Journal of Undergraduate Research
Volume 6, Issue 2 - October 2004

The Effects of J113397, an Orphanin/Nociceptin FQ Receptor Antagonist, on the Limbic-Hypothalamic-Pituitary-Adrenal Axis

Martina Bauer

ABSTRACT

Orphanin FQ (N/OFQ) is a neuropeptide structurally similar to endogenous opioids which plays a functional role in the limbic-hypothalamic-pituitary-adrenal (LHPA) axis. This axis is a self-limiting neuroendocrine pathway important for the processing of emotionally salient stressors. Interestingly, intracerebroventricular (i.c.v.) administration of N/OFQ increases anxiety-related behavior in rats. The goal of the present study was to analyze the effects of a new N/OFQ receptor antagonist, J113397, alone and in combination with N/OFQ in order to assess its effect as an N/OFQ receptor antagonist. In this experiment, rats were treated with i.c.v. injections of artificial extracellular fluid (aECF), or N/OFQ alone, or aECF containing various concentrations of J113397, or a mixture of N/OFQ and J113397. The rats’ anxiety-related behavior was then measured using the open field test, a neophobic test of anxiety in rats. The results show that in accordance with previous studies, N/OFQ increased anxiety-related behavior. In fact, at higher administered doses, J113397 seemed to exert partial agonist effects. In conclusion, a further test is needed using a mixture of N/OFQ and J113397 that contains a concentration of J113397 which does not exert agonistic effects on the N/OFQ receptor in hopes of determining the pharmacological profile of J113397.

INTRODUCTION

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide with extensive sequence homology to endogenous opioids. Its receptor, NOP (formerly known as ORL1) also bears sequence homology to opioid receptors. Despite this homology, N/OFQ does not bind with any affinity to any of the opioid receptors nor does NOP bind endogenous opioids with any specificity (Meunier et al., 1995, Reinscheid et al., 1995). The lack of an overlap in these two systems suggests that despite their similarities on a structural level, there may be fundamental functional differences. The NOP receptor is a Gi protein coupled receptor. Like other receptors in this family, the NOP receptor has 7 transmembrane spanning _-helical segments. It is negatively linked to adenylate cyclase, activates inward rectifiying K+ channels, and inhibits Ca2+ channels (Fukuda et al., 1994; Mollereau et al., 1994). Both N/OFQ and NOP are found in many areas of the central nervous system (Mollereau et al., 1994; Neal et al., 1999) suggesting they may play a role in a wide variety of functions (Fernandez et al., 2004). The distribution of both N/OFQ and NOP in limbic areas suggests that the two may play a role in the activation of the HPA axis (Devine et al., 2001). In addition, the two are densely expressed in a wide variety of limbic and limbic-associated brain regions implicating their importance in the processing of emotionally salient events and anxiety regulation (Devine et al., 2003, Fernandez et al., 2004).

In fact, N/OFQ does indeed play a role in the regulation of the HPA axis. Administering N/OFQ to rats exposed to the mild stress of a novel environment enhances stress induced elevations of plasma ACTH and CORT (Devine et al., 2001). These findings are supported by evidence that N/OFQ knockout mice have disregulated HPA function (Reinscheid et al., 2002). Furthermore, N/OFQ has proven to increase the expression of anxiety related behavior in rats (Fernandez et al., 2004).

The LHPA axis is a self limiting neuroendocrine pathway regulating both physiological and behavioral responses to emotionally salient stressors. In the framework of the neurobiological aspects of stress, stressors can generally be categorized as “systemic” or “processive” (Herman et al., 1996; Herman et al., 1997). Systemic stressors are those that pose a serious and immediate threat to an organism’s homeostasis such as extreme temperature or water/food deprivation. Processive stressors encompass the wide array of stimuli that do not pose an immediate threat to homeostatic balance but instead require more cognitive processing on the part of the organism. Examples of processive stressors include restraint in an animal model of stress or occupational and financial stressors in humans. The neurobiological processing of these two classes of stressors is in fact different. Systemic stressors stimulate the paraventricular nucleus (PVN) of the hypothalamus most likely through catecholaminergic projections from the brainstem, whereas processive stressors stimulate the PVN through limbic and forebrain structures. The present study focused on processive stressors due to their applicability in everyday human experience.

Once stimulated by an emotional stressor, the parvocellular cells of the PVN trigger the synthesis and release of corticotrophin releasing hormone (CRH), which in turn stimulates the pituitary gland to release adrenocorticotrophic hormone (ACTH) (Whitnall, 1993). Upon reaching the adrenal cortex, ACTH stimulates the release of cortisol in humans (corticosterone in rats, abbreviated as CORT). These glucocorticoids serve a variety of functions that primarily prepare the body for an immediate response to an acute stressor. They also serve to shut down the LHPA axis in a negative feedback fashion (Jacobson et al., 1991). Acutely elevated levels of these stress hormones are adaptive in that they help quiet the system and maintain homeostasis. However, chronically elevated glucocorticoid levels can have detrimental effects such as the promotion of systemic disease and affective disorders, as well as neurodegenerative disease (Herman et al., 1997). Accordingly, the development of these kinds of problems might in some cases be indicative of a disregulation of the stress response.

The goal of the present study was to analyze the effects of a NOP receptor antagonist, J-113397, alone and in combination with N/OFQ, in hopes of better understanding the potential role of N/OFQ in the LHPA axis and in stress responses. The open field, a neophobic test of anxiety in the rat, was used to measure the behavioral effects of J-113397 and N/OFQ. This test takes advantage of two innate characteristics of rodents. They are foraging creatures that are motivated to explore novel environments, but they tend to avoid open spaces as well as brightly lit areas where they are more vulnerable to predators (Fernandez et al., 2004).

Accordingly, normal rats and mice exhibit an approach-avoidance conflict when placed in the start box of the open field. Rats treated with a drug that humans report to be anxiolytic (i.e., valium) will spend a greater proportion of their time in the open field and in its central region than will untreated rats (Fernandez et al., 2004) when those rats are freely allowed to explore these regions. Furthermore, the valium-treated rats will stray farther from a safe “start box” than will untreated rats (Devine, unpublished data). Conversely, rats treated with anxiogenic drugs (i.e., FG7142) will spend a decreased amount of time in the open field and its central region (Fernandez et al., 2004) and will not stray far from the start box, in comparison with untreated rats (Devine, unpublished data).

METHODS

Animals

Seventy four male Long-Evans rats were obtained from Charles River (Raleigh, NC) and Harlan (Indianapolis,IN). The rats were pair housed until the time of surgery in polycarbonate cages and kept on a 12h/12h light-dark cycle. The rats were housed in a room with lights on at 1200. Food and water were available ad libitum. After acclimation to the housing room, each rat was implanted under ketamine:xylaxine (83.3 mg ketamine/ml; 16.7 mg xylazine/ml; 0.75 ml/kg) with chronic stainless steel guide cannula terminating 0.5 mm above the lateral ventricle in the right hemisphere (0.8 mm posterior to bregma, 1.4 mm lateral to the midsagittal suture, 2.7 mm ventral to dura). Guide cannulae were fastened to the skull by dental cement and microscrews. Stainless steel blockers were placed in the guide cannulae and removed to allow for drug injection. After surgery, the rats were singly housed and allowed to recover for 7-14 days before anxiety testing ensued.

Apparatus

The open field is a behavioral testing apparatus previously validated for use in the measurement of anxiety related behaviors (Fernandez et al., 2004). It consists of an open acrylic box (90cm x 90cm) with an attached open start box (20cm x 30cm), which is separated from the main area by a guillotine door. This door was opened outside the testing room via a rope and pulley system.

Procedure

Prior to testing in the open field, each rat was handled for three consecutive days and then allowed one day free of handling. The rats were tested in the afternoon between 1:00 and 4:00. Testing time was consistent with the light cycle present in the rats’ housing room, such that all the rats were tested within 5 hours of the beginning of the light part of the daily cycle. All testing was done by an experimenter blind to the testing conditions. Each rat was injected through the guide cannula with 1 µl artificial extracellular fluid (aECF) , or 1 µl aECF containing 0.01 nmole J113397, 0.1 nmole J113397, or 1 nmole J113397, or a 1 µl cocktail of 0.1 nmole N/OFQ + 1 nmole J113397. Each injection was administered using a 5 µl Hamilton syringe with a Harvard syringe pump over a 2 minute period. After each injection, the injector was kept in the cannula for an additional 1 minute to allow for drug dispersion. Five minutes after the beginning of the injection, each rat was placed into the start box of the open field apparatus where it was allowed to acclimate for 1 minute. After 1 minute, the guillotine door was opened from outside the testing room and rats had free access to the entire open field for 5 minutes. The rats’ exploratory behavior was recorded using a video camera mounted on the ceiling directly above the apparatus. All testing was done with 22-52 lux illumination. Each rat was removed from the open field after 5 minutes and placed back in its home cage. Subsequently, each rat was sacrificed by rapid decapitation at 30 minutes after the injection. The brain of each rat was removed and frozen in 2-methyl butane at -40°C and later sectioned at 30 mm in the coronal plane to verify cannula placements.

The videotapes were then scored by a trained viewer who was blind to the testing conditions. In order to collect data, the video image of the open field was partitioned into 25 equal sized squares—16 peripheral squares and 9 inner squares. Using this grid system, four zones were assigned and used to assess anxiety levels. The start box was Zone 0. The 7 peripheral squares closest to the start box made up Zone 1, with the 9 peripheral squares farthest from the start box making up Zone 2. Finally, Zone 3 was made up of the 9 inner squares (See Figure 1). Latency to enter the open field as well as latency to enter each of zones 1-3 was measured. In addition, total time spent in each zone was calculated. Each entry into a specific area was recorded when all four paws were placed in that area.

Statistical Analyses

An exploration score was calculated for each rat. This score was calculated by multiplying the number of seconds the rat spent in each zone by the number assigned to that zone and adding the products of those multiplications. Accordingly, if the rat spent all 300 seconds in the start box (Zone 0), the total exploration score would be zero. If the rat spent 200 seconds in the start box and 100 seconds in Zone 1, the score would be (0 X 200) + (1 X 100) = 100. In this manner, higher exploration scores indicate lower expression of anxiety-related behavior.

Between-group differences in exploration score, open field (Zones 1-3) time, inner zone (Zone 3) time, latency to enter the open field, latency to enter the inner zone, open field entries, and inner zone entries were analyzed for the vehicle-treated rats and the J113397-treated rats using a one way analysis of variance (ANOVA). All significant effects were further analyzed using a Student-Newman-Keuls posttest. Between-group differences in all measures were analyzed for the vehicle-treated rats and the rats treated with N/OFQ only using an independent samples T-Test.

RESULTS

When compared to the aECF-treated rats, the rats treated with 0.1 nmole N/OFQ exhibited significantly lower exploration scores (T21 = 2.107, p < 0.05), indicating greater expression of anxiety-related behavior in the N/OFQ-treated rats (Fig 2). Latency to enter both the open field (T21 = -2.136, p < 0.05) and the inner zone (T21 = -2.989, p < 0.05) were significantly higher in the N/OFQ treated rats compared to the aECF-treated rats. Additionally, the N/OFQ-treated rats exhibited significantly fewer open field entries than aECF-treated rats (T21 = 2.515, P < 0.05). Total open field time (T21 = 1.869, p = 0.076), total inner zone time (T21 = 0.672, p = 0.509), and total number of inner zone entries (T21 = 1.93, p = 0.067) did not differ significantly between aECF-treated and 0.1 nmole N/OFQ-treated rats.

None of the behavioral measures (exploration score {F4,61 = 1.447, p = 0.230}, total open field time { F4,61 = 0.881, p = 0.481}, total inner zone time { F4,61 = 1.222, p = 0.312}, open field latency { F4,61 = 1.906, p = 0.122} or , inner zone latency { F4,61 = 1.97, p = 0.111, open field entries { F4,61 = 1.429, p = 0.235}, inner zone entries { F4,61 = 1.071, p = 0.379} differed significantly between the aECF-treated rats and the rats treated with J113397 (0.01 nmole, 0.1 nmole, 1 nmole, 1 nmole J113397 + .1 nmole N/OFQ).

Figure 2. N/OFQ administration decreased the amount of exploratory behavior and is thus associated with an elevation in anxiety-related behavior in these rats. Administration of J113397 did not significantly affect exploratory behavior. While not statistically significant, the trends observed in exploration score are consistent across other measures. There may still be a possibility that J113397 is a partial agonist of N/OFQ.

Figure 3. The effects of J113397 alone and in combination with N/OFQ were analyzed using behavioral measures of anxiety-related behavior. The administration of N/OFQ caused an increase in anxiety-related behavior as indicated by a significantly higher open field latency (b), significantly higher inner zone latency (e), and significantly fewer open field entries (c). A general trend which did not reach statistical significance appears in the measurement of anxiety-related behavior across the range of administered doses of J113397. The lowest administered dose (0.01 nmole) does not appear to affect the expression of anxiety-related behavior as compared to aECF. However, the administration of higher doses of J113397 may exert variable partial agonist effects on the NOP receptor.

DISCUSSION

In accordance with previous findings (Fernandez et al., 2004), N/OFQ increased the expression of anxiety-related behavior. The rats treated with N/OFQ exhibited lower exploration scores than did the aECF-treated controls, indicating greater anxiety-related behavior. Accordingly, latency to enter both the open field and inner zone was higher in N/OFQ-treated animals, a further indication of its anxiogenic effects.

In contrast, the effects of J113397 are less straightforward. The results suggest that J119937 does not exert purely antagonistic actions at the NOP receptor. At the lowest administered dose (0.01 nmole), anxiety-related behavior was not significantly affected. Interestingly, at the two higher doses (0.1 nmole, 1.0 nmole), J113397 appeared to be exerting a partial agonist effect. Within this range of doses, J13397 actually seemed to weakly resemble the results expected of an anxiogenic drug, although this did not reach statistical significance.

These results demonstrate a need for further analysis in order to fully characterize the pharmacological profile of J113397. In the present study, we challenged 0.1 nmole N/OFQ with 1.0 nmole J113397 in an attempt to assess the ability of J11339 to antagonize N/OFQ. The tenfold difference in N/OFQ and J113397 was necessary in order to overcome the greater affinity of N/OFQ for the NOP receptor. However, at this concentration, J113397 was not capable of attenuating the anxiogenic effects of N/OFQ, perhaps due to partial agonist effects at this high dose. For this reason, we will re-assess the effects of J113397 using a cocktail that contains lower concentrations of both N/OFQ (0.001 nmoles/_l) and J113397 (0.01 nmoles/_l). Previous findings have shown that N/OFQ increases anxiety-related behavior at doses as low as 0.001 nmole/_l (the threshold dose) when administered i.c.v. At 0.01 nmole, J113397-treated rats do not differ significantly from controls in expression of anxiety-related behavior. Accordingly, the combination of these doses of N/OFQ and J113397 may reveal whether J113397 can specifically antagonize the anxiogenic actions of N/OFQ

Another possibility is that J113397 is in fact a partial agonist of the NOP receptor. Recently, a variety of potential N/OFQ antagonists have been introduced. Previous work with some of these N/OFQ analogues has provided evidence that while they exert antagonistic effects in vitro, they act as partial agonists in vivo (Chiou 1999; Devine et al., 2001). While many of these drugs are substituted N/OFQ peptides, J113397 is a non-peptide drug. Because of this distinction it was thought that J113397 might be a better NOP receptor antagonist. Instead, the results suggest that J113397, like previously identified NOP receptor antagonists, may also be a partial agonist of the NOP receptor.

In conclusion, we need to test a dose of J113397 that lacks substantial agonist activity. This dose, as identified in the present study, was 0.01 nmole. The potential that the disregulation of the N/OFQ and NOP system plays a role in emotional disorders makes this an important avenue for further research. Identification of effective antagonists of this system is needed in order to better understand the system as a whole and to address of the possibility of future therapeutic applications.

---

REFERENCES

1. Meunier, J. C.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand, C.; Alvinerie, P.; Butour, J-C.; Guillemot, J-C.; Ferrara, P.; Monsarrat, B.; Mazargull, H.; Vassart, G.; Parmentier, M.; Costentin J. Isolation and structure of the endogenous opioid receptor-like ORL1 receptor. Nature 1995, 377, 532-535.
   
2. Reinscheid, R. K.; Nothacker, H. P.; Bourson, A.; Ardati, A.; Henningsen, R. A.; Bunzow, J. R; Grandy, D. K.; Langen, H.; Monsma, F. J.; Civelli, O. Orphanin FQ: a neuropeptide that activates an opioid-like G-protein-coupled receptor. Science 1995, 270, 792-794.
   
3. Fukuda, K.; Kato, S.; Mori, K.; Nishi, M.; Takeshima, H.; Iwabe, N. Miyata, T.; Houtani, T.; Sugimoto, T. cDNA cloning and regional distribution of a novel member of the opioid receptor family. Fedn Eur. Biochem. Socs Lett. 1994, 343, 42-46
   
4. Mollereau, C.; Parmentier, M.; Mailleux, P.; Butour, J. L.; Mousand, C.; Chalon, P.; Caput, D.; Vassart, G.; Meunier, J. C. ORL1, a novel member of the opioid receptor family—cloning, functional expression, and localization. Fedn Eur. Biochem Socs Lett. 1994, 341, 33-38.
   
5. Nea,l C. R.; Mansour, A.; Reinscheid, R. K.; Nothacker, H. P.; Civelli, O.; Watson, S. J. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J. comp. Neurol. 1999, 406, 503-547.
   
6. Fernandez, F.; Misilmeri, M.; Felger, F.; Devine, D. P. Nociceptin/Orphanin FQ increases anxiety-related behavior and circulating levels of corticosterone during neophobic tests of anxiety. Neuropsychopharmacology 2004, 29, 59-71.
   
7. Devine, D. P.; Watson, S. J.; Akil, H. Orphanin FQ regulates neuroendocrine function of the limbic-hypothalamic-pituitary-adrenal axis. Neuroscience 2001, 102,541-553.
   
8. Devine, D. P.; Hoversten, M. T.; Ueda, Y.; Akil, H. Nociceptin/Orphanin FQ content is decreased in forbrain neurones during acute stress. Journal of Neuroendocrinology 2003, 15, 69-74.
   
9. Reinscheid, R. K.; Civelli, O. The orphanin FQ/nociceptin knockout mouse: A behavioral model for stress responses. Neuropeptides 2002, 36, 72-76.
   
10. Herman, J. P.; Prewitt, C. M.; Cullinan, W. E. Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis. Crit Rev Neurobiol. 1996, 10, 371-394.
    
11. Herman, J. P.; Cullinan, W. E. Neurocircuitry of stress: central control of the hypothalamic-pituitary-adrenocortical axis. Trends Neurosci. 1997, 20, 78-84.
    
12. Jacobson, L.; Sapolsky, R. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis. Endocrine Review 1991, 12, 118-134.
    
13. Chiou, L. C. [Phe1_ (CH2-NH)Gly2]nociceptin-(1-13)-NH2 activation of an inward rectifier as a partial agonist of ORL1 receptors in rat periaqueductal gray. British Journal of Pharmacology 1999, 128, 103-107.

---

--top--

Back to the Journal of Undergraduate Research