; 5:1 ratio) and implanted with a guide cannula for the microdialysis probe (CMA/12, BAS Co., Lafayette, IN). The guide cannula was implanted into the POA at coordinates A:7.8, L:10, and H:9.0 (plus 2 mm length of probe tip), and a dummy cannula was inserted and left in place until the day of the experiment. On the day of the experiment (day 65), the dummy cannula was removed from the guide cannula and the microdialysis probe (CMA/12, BAS Co., ID = 400 µm, OD = 500 µm, tip length = 2 mm) was inserted and perfusion buffer pumped using a model 3 peristaltic pump (Gilson, Middleton, WI). At least 2 h of pumping was performed before collecting the perfusate to allow for probe equilibration. The perfusion media, modified Krebs-Ringer-Phosphate (KRP) medium, consisted of 123 mM NaCl, 2.4 mM KCl, 1.2 mM MgSO4, 1.2 mM CaCl, 0.9 mM NaH2PO4 and 1.4 mM Na2HPO4 (pH 7.4). The flow rate was 1.5 µl/min. After the equilibration, naloxone (2.5 mg/kg BW) was injected sc, and the time of injection was taken as zero time point and the perfusate was collected at -30, -15, 0, 7.5, 15, 30, and 60 min, that is, both before and after the naloxone injection. Microdialysis was performed in unanesthetized, unrestrained animals in an open-top plastic cage in which the animals were free to move, eat, drink, and rest. The levels of glutamate and serine in preoptic dialysate samples were determined using HPLCy (HPLC) as described previously (46). Amino acids in dialysate samples (20 µl) were derivatized with 5 µl of fluoraldehyde (Orthophthaldehyde, Pierce Chemical Co., Rockford, IL) for exactly 1 min, injected into a reverse-phase column (Econosphere C-18, 5 µM, 250 x 4.6 mM) and separated isocratically. Standards of known concentration were treated in exactly the same way to circumvent the problem of adduct degradation during the elution process. The HPLC (Beckman Instruments) consisted of a model 421 microprocessor, model 110A pumps, an injector with a 20-µl sample loop and a fluorescence detector (Gilson Spectra Glo), coupled to a recording integrator (Altex, C-RIA). The fluorescence detector used a standard flow cell, a 7–51 excitation filter and a 3–72 M emission filter. The mobile phase used for separation consisted of 18% acetonitrile in 0.1 M sodium phosphate (NaH2PO4, vol/vol) at a pH of 5.7 and a flow rate of 1.0 ml/min. Quantification of sample peaks was accomplished by comparing peak areas with those of known concentrations of standards. The data were expressed as percent control with the zero timepoint set as 100%.

RIA of LH
The concentrations of LH in serum samples were analyzed by a double antibody RIA method as described previously by Brann and Mahesh (42). The purified hormones and standards and the first antibody for LH (NIAMDD-YLH-S-10 (rabbits)) were obtained from NIDDK (National Hormone Pituitary Program, Baltimore, MD). The purified hormone was iodinated with 125I (Amersham) by the chloramine-T method. The second antibody was purchased from Arnell, Inc. (Brooklyn, NY) and used at a 1:250 dilution. The assay was linear at 4–128 ng/tube for LH. Hormone levels are expressed in terms of NIAMDD-RP-3 standard for LH. Intraassay and interassay variabilities for the LH assay were 9% and 10.4%, respectively.

Statistical analysis
The data were expressed as mean ± SE, with 4–6 animals used per group. The experiments were repeated for verification of results. The results were analyzed by one-way ANOVA and significance of differences was determined by the Student-Newman-Keuls test. When only two treatment groups were being compared, the Student t test was employed. A P value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go illustrates the effect of naloxone upon LH levels (Fig. 1AGo) and NOS activity in the POA (Fig. 1BGo) and MBH (Fig. 1CGo) of the male rat killed 20 min after naloxone injection. The effect of the glutamate (NMDA) receptor antagonist, MK-801 is also shown. The insert for each figure shows the results of a second experiment to confirm the naloxone effects and to determine whether MK-801 affects basal LH levels or NOS activity levels. As shown in Fig. 1Go, naloxone treatment significantly increased LH serum levels (Fig. 1AGo) and NOS activity in the POA (Fig. 1BGo) and MBH (Fig. 1CGo) 20 min after its injection. Of significant interest, pretreatment with the glutamate (NMDA) receptor antagonist, MK-801 (0.2 mg/kg), significantly attenuated the ability of naloxone to increase POA NOS activity (Fig. 1BGo) and MBH NOS activity (Fig. 1CGo), and correspondingly abolished the naloxone-induced LH surge (Fig. 1AGo), suggesting that opioids act on glutamate neurons, which are upstream of NO neurons. As shown in the Fig. 1Go inserts, the naloxone effects on LH levels (Fig. 1AGo, inset) and NOS activity are reproducible (Fig. 1Go, B and C, insets), and MK-801 has no effect on basal LH levels even though it causes a slight lowering of POA NOS activity levels (Fig. 1BGo, inset) with no effect on MBH NOS activity levels (Fig. 1CGo, inset) when administered alone.



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  		Figure 1. Effect of naloxone (2.5 mg/kg) upon serum LH levels (A) and nitric oxide synthase (NOS) activity in the preoptic area (B) and medial basal hypothalamus (C) of the adult male rat. The effect of a glutamate receptor antagonist (MK-801; 0.2 mg/kg) on naloxone-induced elevations of serum LH levels and NOS activity in the POA and MBH is also shown. MK-801 was administered 20 min before naloxone. The animals were killed 20 min after naloxone treatment. The results of a second experiment to confirm naloxone effects on serum LH levels and NOS activity levels and to examine the effect of MK-801 upon basal LH and NOS activity levels is presented in the insets for Fig. 1Go, A–C. VEH, Vehicle; NAL, naloxone; MK, MK-801. Groups with different subscripts are significantly different (P < 0.05).

 
Because the above results suggested that opioid inhibition is mediated on glutamate neurons that are upstream of NO neurons, additional experiments were performed to determine: 1) whether glutamate release rates in the POA are increased after naloxone treatment, and 2) whether activation of glutamate (NMDA) receptors in the hypothalamus actually can enhance POA and MBH NOS activity. With respect to the first experiment, Fig. 2Go illustrates the results of microdialysis studies measuring glutamate and serine (control amino acid) release rates in the POA before and after naloxone administration in conscious, unanesthetized, freely moving adult male rats. As shown in Fig. 2AGo, naloxone treatment induced a significant elevation in POA glutamate release rates 15 min after its administration, which immediately preceded the increase in hypothalamic NOS activity and the surge in LH levels that occurred at 20 min post naloxone injection (see Fig. 1Go, A–C). The increase in glutamate release rates was transient as glutamate release rates returned to preinjection levels by 30 min post injection. Note also that the preinjection glutamate release rates were very stable, showing little fluctuation. As shown in Fig. 2BGo, release rates for the control amino acid, serine did not show significant change following naloxone injection, verifying the specificity of the naloxone effect on glutamate release rates.



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  		Figure 2. Effect of naloxone on glutamate (A) and serine (B) release rates in the preoptic area (POA) of conscious, unanesthetized adult male rats as determined by microdialysis. Groups with different subscripts are significantly different (P < 0.05).

 
Because the above data suggested that releasing the opioid brake led to activation of glutamate neurons with subsequent activation of NO neurons via the NMDA receptor, it was important to confirm that activation of the NMDA receptor actually increases hypothalamic NOS activity. Therefore, the effect of NMDA (50 mM) on NOS activity in POA and MBH fragments in vitro was examined. This dose of NMDA has been shown previously by our laboratory and others to stimulate LHRH release from hypothalamic fragments in vitro (36, 47). As shown in Fig. 3Go, A and B, NMDA treatment induced a significant elevation of POA and MBH NOS activity, thus verifying that activation of the NMDA receptor does lead to activation of NO neurons. The effect of NMDA on NOS activity was specific and mediated by the NMDA receptor as evidenced by the fact that the NMDA effect was completely blocked by the NMDA receptor antagonist, MK-801.



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  		Figure 3. Effect of N-methyl-D-aspartate (NMDA) (50 mM) on NOS activity in male rat POA and MBH fragments incubated in vitro. The ability of the NMDA receptor antagonist, MK-801 (100 µM) to block the effect of NMDA is also shown. VEH, Vehicle; MK,MK-801. Groups with different subscripts are significantly different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been known for some time that opioids constitute a major component of the CNS inhibitory brake that tonically restrains LHRH secretion (see Refs. 23, 24, and 48 for review). For instance, administration of the opioid antagonist, naloxone rapidly elevates LHRH and LH release in male and female animals (23, 49, 50, 51), whereas opioid agonists such as morphine markedly suppresses LHRH and LH secretion (49, 51, 52). The tonic opioid inhibition of LHRH neurosecretion appears to be mediated by ß-endorphin neurons that are located in the arcuate nucleus and that send projections to virtually all hypothalamic nuclei (23, 24, 53). The majority of evidence accumulated to date suggests that the µ opioid receptor is responsible for mediating the tonic inhibitory opioid brake on LHRH neurosecretion (54, 55). While the presence of the opioid inhibitory brake has been well established, it is unclear whether the opioid brake is mediated directly on the LHRH neuron or occurs through an indirect mechanism. In potential support of a direct mechanism, ß-endorphin neurons have been reported to synapse on LHRH neurons in the rat (56). Evidence supporting an indirect mechanism has come from studies which demonstrated that immortalized GnRH (GT1–1) cells do not express the µ opioid receptor (57), and that µ opioid agonists do not inhibit LHRH release from GT1–7 cells in vitro (58). Furthermore, Sannella and Peterson (41) recently demonstrated that µ, k, and {delta} opioid receptor transcripts are not expressed in LHRH neurons in vivo in the rat. This led Sannella and Peterson to suggest that opioids restrain LHRH neurosecretion by restraining excitatory "accelerator" components.

The present study provides further clarity to this issue by providing evidence that suggests that opioid neurons restrain LHRH neurosecretion by tonically inhibiting two major components of the "accelerator" mechanism, glutamate and NO neurons. Along these lines, opioid disinhibition resulting from naloxone treatment was shown to result in a significant elevation of NOS activity in the POA and MBH of the male rat 20 min after naloxone treatment. The increased NOS activity was essential for production of the LH surge as administration of the glutamate (NMDA) receptor antagonist, MK-801, significantly attenuated the naloxone-induced elevation in NOS activity in the POA and MBH, with a corresponding block of the elevation of serum LH levels induced by naloxone.

The finding that the NMDA receptor antagonist attenuates naloxone-induced elevations of hypothalamic NOS activity levels and serum LH levels suggests that glutamate neurons are activated first upon opioid disinhibition, and that glutamate release subsequently activates downstream NO neurons, which serves to relay the stimulatory signal to the LHRH neuron. That opioid disinhibition actually increases glutamate release rates in the POA was demonstrated by microdialysis studies, which demonstrated that POA glutamate release rates increase significantly 15 min after naloxone treatment, which immediately precedes the elevation of hypothalamic NOS activity and LH secretion. The released glutamate is proposed to activate NMDA receptors on hypothalamic NO neurons, leading to calcium influx that activates NOS and thus enhances NO release. In support of this suggestion, previous work by our laboratory has demonstrated that hypothalamic NO neurons contain NMDA receptors and that an NOS inhibitor blocks NMDA-induced LH release (31). Additionally, glutamate and NMDA-induced GnRH release from hypothalamic fragments in vitro have also been shown to be blocked by pretreatment with an NOS inhibitor or by administration of hemoglobin, an NO scavenger (34). The presence of a glutamate-NO signaling pathway in the hypothalamus was further confirmed by demonstration in the present study for the first time that NMDA actually enhances POA and MBH NOS activity in vitro. Treatment with the NMDA receptor antagonist, MK-801 prevented the effect of NMDA upon hypothalamic NOS activity levels, verifying that the effect of NMDA was specific and mediated through the NMDA receptor.

In addition to increasing glutamate release in the POA as observed in the present study, recent work by several laboratories has also shown that opioid disinhibition increases glutamate release in the locus coeruleus of the rat (59, 60, 61). This is intriguing, as the locus coeruleus is the site of a major catecholamine cell body group that provides extensive innervation of the hypothalamus, and catecholamines are well known to stimulate LHRH secretion (see Ref. 22 for review). Interestingly, there is evidence that catecholamine neurons in the locus coeruleus are regulated by glutamate neurons. For instance, catecholamine neurons in the locus coeruleus have been shown to possess NMDA receptors (62) and express c-Fos protein after NMDA administration (20). Furthermore, NMDA administration increases catecholamine levels in the hypothalamus (39, 40), and catecholamine receptor antagonists and synthesis inhibitors have been shown to block NMDA-induced LH and LHRH release (28, 63, 64). Based on the above, it is conceivable that opioid disinhibition could lead to activation of glutamate neurons in the locus coeruleus in addition to the hypothalamus and that glutamate could drive both NO and catecholamine accelerator components for induction of LHRH and LH release. Further work on this issue is needed, especially with respect to the postulated opioid-glutamate-catecholamine signaling link and its potential role in the control of LHRH release.

Taken as a whole, the findings of the present study suggest that opioid neurons act to restrain LHRH neurons by exerting tonic inhibition over glutamate neurons in the hypothalamus, a principal component of the accelerator mechanism. Opioid restrainment of glutamate neurons also serves to restrain NO neurons, which are downstream of glutamate neurons and are known to relay/mediate glutamate excitatory signals to the LHRH neuron. The existence of an opioid-glutamate-NO signaling pathway in the control of LHRH neurons provides a clearer understanding of how LHRH neurosecretion can be regulated and further demonstrates the importance of brake-accelerator component interactions in the dynamic control of LHRH and LH secretion in vivo.


   Footnotes
 
1 This work was supported by a Research Grant (R01-HD-28964) (to D.W.B.) from the National Institute of Child Health and Human Development, NIH, U.S. Public Health Service. Parts of this work was presented in abstract form at the Tenth International Congress of Endocrinology held in San Francisco, California, June 12–16, 1996. Back

Received October 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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