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

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The Effects of J113397, an Orphanin/Nociceptin FQ Receptor Antagonist, on the Limbic-Hypothalamic-Pituitary-Adrenal Axis
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Bauer, Martina
Devine, Darragh ( Mentor )
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University of Florida
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The Effects of J113397, an Orphanin/Nociceptin FQ Receptor Antagonist,
on the Limbic-Hypothatamic-Pituitary-Adrenat 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 pl artificial extracellular fluid (aECF) , or 1 pl aECF containing

0.01 nmole J113397, 0.1 nmole J113397, or 1 nmole J113397, or a 1 pl cocktail of 0.1 nmole N/OFQ + 1





nmole J113397. Each injection was administered using a 5 pl 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 -400C 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.



t

zone 2 zone 2 zone 2 zone 2 zone 2


zone 2 zone 3 zone 3 zone 3 zone 2


zone 2 zone 3 zone 3 zone 3 zone 2


zone 1 zone 3 zone 3 zone 3 zone 1


zone 1 zone 1 zone 1 zone 1 zone 1







Figure 1. Open Field Zone Classifications.



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).




" 150

t
a 100-


P
X

aECF .10 1 j0 .10 1.0
NOFQ J11 NOFQ

Treatment (nmole) J11


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 3113397 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 3113397

is a partial agonist of N/OFQ.


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T
iO C


TrbMlm (vnDoom JlI


rjoFQ ill NCF Q
"rr-0-4 "MONO il


'0 00o ' L0 10 I 0- 10
?OFO J1 NOFO
Treobu im. j11


Figure 3. The effects of 3113397 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 3113397. 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 3113397 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/_I) and J113397 (0.01 nmoles/_I). 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





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