Citation
The Effects of Environmental Challenges on Nociceptin/Orphanin FQ-Induced Anxiety

Material Information

Title:
The Effects of Environmental Challenges on Nociceptin/Orphanin FQ-Induced Anxiety
Creator:
Hersh, Carrie M.
Devine, Darragh
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Journal of Undergraduate Research
Genre:
serial ( sobekcm )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.

Downloads

This item has the following downloads:


Full Text




journal orf ILn.err.3du.3- e :.- Rese.arch

..Oluinie 6, issue - - [..3, june l I.u''L



The Effects of Environmental Challenges on Nociceptin/Orphanin FQ-
Induced Anxiety

Carrie M. Hersh


ABSTRACT


Nociceptin/orphanin FQ (N/OFQ) is an opioid-related neurotransmitter that is the endogenous ligand for

the nociceptin/orphanin FQ peptide (NOP) receptor. This neurotransmitter is released from forebrain neurons

during exposure to acute stress and increases circulating concentrations of adrenocorticotrophic hormone

and corticosterone in unstressed and mildly-stressed rats after i.c.v. injection. Furthermore, i.c.v. microinjections

of N/OFQ increase anxiety-related behavior in rats during neophobic tests of anxiety. However, some studies

have reported that N/OFQ is anxiolytic in mice and rats. One possible reason for the differences in findings is that

the basal stress levels of the rats may have differed across studies prior to drug administration. We tested

this hypothesis by exposing some rats to the stress of restraint and found that the stressed and unstressed N/

OFQ-treated rats exhibited equivalent high expression of anxiety-related behavior.



INTRODUCTION


Acute stress arises from physiological and emotional challenges to homeostasis (Herman and Cullinan, 1997),

where these challenges cause temporary changes in neurotransmitter release and increases in

circulating concentrations of adrenocorticotrophic hormone (ACTH) and corticosterone (CORT). Chronic stress, on

the other hand, causes enduring alterations in the regulation of the neurotransmitters and hormones.



Stress can be subdivided into two functional types: "processive" or emotional and "systemic" or somatic

stress (Herman et al., 1997; Rodriguez Enchandia et al., 1998). Processive stressors, appear to be mediated

through limbic forebrain inputs to the parvocellular neurons of the paraventricular nucleus (PVN) of

the hypothalamus. These stressors do not pose an immediate threat to biological homeostatic systems.

These stressors consist of emotionally threatening stimuli (Johnson et al., 1992; Roozendaal et al., 1996).

Systemic stressors, on the other hand, represent immediate physiological challenges to an organism, sending

direct inputs to the PVN from brainstem regions. Unlike processive stressors, systemic stressors may not

require higher brain functioning (Herman et al., 1997). Nociceptive stimulation, immune system threats, exposure

to extreme temperatures, and food deprivation are common examples of systemic stressors.



During exposure to processive stress, limbic regions of the brain signal neurons of the PVN, initiating a neuronal





and hormonal cascade involving the limbic hypothalamic-pituitary adrenal (LHPA) axis. Parvocellular neurons of

the PVN synthesize and release corticotrophin releasing hormone (CRH) into the hypophyseal portal system. CRH

acts on the corticotrophs of the anterior pituitary following stimulation of the limbic system. The corticotrophs

then release ACTH into the systemic circulation. ACTH reaches the adrenal glands through the circulation, where

it acts on high-affinity Gs-coupled ACTH receptors to synthesize and secrete CORT, the most significant steroid

in rodents. In normal functioning organisms, circulating corticosterone then acts on higher brain systems, such as

the hippocampus, as a negative feedback system to directly limit the amount of CRF secreted into the

anterior pituitary gland, making the LHPA axis a self limiting pathway.



Nociceptin/orphanin FQ (N/OFQ) is released from forebrain neurons during exposure to acute stress,

implicating endogenous N/OFQ neurotransmission in physiological responses to stress (Devine et al., 2003). N/OFQ

is an endogenous 17-amino acid neuropeptide that exhibits amino acid sequence homology with opioid

peptide neurotransmitters (Reinscheid et al., 1995; Meunier et al., 1995), indicating close evolutionary

homology between the opioid and N/OFQ systems. However, there are important distinctions between these

systems. Opioids function by binding to specific opioid receptors in the Central (CNS) and in the Peripheral

(PNS) Nervous Systems. N/OFQ lacks classical opioid activity in that it does not bind to the opioid receptors.

Instead, it displays high affinity and selectivity for the nociceptin/orphanin FQ peptide (NOP) receptor (Reinscheid

et al., 1995; Meunier et al., 1995), which makes this system functionally distinct from the opioid systems. The

NOP receptor is a 7-transmembrane Gi-protein coupled receptor. NOP exhibits structural and functional similarities

to the opioid receptors but does not selectively bind to the classical opioid ligands (Bunzow et al., 1994; Lachowicz

et al., 1995; Mollereau et al., 1994). When activated, it inhibits adenylyl cyclase activity and modulates K+ and Ca2

+ conductance (Fukuda et al., 1994; Mollereau et al., 1994).



N/OFQ and its receptor are abundantly expressed in limbic brain regions that regulate emotional states (Neal et

al., 1999a, 1999b). Hence, N/OFQ may regulate emotional behavior. In support of this hypothesis, it has been

found that N/OFQ administration increases hormonal measures of stress (Devine et al., 2001). Nevertheless, there

is a controversy regarding the role of N/OFQ in regulation of fear and anxiety. Previous studies have reported that

N/OFQ is anxiolytic in mice and rats (Jenck et al., 1997; Gavioli et al., 2002). In contrast, Devine and

colleagues found that i.c.v. N/OFQ caused increases in anxiety-related behaviors (Devine et al., 2004) that

were similar to those produced by FG 7142, a benzodiazepine receptor inverse agonist, and contrasted with

the anxiolytic effects of diazepam. Increased plasma concentrations of ACTH and CORT were observed with each

drug treatment during exposure to three tests of anxiety: Open-Field Test, Elevated Plus Maze, and Dark-Light

Test. This suggests that during tests of anxiety, administration of N/OFQ, diazepam, and FG 7142 each elevated

the responsiveness of the LHPA axis. The dose ranges overlapped in the studies that reported conflicting

anxiolytic and anxiogenic actions, indicating that the discrepancy in findings was not due to differences in

drug dosages.



The contrasting reports of anxiolytic and anxiogenic actions of N/OFQ are perplexing. One factor, which may

have contributed to the conflicting results, is the rats' basal stress levels prior to neophobic tests. The methods





used in the earlier reports may have induced psychological stress on tested rodents prior to drug

administration. Indeed, the expression of high amounts of anxiety-related behavior in the vehicle-treated rats

in those studies (Jenk et al., 1997, Gavioli et al., 2002) suggests that the rats were stressed at the time of

anxiety testing (Fernandez et al., 2004). Accordingly, one purpose of the present study was to evaluate whether

the recent stress history of the rat affected the rat's emotional behavior during drug treatment and

neophobic testing. The effects of N/OFQ administration on anxiety-related behavior in rats exposed to the

emotional stress of restraint shortly prior to drug administration and testing were evaluated.



MATERIALS AND METHODS


Animals


Forty-four male Long-Evans rats (Harlan, Indianapolis, IN) were pair-housed in 43 cm x 21.5 cm x 25.5

cm polycarbonate cages. They were kept in a 12hr/12hr light-dark cycle (lights on at 12:00 pm), and with rat

chow and tap water available ad libitum. Following 7-days acclimation, each rat was anesthetized with

ketamine:xylazine (62.5 mg/kg ketamine, 12.5 mg/kg xylazine) and implanted with a 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 matter). Each cannula was fastened to the

skull with dental cement and microscrews. Following surgery, a stainless steel obturator was placed in the

guide cannula, only to be removed for drug injection. The obturator extended 1.0 mm beyond the tip of the

guide cannula. The rats were then singly housed in the original rat room and allowed 7-days recovery prior to

anxiety testing.



Apparatus


The open field apparatus is an open acrylic box (90cm x 90cm x 59cm) with an attached start box (20cm x 30

cm). The start box is separated from the open arena by means of a guillotine door that was lifted and lowered

from outside of the testing room with a rope. A camera suspended over the center of the field was used to

videotape the rats. The videotaped images were then scored using a lined grid (divided into 25 equal-sized

squares) taped to a television screen. The open field was previously validated to measure behavioral indices

of anxiety in rats (Fernandez et al., 2004).



Procedures


Each rat was handled for five minutes on each of three consecutive days, followed by a day without disturbance.

On the fifth day, each of 22 rats was individually placed into a plastic restraint tube with velcro straps for 5

minutes, one half hour prior to administering 1.0 _1 of artificial extracellular fluid (aEFC) or 0.10 nmole N/OFQ in

1.0 _1 aECF vehicle (n = 11 rats per group). The microinjection was administered through the guide cannula into

the lateral ventricle, over 120 sec, using a Harvard syringe pump. Twenty-two rats were not restrained, and

these rats were each injected with aECF or 0.10 nmole N/OFQ in the same manner as the restrained rats were (n






= 11 rats per group).


Five minutes after the start of the microinjection, each rat was placed into a start box attached to the open field

for one minute to allow for acclimation to a novel environment. At the end of the acclimation period, the door

was lifted so that the rat had free access to the open field for five minutes. At the end of the five-minute access

to the open field, each rat was returned to its home cage. Then, thirty minutes from the start of drug

microinjection the rat was sacrificed by rapid decapitation. Trunk blood was collected and stored into

chilled polypropylene tubes on ice, where the tubes contained EDTA. Each tube was centrifuged at 1000

times gravity. The blood was aliquotted, frozen on dry ice, and stored at -800C. The brain of each rat was

removed and frozen in 2-methyl butane at -400C, and stored at -800C. Each brain was later sectioned at 30 mm

in the coronal plane to verify the cannula placement.



Exploration Scores


The rats' exploratory behaviors were assessed from the videotapes of the open field test sessions. The time spent

in the exposed arena of the open field, and in the very exposed central area of the open field (see below),

was measured as indices of the anxiety state in each rat.



The video image of the open field arena was partitioned into 25 equal-size squares: 16 peripheral or outer

zone squares and 9 inner zone squares (Figure 1).


Figure 1. Open Field Zone Classifications


The open field time and latency to enter the open field were measured during the 300 s session. Entrance into

each zone was determined when all four paws entered the area. A latency score of 300 s was assigned for any zone

if the rat did not enter that zone.


Radioimmunoassay






Plasma CORT levels were evaluated with a highly specific solid-phase radioimmunoassay (RIA, Diagnostic

Products Corp., Los Angeles, CA).



Statistical Analyses


Between-group differences in open field time, latency to enter the open field, time spent in the inner zone, latency

to enter the inner zone, and number of inner zone entries were each evaluated in the restrained and

unrestrained groups of rats using t-tests. The circulating CORT concentrations were also evaluated with t-tests.



RESULTS


The restrained aECF vehicle-treated group spent significantly less time in the open field (t(20) = 3.146,

p<0.01; Figure 2a), exhibited longer latencies to enter the open field (t(2o) = 3.540, p<0.01; Figure 2b), spent

less time in the inner zone of the open field (t(2o) = 2.667, p<0.05; Figure 2c), exhibited longer latencies to enter

the inner zone (t(2o) = -2.460, p<0.05; Figure 2d), and exhibited fewer entries into the inner zone (t(2o) =

2.609, p<0.05; Figure 2e), compared with the behaviors of the unrestrained aECF vehicle-treated controls.



The unrestrained N/OFQ-treated group spent significantly less time in the open field (t(20) = 2.604, p<0.05;

Figure 2a), exhibited longer latencies to enter the open field (t(20) = 3.390, p<0.01; Figure 2b), spent less time

in the inner zone of the open field (t(20) = 2.667, p<0.05; Figure 2c), exhibited longer latencies to enter the

inner zone (t(2o) = -2.460, p<0.05; Figure 2d), and exhibited fewer entries into the inner zone (t(2o) =

2.609, p<0.05; Figure 2e), compared with the behaviors of the unrestrained aECF vehicle-treated controls.



The unrestrained N/OFQ-treated group did not significantly differ in open field time (t(2o) = 0.09141, p>0.05;

Figure 2a) or latency to enter the open field (t(2o) = 1.230, p>0.05; Figure 2b), compared with the behaviors of

the restrained N/OFQ-treated group. These groups did not enter the inner zone at all. Accordingly, the

statistics cannot be computed (due to a complete lack of variance within all groups), but there were no

differences between the groups in inner zone time, latency to enter the inner zone, and number of inner zone entries.



The restrained vehicle-treated group also did not significantly differ in open field time (t(2o) = 0.6422, p>0.05;

Figure 2a) or latency to enter the open field (t(2o) = 1.312, p>0.05; Figure 2b), compared with the behaviors of

the restrained N/OFQ-treated group. These groups did not enter the inner zone at all. Accordingly, the

statistics cannot be computed (due to a complete lack of variance within all groups), but there were no

differences between the groups in inner zone time, latency to enter the inner zone, and number of inner zone entries.






n.5.
a


ns.
n.5.


,jQ


Tredsrnt



MLs.
C


1 A

F

I


n.s.
n.5.







0tl


Treutment


nLs.


[I


Treutluert


/


S ___ n.s.





0.2-
J -J--


,~~P


Figure 2. The expression of anxiety-related behaviors are depicted as follows: (a) time spent in the
open field, (b) latencies to enter the open field, (c) time spent in the inner zone, (d) latencies to
enter the inner zone, and (e) inner zone entries (n = 11 rats per group, NR = not restrained, R
= restrained). Values expressed are group means � the standard error of the mean (SEM). End points
of lines indicate which groups were compared. Significant differences between the relevant groups
are depicted as follows: *p<0.05, **p<0.01, n.s. = not significant.



Plasma CORT concentrations of the unrestrained rats treated with N/OFQ were significantly higher than the
CORT concentrations of the unrestrained aECF vehicle-treated controls (t(20) = 2.568, p<0.05; Figure 3).


13 - . . . .


O/


I/
t^ p





The restrained aECF vehicle-treated group did not differ significantly in plasma CORT concentrations (t(20) =

0.2940, p>0.05; Figure 3), compared to the CORT levels of the unrestrained vehicle-controls. The restrained N/

OFQ treated-group exhibited significantly elevated plasma CORT concentrations (t(2o) = 2.220, p<0.05; Figure

3), compared to the CORT concentrations of the restrained vehicle-treated group. The unrestrained N/OFQ-

treated group did not exhibit significant differences in CORT concentrations (t(20) = 0.2281, p>0.05; Figure

3), compared to the CORT levels of the restrained N/OFQ-treated group.





n.s.

400- -

300
S





0
# A





Treatment



Figure 3. Plasma CORT concentrations in the unrestrained rats treated with 0.1 nmole N/OFQ

were significantly higher, compared to the CORT levels of the aECF vehicle-treated controls.

No significant differences in circulating plasma CORT concentrations were observed between the

aECF vehicle controls and restrained aECF vehicle-treated group. There were also no

significant differences in CORT levels between the restrained and unrestrained N/OFQ-treated

groups. Plasma CORT concentrations in the restrained rats treated with N/OFQ were significantly

higher, compared to the CORT concentrations of the restrained aECF-treated group. Values expressed

are group means � the standard error of the mean (SEM, n = 11 rats per group, NR = not restrained, R

= restrained). End points of lines indicate which groups were compared. Significant differences

between the relevant groups are depicted as follows: *p<0.05, n.s. = no significant effects.




DISCUSSION



The results demonstrate that the rats treated with N/OFQ, without the stress of restraint, expressed more

anxiety-related behaviors, compared to the behaviors of the unrestrained aECF vehicle-treated controls in the

open field test. Specifically, the N/OFQ-treated rats exhibited longer latencies to enter the open field,

spent significantly less time in the open field and exhibited total avoidance of the inner zone, compared to

the behaviors of the unrestrained aECF-treated rats. These behaviors resembled the behaviors of FG 7142-

treated rats and contrasted with the behaviors that were exhibited by the rats treated with diazepam in a

previous report (Fernandez et al., 2004). Restrained rats treated with aECF also exhibited elevations in






anxiety-related behaviors (i.e., shorter time spent and longer latencies to enter the open field and

complete avoidance of the inner zone), compared to the behaviors of the unrestrained aECF-vehicle treated

group. So, overall it appears that both restraint and N/OFQ administration both activate increases in expression

of anxiety-related behavior. Furthermore, there were no significant differences between the restrained vehicle-

treated and restrained N/OFQ-treated groups in each behavioral measure. This comparison illustrates that N/

OFQ does not alter the anxiety response to an emotional stressor, so the effect of N/OFQ does not depend on

the recent stress history of the animal. Accordingly, N/OFQ increases anxiety in rats that are mildly stressed by

the testing environment, but it cannot alter the anxiogenic effect of restraint (Figure 2), and the discrepancy

between the recent report by Fernandez et al. (2004) and the previous reports (Jenk et al., 1997; Gavioli et

al., 2002) does not seem to arise from differences in the emotional states of the animals at the time of testing.



Exposure of the rats to the open field produced moderate elevations in the circulating concentrations of CORT in

the unrestrained vehicle-treated rats (Figure 3). These circulating CORT responses to the mild stress of the open

field were significantly enhanced by the N/OFQ treatment in the unrestrained rats when these hormonal

responses were measured 30 min after the N/OFQ administration. This enhancement of the stress-induced

elevations in CORT concur with previous reports that N/OFQ activates the HPA axis in unstressed rats and

increases hormonal responses in the presence of a mild stressor to a novel environment (Devine et al.,

2001). Accordingly, the anxiogenic actions of N/OFQ administration are accompanied by enhancement of the

LHPA axis response to the open field. Nevertheless, the relationship between N/OFQ-induced HPA axis activity and

N/OFQ-induced anxiety may not be straightforward. A previous study reported that 1.0 pmole dose of N/

OFQ produced significant anxiety-related behavior in the open field test, but it did not significantly alter

the circulating CORT response. This attests that the CORT and anxiety responses are dissociable. Thus it is likely

that the anxiogenic actions of N/OFQ arise from central actions of N/OFQ following i.c.v. microinjections, rather

than an indirect response involving circulating CORT (Fernandez et al., 2004).



An interesting finding was that the restrained aECF vehicle-treated rats did not exhibit significant differences

in circulating CORT concentrations, compared to the CORT levels in the unrestrained aECF vehicle-treated

controls (Figure 3). The time course of the experiment was designed so that restraint occurred at time 0

(the unrestrained rats sat in their cages during this time), drug injection occurred at time 30, the start of the

open field test occurred at time 35, and sacrifice occurred at time 60. One possibility that may account for

the moderately low CORT response in the restrained vehicle-treated rats is that the increases in circulating

CORT from the stress of restraint at time 0 induced a negative feedback response on the cortical limbic region,

the PVN of the hypothalamus, and the pituitary gland, which then diminished the CORT response to the

novel environment exposure during anxiety testing. In contrast, the rats that were treated with N/OFQ and

restrained exhibited highly elevated plasma CORT concentrations, suggesting that N/OFQ exerts direct

activating effects on HPA axis regulation and/or interfered with the negative feedback effect of the restraint on

the subsequent stressful exposure to a novel environment - these restrained N/OFQ-treated rats did not exhibit

the blunted CORT responses observed in the aECF-treated group.





There were no significant differences in the CORT response, measured 60 min following restraint (30 min

following drug administration), between the restrained and unrestrained N/OFQ-treated rats (Figure 3).

This comparison indicates that N/OFQ enhancement of the LHPA axis does not alter the CORT response in

moderately stressed rats prior to N/OFQ administration. A previous study reported similar findings in the

hormonal data, demonstrating that N/OFQ exaggerates and prolongs the ACTH/CORT response to a mild stressor,

but it cannot alter the LHPA axis activity in the presence of a restraint stress (Devine et al., 2001).

The current findings indicate that both the behavioral and hormonal effects of N/OFQ administration resemble

the effects of FG 7142 (Pellow and File, 1985, 1986) and caffeine (Spindel et al., 1983; Pellow et al., 1985). All

of these drugs are anxiogenic in neophobic tests of anxiety, and all of these drugs elevate circulating ACTH and/

or CORT following acute administration.



In this experiment we tested the possibility that the anxiety-modulating effects of N/OFQ may differ depending on

the basal state or emotional tone of the rat, a possibility that could reconcile the contradictory findings of

Fernandez et al. (2004) and Jenk et al. (1997). However, the behavioral and hormonal results of the study

suggest that the initial emotional status of the rats prior to drug administration did not affect the anxiogenic effects

of N/OFQ. The reason for the contradictory results remains unexplained.






REFERENCES



1. Bunzow J. R., Saez C., Mortrud M., Bouvier C., Williams J. T., and Low M. (1994) Molecular Cloning and

tissue distribution of a putative member of the rat opioid receptor gene family that is not a , _, or _ opioid

receptor type. FEBS Lett 347: 284-288.

2. Devine D. P., Hoversten M.T., Ueda Y., and Akil H. (2003) Nociceptin/Orphanin FQ content is decreased in

forebrain neurons during acute stress. J Neuroendocrinol 15: 69-74.

3. Devine D. P., Watson S. J., and Akil H. (2001) Nociceptin/Orphanin FQ regulates neuroendocrine function of

the limbic-hypothalamic-pituitary-adrenal axis. Neurosci 102: 541-553.

4. Fernandez F., Misilmeri M.A., Felger J. C., and Devine D. P. (2004) Nociceptin/Orphanin FQ increases anxiety-

related behavior and circulating levels of corticosterone during neophobic tests of anxiety.

Neuropsychopharmocol 29: 59-71.

5. Fukuda K., Kato S., Mori K., Nishi M., Takeshima H. et al. (1994) cDNA cloning and regional distribution of a

novel member of the opioid receptor family. FEBS Lett 343: 42-46.

6. Gavioli E. C., Rae G. A., Calo G., Guerrini R., and De Lima T. C. (2002) Central injections of nocistatin or its

C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test. BrJ Pharmacol

136: 764-772.


7. Herman J. P. and Cullinan W. E. (1997) Neurocircuitry of stress: Central control of the hypothalamo-





pituitary-adrenocortical axis. Trends Neurosci 20: 78-84.


8. Jenk F., Moreau J.-L., Martin J. R., Kilpatrick G. J., Reinscheid R. K., et al. (1997) Orphanin FQ acts as an anxiolytic

to attenuate behavioral responses to stress. Proc Natn Acad Sci USA 94: 14854-14858.

9. Johnson E. 0., Kamilaris T. C., Chrousos G. P., and Gold P. W. (1992) Mechanisms of stress: a dynamic overview

of hormonal and behavioral homeostasis. Neurosci Biobehav Rev 16: 115-130.

10. Lachowicz J. E., Shen Y., Monsma Jr. F. J., and Sibley D. R. (1995) Molecular cloning of a novel G protein-

coupled receptor related to the opiate receptor family. J Neurochem 64: 34-40.

11. Mollereau C. Parmentier M., Mailleux P., Butour J.-L., Moisand C., and Chalon P. (1994) ORL 1, a novel member

of the opioid receptor family - Cloning, functional expression and localization. FEBS Lett 341: 33-38.

12. Meunier J. C., Mollereau C., Toll L. et al. (1995) Isolation and structure of the endogenous agonist of opioid

receptor-like ORL1 receptor. Nature 377: 532-535.

13. Neal C. R. Jr., Mansour A., Reinscheid R., Nothacker H.-P., Civelli 0., and Watson S. J. Jr. (1999) Localization

of Orphanin FQ (Nociceptin) peptide and messenger RNA in the central nervous system of the rat. 3 Comp

Neurol 406: 503-547.

14. Pellow S. and File S. E. (1985) The effects of putative anxiogenic compounds (FG 7142, CGS 8216 and Ro 15-788)

on the rat corticosterone response. Physiol Behav 35: 587-590.

15. Pellow S. and File S. E. (1986) Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated

plus-maze: a novel test of anxiety in the rat. Pharmacol Biochem Behav 24: 525-529.

16. Reinscheid R. K., Nothacker H.-P., Bourson A., Ardati A., et al. (1995) Orphanin FQ: a neuropeptide that activates

an opioidlike G protein-coupled receptor. Sci 270: 792-793.

17. Rodriguez Enchandia E. L., Gonzalez A. S., Cabrera R., and Fracchia L. N. (1988) A further analysis of behavioral

and endocrine effects of unpredictable chronic stress. Physiol Behav 43: 789-795.

18. Roozendaal B. and McGaugh J. L. (1996) Amygdaloid nuclei lesions differentially affect glucocorticoid-

induced memory enhancement in an inhibitory avoidance task. Neurobiol Learn Mem 65: 1-8.

19. Spindel E., Griffith L. Wurtman R. J. (1983) Neuroendocrine effects of caffeine. II. Effects on thyrotropin

and corticosterone secretion. J Pharmacol Exp Ther 225: 346-350.





--top--



Back to the Journal of Undergraduate Research




College of Liberal Arts and Sciences I University Scholars Program I University of Florida I UNIVERSITY of
U L FLORIDA
The Thi~ffh'mf r ( - -k M'rNhf




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EPA07KZEG_ASVOV5 INGEST_TIME 2011-05-31T19:49:41Z PACKAGE UF00091523_00330
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

Journal of Undergraduate Research Volume 6, Issue 7 May June 2005The Effects of Environmental Challenges on Nociceptin/Orphanin FQInduced AnxietyCarrie M. Hersh ABSTRACTNociceptin/orphanin FQ (N/OFQ) is an opioid-related neurotransmitter that is the endogenous ligand for the nociceptin/orphanin FQ peptide (NOP) receptor. This neurotransmitter is released from forebrain neurons during exposure to acute stress and increases circulating concentrations of adrenocorticotrophic hormone and corticosterone in unstressed and mildly-stressed rats after i.c.v. injection. Furthermore, i.c.v. microinjections of N/OFQ increase anxiety-related behavior in rats during neophobic tests of anxiety. However, some studies have reported that N/OFQ is anxiolytic in mice and rats. One possible reason for the differences in findings is that the basal stress levels of the rats may have differed across studies prior to drug administration. We tested this hypothesis by exposing some rats to the stress of restraint and found that the stressed and unstressed N/ OFQ-treated rats exhibited equivalent high expression of anxiety-related behavior.INTRODUCTIONAcute stress arises from physiological and emotional challenges to homeostasis (Herman and Cullinan, 1997), where these challenges cause temporary changes in neurotransmitter release and increases in circulating concentrations of adrenocorticotrophic hormone (ACTH) and corticosterone (CORT). Chronic stress, on the other hand, causes enduring alterations in the regulation of the neurotransmitters and hormones. Stress can be subdivided into two functional types: processive or emotional and systemic or somatic stress (Herman et al., 1997; Rodriguez Enchandia et al., 1998). Processive stressors, appear to be mediated through limbic forebrain inputs to the parvocellular neurons of the paraventricular nucleus (PVN) of the hypothalamus. These stressors do not pose an immediate threat to biological homeostatic systems. These stressors consist of emotionally threatening stimuli (Johnson et al., 1992; Roozendaal et al., 1996). Systemic stressors, on the other hand, represent immediate physiological challenges to an organism, sending direct inputs to the PVN from brainstem regions. Unlike processive stressors, systemic stressors may not require higher brain functioning (Herman et al., 1997). Nociceptive stimulation, immune system threats, exposure to extreme temperatures, and food deprivation are common examples of systemic stressors. During exposure to processive stress, limbic regions of the brain signal neurons of the PVN, initiating a neuronal

PAGE 2

and hormonal cascade involving the limbic hypothalamic-pituitary adrenal (LHPA) axis. Parvocellular neurons of the PVN synthesize and release corticotrophin releasing hormone (CRH) into the hypophyseal portal system. CRH acts on the corticotrophs of the anterior pituitary following stimulation of the limbic system. The corticotrophs then release ACTH into the systemic circulation. ACTH reaches the adrenal glands through the circulation, where it acts on high-affinity Gs-coupled ACTH receptors to synthesize and secrete CORT, the most significant steroid in rodents. In normal functioning organisms, circulating corticosterone then acts on higher brain systems, such as the hippocampus, as a negative feedback system to directly limit the amount of CRF secreted into the anterior pituitary gland, making the LHPA axis a self limiting pathway. Nociceptin/orphanin FQ (N/OFQ) is released from forebrain neurons during exposure to acute stress, implicating endogenous N/OFQ neurotransmission in physiological responses to stress (Devine et al., 2003). N/OFQ is an endogenous 17-amino acid neuropeptide that exhibits amino acid sequence homology with opioid peptide neurotransmitters (Reinscheid et al., 1995; Meunier et al., 1995), indicating close evolutionary homology between the opioid and N/OFQ systems. However, there are important distinctions between these systems. Opioids function by binding to specific opioid receptors in the Central (CNS) and in the Peripheral (PNS) Nervous Systems. N/OFQ lacks classical opioid activity in that it does not bind to the opioid receptors. Instead, it displays high affinity and selectivity for the nociceptin/orphanin FQ peptide (NOP) receptor (Reinscheid et al., 1995; Meunier et al., 1995), which makes this system functionally distinct from the opioid systems. The NOP receptor is a 7-transmembrane Gi-protein coupled receptor. NOP exhibits structural and functional similarities to the opioid receptors but does not selectively bind to the classical opioid ligands (Bunzow et al., 1994; Lachowicz et al., 1995; Mollereau et al., 1994). When activated, it inhibits adenylyl cyclase activity and modulates K+ and Ca2+ conductance (Fukuda et al., 1994; Mollereau et al., 1994). N/OFQ and its receptor are abundantly expressed in limbic brain regions that regulate emotional states (Neal et al., 1999a, 1999b). Hence, N/OFQ may regulate emotional behavior. In support of this hypothesis, it has been found that N/OFQ administration increases hormonal measures of stress (Devine et al., 2001). Nevertheless, there is a controversy regarding the role of N/OFQ in regulation of fear and anxiety. Previous studies have reported that N/OFQ is anxiolytic in mice and rats (Jenck et al., 1997; Gavioli et al., 2002). In contrast, Devine and colleagues found that i.c.v. N/OFQ caused increases in anxiety-related behaviors (Devine et al., 2004) that were similar to those produced by FG 7142, a benzodiazepine receptor inverse agonist, and contrasted with the anxiolytic effects of diazepam. Increased plasma concentrations of ACTH and CORT were observed with each drug treatment during exposure to three tests of anxiety: Open-Field Test, Elevated Plus Maze, and Dark-Light Test. This suggests that during tests of anxiety, administration of N/OFQ, diazepam, and FG 7142 each elevated the responsiveness of the LHPA axis. The dose ranges overlapped in the studies that reported conflicting anxiolytic and anxiogenic actions, indicating that the discrepancy in findings was not due to differences in drug dosages. The contrasting reports of anxiolytic and anxiogenic actions of N/OFQ are perplexing. One factor, which may have contributed to the conflicting results, is the rats basal stress levels prior to neophobic tests. The methods

PAGE 3

used in the earlier reports may have induced psychological stress on tested rodents prior to drug administration. Indeed, the expression of high amounts of anxiety-related behavior in the vehicle-treated rats in those studies (Jenk et al., 1997, Gavioli et al., 2002) suggests that the rats were stressed at the time of anxiety testing (Fernandez et al., 2004). Accordingly, one purpose of the present study was to evaluate whether the recent stress history of the rat affected the rats emotional behavior during drug treatment and neophobic testing. The effects of N/OFQ administration on anxiety-related behavior in rats exposed to the emotional stress of restraint shortly prior to drug administration and testing were evaluated. MATERIALS AND METHODSAnimals Forty-four male Long-Evans rats (Harlan, Indianapolis, IN) were pair-housed in 43 cm x 21.5 cm x 25.5 cm polycarbonate cages. They were kept in a 12hr/12hr light-dark cycle (lights on at 12:00 pm), and with rat chow and tap water available ad libitum. Following 7-days acclimation, each rat was anesthetized with ketamine:xylazine (62.5 mg/kg ketamine, 12.5 mg/kg xylazine) and implanted with a 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 matter). Each cannula was fastened to the skull with dental cement and microscrews. Following surgery, a stainless steel obturator was placed in the guide cannula, only to be removed for drug injection. The obturator extended 1.0 mm beyond the tip of the guide cannula. The rats were then singly housed in the original rat room and allowed 7-days recovery prior to anxiety testing. Apparatus The open field apparatus is an open acrylic box (90cm x 90cm x 59cm) with an attached start box (20cm x 30 cm). The start box is separated from the open arena by means of a guillotine door that was lifted and lowered from outside of the testing room with a rope. A camera suspended over the center of the field was used to videotape the rats. The videotaped images were then scored using a lined grid (divided into 25 equal-sized squares) taped to a television screen. The open field was previously validated to measure behavioral indices of anxiety in rats (Fernandez et al., 2004). Procedures Each rat was handled for five minutes on each of three consecutive days, followed by a day without disturbance. On the fifth day, each of 22 rats was individually placed into a plastic restraint tube with velcro straps for 5 minutes, one half hour prior to administering 1.0 _l of artificial extracellular fluid (aEFC) or 0.10 nmole N/OFQ in 1.0 _l aECF vehicle (n = 11 rats per group). The microinjection was administered through the guide cannula into the lateral ventricle, over 120 sec, using a Harvard syringe pump. Twenty-two rats were not restrained, and these rats were each injected with aECF or 0.10 nmole N/OFQ in the same manner as the restrained rats were (n

PAGE 4

= 11 rats per group). Five minutes after the start of the microinjection, each rat was placed into a start box attached to the open field for one minute to allow for acclimation to a novel environment. At the end of the acclimation period, the door was lifted so that the rat had free access to the open field for five minutes. At the end of the five-minute access to the open field, each rat was returned to its home cage. Then, thirty minutes from the start of drug microinjection the rat was sacrificed by rapid decapitation. Trunk blood was collected and stored into chilled polypropylene tubes on ice, where the tubes contained EDTA. Each tube was centrifuged at 1000 times gravity. The blood was aliquotted, frozen on dry ice, and stored at 80C. The brain of each rat was removed and frozen in 2-methyl butane at -40C, and stored at 80C. Each brain was later sectioned at 30 mm in the coronal plane to verify the cannula placement. Exploration Scores The rats exploratory behaviors were assessed from the videotapes of the open field test sessions. The time spent in the exposed arena of the open field, and in the very exposed central area of the open field (see below), was measured as indices of the anxiety state in each rat. The video image of the open field arena was partitioned into 25 equal-size squares: 16 peripheral or outer zone squares and 9 inner zone squares (Figure 1). Figure 1. Open Field Zone Classifications The open field time and latency to enter the open field were measured during the 300 s session. Entrance into each zone was determined when all four paws entered the area. A latency score of 300 s was assigned for any zone if the rat did not enter that zone. Radioimmunoassay

PAGE 5

Plasma CORT levels were evaluated with a highly specific solid-phase radioimmunoassay (RIA, Diagnostic Products Corp., Los Angeles, CA). Statistical Analyses Between-group differences in open field time, latency to enter the open field, time spent in the inner zone, latency to enter the inner zone, and number of inner zone entries were each evaluated in the restrained and unrestrained groups of rats using t-tests. The circulating CORT concentrations were also evaluated with t-tests. RESULTSThe restrained aECF vehicle-treated group spent significantly less time in the open field (t(20) = 3.146, p<0.01; Figure 2a), exhibited longer latencies to enter the open field (t(20) = 3.540, p<0.01; Figure 2b), spent less time in the inner zone of the open field (t(20) = 2.667, p<0.05; Figure 2c), exhibited longer latencies to enter the inner zone (t(20) = -2.460, p<0.05; Figure 2d), and exhibited fewer entries into the inner zone (t(20) = 2.609, p<0.05; Figure 2e), compared with the behaviors of the unrestrained aECF vehicle-treated controls. The unrestrained N/OFQ-treated group spent significantly less time in the open field (t(20) = 2.604, p<0.05; Figure 2a), exhibited longer latencies to enter the open field (t(20) = 3.390, p<0.01; Figure 2b), spent less time in the inner zone of the open field (t(20) = 2.667, p<0.05; Figure 2c), exhibited longer latencies to enter the inner zone (t(20) = -2.460, p<0.05; Figure 2d), and exhibited fewer entries into the inner zone (t(20) = 2.609, p<0.05; Figure 2e), compared with the behaviors of the unrestrained aECF vehicle-treated controls. The unrestrained N/OFQ-treated group did not significantly differ in open field time (t(20) = 0.09141, p>0.05; Figure 2a) or latency to enter the open field (t(20) = 1.230, p>0.05; Figure 2b), compared with the behaviors of the restrained N/OFQ-treated group. These groups did not enter the inner zone at all. Accordingly, the statistics cannot be computed (due to a complete lack of variance within all groups), but there were no differences between the groups in inner zone time, latency to enter the inner zone, and number of inner zone entries. The restrained vehicle-treated group also did not significantly differ in open field time (t(20) = 0.6422, p>0.05; Figure 2a) or latency to enter the open field (t(20) = 1.312, p>0.05; Figure 2b), compared with the behaviors of the restrained N/OFQ-treated group. These groups did not enter the inner zone at all. Accordingly, the statistics cannot be computed (due to a complete lack of variance within all groups), but there were no differences between the groups in inner zone time, latency to enter the inner zone, and number of inner zone entries.

PAGE 6

Figure 2. The expression of anxiety-related behaviors are depicted as follows: (a) time spent in the open field, (b) latencies to enter the open field, (c) time spent in the inner zone, (d) latencies to enter the inner zone, and (e) inner zone entries (n = 11 rats per group, NR = not restrained, R = restrained). Values expressed are group means the standard error of the mean (SEM). End points of lines indicate which groups were compared. Significant differences between the relevant groups are depicted as follows: *p<0.05, **p<0.01, n.s. = not significant. Plasma CORT concentrations of the unrestrained rats treated with N/OFQ were significantly higher than the CORT concentrations of the unrestrained aECF vehicle-treated controls (t(20) = 2.568, p<0.05; Figure 3).

PAGE 7

The restrained aECF vehicle-treated group did not differ significantly in plasma CORT concentrations (t(20) = 0.2940, p>0.05; Figure 3), compared to the CORT levels of the unrestrained vehicle-controls. The restrained N/ OFQ treated-group exhibited significantly elevated plasma CORT concentrations (t(20) = 2.220, p<0.05; Figure 3), compared to the CORT concentrations of the restrained vehicle-treated group. The unrestrained N/OFQtreated group did not exhibit significant differences in CORT concentrations (t(20) = 0.2281, p>0.05; Figure 3), compared to the CORT levels of the restrained N/OFQ-treated group. Figure 3. Plasma CORT concentrations in the unrestrained rats treated with 0.1 nmole N/OFQ were significantly higher, compared to the CORT levels of the aECF vehicle-treated controls. No significant differences in circulating plasma CORT concentrations were observed between the aECF vehicle controls and restrained aECF vehicle-treated group. There were also no significant differences in CORT levels between the restrained and unrestrained N/OFQ-treated groups. Plasma CORT concentrations in the restrained rats treated with N/OFQ were significantly higher, compared to the CORT concentrations of the restrained aECF-treated group. Values expressed are group means the standard error of the mean (SEM, n = 11 rats per group, NR = not restrained, R = restrained). End points of lines indicate which groups were compared. Significant differences between the relevant groups are depicted as follows: *p<0.05, n.s. = no significant effects.DISCUSSIONThe results demonstrate that the rats treated with N/OFQ, without the stress of restraint, expressed more anxiety-related behaviors, compared to the behaviors of the unrestrained aECF vehicle-treated controls in the open field test. Specifically, the N/OFQ-treated rats exhibited longer latencies to enter the open field, spent significantly less time in the open field and exhibited total avoidance of the inner zone, compared to the behaviors of the unrestrained aECF-treated rats. These behaviors resembled the behaviors of FG 7142treated rats and contrasted with the behaviors that were exhibited by the rats treated with diazepam in a previous report (Fernandez et al., 2004). Restrained rats treated with aECF also exhibited elevations in

PAGE 8

anxiety-related behaviors (i.e., shorter time spent and longer latencies to enter the open field and complete avoidance of the inner zone), compared to the behaviors of the unrestrained aECF-vehicle treated group. So, overall it appears that both restraint and N/OFQ administration both activate increases in expression of anxiety-related behavior. Furthermore, there were no significant differences between the restrained vehicletreated and restrained N/OFQ-treated groups in each behavioral measure. This comparison illustrates that N/ OFQ does not alter the anxiety response to an emotional stressor, so the effect of N/OFQ does not depend on the recent stress history of the animal. Accordingly, N/OFQ increases anxiety in rats that are mildly stressed by the testing environment, but it cannot alter the anxiogenic effect of restraint (Figure 2), and the discrepancy between the recent report by Fernandez et al. (2004) and the previous reports (Jenk et al., 1997; Gavioli et al., 2002) does not seem to arise from differences in the emotional states of the animals at the time of testing. Exposure of the rats to the open field produced moderate elevations in the circulating concentrations of CORT in the unrestrained vehicle-treated rats (Figure 3). These circulating CORT responses to the mild stress of the open field were significantly enhanced by the N/OFQ treatment in the unrestrained rats when these hormonal responses were measured 30 min after the N/OFQ administration. This enhancement of the stress-induced elevations in CORT concur with previous reports that N/OFQ activates the HPA axis in unstressed rats and increases hormonal responses in the presence of a mild stressor to a novel environment (Devine et al., 2001). Accordingly, the anxiogenic actions of N/OFQ administration are accompanied by enhancement of the LHPA axis response to the open field. Nevertheless, the relationship between N/OFQ-induced HPA axis activity and N/OFQ-induced anxiety may not be straightforward. A previous study reported that 1.0 pmole dose of N/ OFQ produced significant anxiety-related behavior in the open field test, but it did not significantly alter the circulating CORT response. This attests that the CORT and anxiety responses are dissociable. Thus it is likely that the anxiogenic actions of N/OFQ arise from central actions of N/OFQ following i.c.v. microinjections, rather than an indirect response involving circulating CORT (Fernandez et al., 2004). An interesting finding was that the restrained aECF vehicle-treated rats did not exhibit significant differences in circulating CORT concentrations, compared to the CORT levels in the unrestrained aECF vehicle-treated controls (Figure 3). The time course of the experiment was designed so that restraint occurred at time 0 (the unrestrained rats sat in their cages during this time), drug injection occurred at time 30, the start of the open field test occurred at time 35, and sacrifice occurred at time 60. One possibility that may account for the moderately low CORT response in the restrained vehicle-treated rats is that the increases in circulating CORT from the stress of restraint at time 0 induced a negative feedback response on the cortical limbic region, the PVN of the hypothalamus, and the pituitary gland, which then diminished the CORT response to the novel environment exposure during anxiety testing. In contrast, the rats that were treated with N/OFQ and restrained exhibited highly elevated plasma CORT concentrations, suggesting that N/OFQ exerts direct activating effects on HPA axis regulation and/or interfered with the negative feedback effect of the restraint on the subsequent stressful exposure to a novel environment these restrained N/OFQ-treated rats did not exhibit the blunted CORT responses observed in the aECF-treated group.

PAGE 9

There were no significant differences in the CORT response, measured 60 min following restraint (30 min following drug administration), between the restrained and unrestrained N/OFQ-treated rats (Figure 3). This comparison indicates that N/OFQ enhancement of the LHPA axis does not alter the CORT response in moderately stressed rats prior to N/OFQ administration. A previous study reported similar findings in the hormonal data, demonstrating that N/OFQ exaggerates and prolongs the ACTH/CORT response to a mild stressor, but it cannot alter the LHPA axis activity in the presence of a restraint stress (Devine et al., 2001). The current findings indicate that both the behavioral and hormonal effects of N/OFQ administration resemble the effects of FG 7142 (Pellow and File, 1985, 1986) and caffeine (Spindel et al., 1983; Pellow et al., 1985). All of these drugs are anxiogenic in neophobic tests of anxiety, and all of these drugs elevate circulating ACTH and/ or CORT following acute administration. In this experiment we tested the possibility that the anxiety-modulating effects of N/OFQ may differ depending on the basal state or emotional tone of the rat, a possibility that could reconcile the contradictory findings of Fernandez et al. (2004) and Jenk et al. (1997). However, the behavioral and hormonal results of the study suggest that the initial emotional status of the rats prior to drug administration did not affect the anxiogenic effects of N/OFQ. The reason for the contradictory results remains unexplained. REFERENCES1. Bunzow J. R., Saez C., Mortrud M., Bouvier C., Williams J. T., and Low M. (1994) Molecular Cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a _, _, or _ opioid receptor type. FEBS Lett 347: 284-288. 2. Devine D. P., Hoversten M.T., Ueda Y., and Akil H. (2003) Nociceptin/Orphanin FQ content is decreased in forebrain neurons during acute stress. J Neuroendocrinol 15: 69-74. 3. Devine D. P., Watson S. J., and Akil H. (2001) Nociceptin/Orphanin FQ regulates neuroendocrine function of the limbic-hypothalamic-pituitary-adrenal axis. Neurosci 102: 541-553. 4. Fernandez F., Misilmeri M.A., Felger J. C., and Devine D. P. (2004) Nociceptin/Orphanin FQ increases anxietyrelated behavior and circulating levels of corticosterone during neophobic tests of anxiety. Neuropsychopharmocol 29: 59-71. 5. Fukuda K., Kato S., Mori K., Nishi M., Takeshima H. et al. (1994) cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett 343: 42-46. 6. Gavioli E. C., Rae G. A., Calo G., Guerrini R., and De Lima T. C. (2002) Central injections of nocistatin or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test. Br J Pharmacol 136: 764-772. 7. Herman J. P. and Cullinan W. E. (1997) Neurocircuitry of stress: Central control of the hypothalamo-

PAGE 10

pituitary-adrenocortical axis. Trends Neurosci 20: 78-84. 8. Jenk F., Moreau J.-L., Martin J. R., Kilpatrick G. J., Reinscheid R. K., et al. (1997) Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natn Acad Sci USA 94: 14854-14858. 9. Johnson E. O., Kamilaris T. C., Chrousos G. P., and Gold P. W. (1992) Mechanisms of stress: a dynamic overview of hormonal and behavioral homeostasis. Neurosci Biobehav Rev 16: 115-130. 10. Lachowicz J. E., Shen Y., Monsma Jr. F. J., and Sibley D. R. (1995) Molecular cloning of a novel G proteincoupled receptor related to the opiate receptor family. J Neurochem 64: 34-40. 11. Mollereau C. Parmentier M., Mailleux P., Butour J.-L., Moisand C., and Chalon P. (1994) ORL 1, a novel member of the opioid receptor family Cloning, functional expression and localization. FEBS Lett 341: 33-38. 12. Meunier J. C., Mollereau C., Toll L. et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377: 532-535. 13. Neal C. R. Jr., Mansour A., Reinscheid R., Nothacker H.-P., Civelli O., and Watson S. J. Jr. (1999) Localization of Orphanin FQ (Nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol 406: 503-547. 14. Pellow S. and File S. E. (1985) The effects of putative anxiogenic compounds (FG 7142, CGS 8216 and Ro 15-788) on the rat corticosterone response. Physiol Behav 35: 587-590. 15. Pellow S. and File S. E. (1986) Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol Biochem Behav 24: 525-529. 16. Reinscheid R. K., Nothacker H.-P., Bourson A., Ardati A., et al. (1995) Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Sci 270: 792-793. 17. Rodriguez Enchandia E. L., Gonzalez A. S., Cabrera R., and Fracchia L. N. (1988) A further analysis of behavioral and endocrine effects of unpredictable chronic stress. Physiol Behav 43: 789-795. 18. Roozendaal B. and McGaugh J. L. (1996) Amygdaloid nuclei lesions differentially affect glucocorticoidinduced memory enhancement in an inhibitory avoidance task. Neurobiol Learn Mem 65: 1-8. 19. Spindel E., Griffith L. Wurtman R. J. (1983) Neuroendocrine effects of caffeine. II. Effects on thyrotropin and corticosterone secretion. J Pharmacol Exp Ther 225: 346-350. --top-Back to the Journal of Undergraduate Research College of Liberal Arts and Sciences | University Scholars Program | University of Florida |

PAGE 11

University of Florida, Gainesville, FL 32611; (352) 846-2032.