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N/OFQ-Mediated Anxiety: Role of the Bed Nucleus of Stria Terminalis

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N/OFQ-Mediated Anxiety: Role of the Bed Nucleus of Stria Terminalis
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Barbieri, Emily
Devine, Darragh ( Mentor )
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Gainesville, Fla.
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University of Florida
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N/OFQ-Mediated Anxiety: Role of the Bed Nucleus of Stria Terminalis

E. V. Barbieri


ABSTRACT


Nociceptin/Orphanin FQ (N/OFQ) has been shown to have a role in stress and anxiety. Intracerebroventricular

(ICV) injections of N/OFQ increase circulating corticosterone (CORT) levels in unstressed rats and prolong

CORT elevations induced by a mild stressor. This effect is mediated by limbic inputs. Unstressed injections of N/

OFQ into the bed nucleus of stria terminalis (BNST), amygdala, and septum, for example, produce elevations

in circulating CORT. ICV injections of N/OFQ also produce anxiogenic behavioral effects in neophobic tests of

anxiety. Rats injected with N/OFQ show longer latencies to enter open or lit areas and decreased total time spent

in open/lit areas. We examined whether these anxiogenic behavioral effects are also limbic-mediated. Therefore,

in the present experiment, we have examined the effects N/OFQ injections into one limbic structure, the BNST.



Twenty-eight male Long Evans rats were implanted with guide cannulae into the right BNST. Each rat received

a microinjection of N/OFQ (0, 0.01, 0.1, or 1.0 nmole). Following injections, the rats were placed in the open field,

a neophobic test of anxiety, and given 5 minutes to freely explore. Latency to enter, total time spent in the open

field and the inner zone, and number of entries into the open field and the inner zone were used as measures

of anxiety. Plasma was collected for analysis of circulating CORT.



N/OFQ injections into the BNST produced increases in anxiety-related behaviors and circulating CORT

concentrations in rats, particularly with the 1.0 nmole dose. However, injections into the BNST were not as potent

as injections into the lateral ventricle. This may be because the BNST is not a primary site of action for the

anxiety-related behavioral effects of N/OFQ. Alternatively, ICV injections may be reaching multiple limbic,

cortical, and brainstem sites that could all be contributing to these effects.



INTRODUCTION


Nociceptin/Orphanin FQ (N/OFQ) is a 17-amino acid peptide related to the family of opioid

neurotransmittersl,. However, N/OFQ does not bind with high affinity to the p, 6, or K-Opioid receptor types. It

does bind saturably and with high affinity to the NOP receptor3, a member of the superfamily of

seven transmembrane4, Gi protein-coupled receptorss5. The NOP receptor is negatively linked to adenylate

cyclase, inhibits N-type Ca2+ channels, and activates inward rectifying K+ channels6. While the NOP receptor has




high amino acid sequence homology with cloned opioid receptor types, it does not selectively bind prototypical

opioid agonists or antagonists7. Therefore, the N/OFQ-NOP system appears to be functionally distinct from the

opioid systems.



N/OFQ, the NOP receptor, and their mRNAs are ubiquitously expressed in the mammalian brain and spinal

cord, consistent with a wide range of functions including pain modulation1, 2, 8, feeding9, and locomotion10. N/

OFQ, NOP, and their mRNAs are relatively abundant within limbic structures11,12. This expression in the limbic

system suggests that N/OFQ may play a role in the processing of emotion. In fact, acute stress exposure

decreases the content of NOF/Q in forebrain neurons, implicating endogenous neurotransmission in

physiological stress responses13. Additionally, intracranial injections of N/OFQ increase anxiety-related behaviors

and hypothalamic-pituitary-adrenal (HPA) axis activity14, 15



In order to study anxiety, tests of neophobia are used. One such test is the open field test. This test takes

advantage of two characteristics of rats. Rats are foraging animals and thus tend to explore novel

environments. However, they are also a prey species and so tend to avoid open, brightly-lit areas where they

are more vulnerable. When placed in the open field rats exhibit thigmotaxis, or exploration restricted to

the periphery16. These behaviors are altered by drugs humans describe as anxiolytic (decreasing anxiety)

or anxiogenic (increasing anxiety). Drugs reported as being anxiolytic, such as diazepam, increase exploration of

the central region of the open field while drugs reported as being anxiogenic, such as FG-7142, decrease

exploration of the central region of the open field5s. We use a modified open field, in which a start box is attached

to one wall of the open field apparatus. This allows us to make additional measures such as latency to enter the

open field, time spent in the open field, and number of entries into the open field. Intracerebroventricular

(ICV) administration of N/OFQ decreases exploration behaviors in this modified open field test. These results

are similar to those produced following injections of FG-7142 suggesting that N/OFQ has anxiogenic actions

following ICV injections16.



ICV injections of N/OFQ also produce increases in plasma corticosterone (CORT) levels in unstressed rats and

prolong these stress-induced elevations of CORT after exposure to an acute stressor14. These elevations are at

least partly mediated by limbic inputs to the paraventricular nucleus of the hypothalamus (PVN). Unstressed N/

OFQ injections into the bed nucleus of stria terminalis (BNST), septum, and amygdala each produce increases

in circulating CORT17,18.



The purpose of this experiment was to determine if the anxiety-related behavioral effects of N/OFQ are also

mediated by limbic structures. Specifically, we examined the effect of N/OFQ on anxiety-related behaviors when it

is injected into the BNST, a structure that is known to participate in the regulation of responses to fear-

inducing stimuli19.



ICV injections of N/OFQ also produce increases in plasma corticosterone (CORT) levels in unstressed rats and

prolong these stress-induced elevations of CORT after exposure to an acute stressor14. These elevations are at





least partly mediated by limbic inputs to the paraventricular nucleus of the hypothalamus (PVN). Unstressed N/

OFQ injections into the bed nucleus of stria terminalis (BNST), septum, and amygdala each produce increases

in circulating CORT17,18.



The purpose of this experiment was to determine if the anxiety-related behavioral effects of N/OFQ are also

mediated by limbic structures. Specifically, we examined the effect of N/OFQ on anxiety-related behaviors when it

is injected into the BNST, a structure that is known to participate in the regulation of responses to fear-

inducing stimuli19.



METHODS


Animals


Thirty-four male Long Evans rats (Harlan, Indianapolis, IN) were pair-housed for a one-week acclimation period

in polycarbonate cages with dimensions 43 cm x 21.5 cm x 25.5 cm. After the acclimation period, each rat

was surgically implanted with a cannula terminating 1.0 mm above the BNST. Then, the rats were singly housed

for the remainder of the experiment. Food and water were supplied ad libitum. The rats were on a 12hr-12 hr

light-dark cycle with lights on at 7:00 am. The temperature of the climate-controlled vivarium was kept between

21-23 OC with humidity between 55-60%.



Drugs


Ketamine, xylazine, ketorolac tromethamine, and AErrane (99.9% isoflurane) were used for surgery and

were purchased from Henry Schein (Melville, NY). N/OFQ was purchased from Sigma-Aldrich (St. Louis, MO).



N/OFQ was dissolved in artificial extracellular fluid (aECF) composed of 2.0 mM Sorenson's phosphate buffer

(pH 7.4): 145 mM Na+, 2.7 mM K+, 1.0 mM Mg2+, 1.2 mM Ca2+, 150 mM Cl-, and 0.2 mM ascorbate. These

ion concentrations replicate the ion concentrations found in the brain's extracellular fluids20. N/OFQ

concentrations were prepared at 0.01, 0.1, and 1 nmole per 0.5 pL of aECF.



Surgery


Surgery was conducted under ketamine/xylazine anesthesia (62.5 mg/kg ketamine and 12.5 mg/kg xylazine, i.p. in

a volume of 0.75 mL/kg). AErrane was used as supplemental anesthesia as needed. The analgesic,

ketorolac tromethamine (2 mg/kg), was injected subcutaneously at the time of surgery.



Twenty-eight rats were each implanted with a 11mm, 22 gauge stainless steel guide cannula into the right BNST

(0.3 mm posterior to bregma, 3.0 mm lateral from midline, 5.4 mm ventral from dura, 140 angle). Six additional

rats served as anatomical controls and were implanted with cannulae aimed at extra-BNST sites. The cannulae

were secured with dental cement anchored to the skull with stainless steel screws (0.8 x 3/32"). After





surgery, obturators 1.2 mm longer than the cannula tips were inserted and remained in the cannulae until the day

of behavioral testing. The rats were given approximately one week to recover from surgery.



Equipment


The open field was made of black acrylic with a 90 x 90 x 60 cm field and a 20 x 30 x 60 cm start box (see figure

1). A guillotine door separated the start box from the open field. A rope and pulley system enabled the

experimenter to open the guillotine door from outside the testing room. Illumination of the start box and the

open field were approximately equal (14-30 lux). A camera was mounted above the open field for recording the

rats' behaviors.



Videos of the open field behaviors were reviewed and the experimenter (blind to treatment) scored the

exploratory behaviors of each rat. To score these behaviors, a grid of 25 squares was superimposed on the

video monitor dividing the open field into the outer zone and the inner zone (see Figure 1). The outer zone

was defined as the 16 squares along the perimeter of the field while the inner zone was defined as the central

9 squares. Latency to enter the open field, latency to enter the inner zone, number of open field entries, number

of inner zone entries, total time in the open field, and total time in the inner zone were used as measures of

anxiety. Movement from the start box into the open field or from one zone into another was counted when all

four paws left one zone and entered another zone (defined by the lines of the grid).


Oute zone



In er
Zone


Figure 1. Photograph of the open field (left) and diagram of the open field zones depicting the

overlying grid (right). The open field is composed of a large open field which is divided into an inner

zone and a peripheral, outer zone. Attached to the open field is a start box. The rats are

individually placed into the start box and, after a one-minute acclimation period, are given 5 minutes

to freely explore the open field.


Experimental Procedure





Beginning 7-10 days after surgery, each rat was handled for 5 minutes on each of 3 days, then given one day with

no disturbance. On the 5th day, each rat was injected with a 0.0, 0.01, 0.1, or 1 nmole dose of N/OFQ dissolved

in aECF (note: none of the anatomical controls received the 0.0 nmole dose). At the time of the injections,

an experimenter who was blind to the doses prepared a 28-gauge stainless steel injector connected by

polyethylene tubing (PE20) to a 1 microliter Hamilton syringe in a syringe pump. The injector was then inserted

into the guide cannula, individually. The injection was administered over 2 minutes using the syringe pump.

The injector tip was then left in place for an additional 3 minutes to allow diffusion. The rats were freely moving

in their cages during the 5 minute period. The injections were carried out 90-210 minutes after lights

on, corresponding to the time when the HPA axis is at its daily nadir21,22.



Upon completion of the injection, each rat was individually placed in the start box of the open field and the door

to the testing room was closed. The rat was given a 1 minute acclimation period and then the guillotine door

was opened and remained open throughout the test. The rat was given 5 minutes to explore the start box and

the open field. These 5 minutes of activity were recorded and then the animal was returned to his cage.



Each rat was rapidly decapitated 30 minutes after the start of the injection. Following decapitation, 6 mL of

trunk blood was collected into polypropylene tubes containing 600 pL of chilled Na2EDTA at 20 pg/pL. The blood

was centrifuged at 1000x gravity at 40C allowing the extraction of the plasma (stored at -800C until

use). Radioimmunoassay was conducted to quantify plasma CORT concentrations using a kit (Diagnostic

Products Corp. Los Angeles, CA).



The spleen, adrenal glands, and thymus glands were extracted (stored at -800C until use) and weighed

for verification of the health status of each rat. The brain was removed and flash frozen at -400C, then frozen at -

800C. The brains were sectioned at 30 pm and then stained with cresyl violet to verify cannula placement.



Scoring and Statistics

Differences between BNST-implanted groups receiving aECF or N/OFQ were analyzed by one-way ANOVAs for

latency to enter the open field and inner zone, number of entries into the open field and inner zone, total time

spent in the open field and inner zone, plasma CORT concentrations, and organ masses. All significant effects

were further analyzed using Newman-Keuls post-tests. Differences between the anatomical controls and the

intra-BNST aECF groups were analyzed by t-tests for each dependent measure.



RESULTS


The N/OFQ-treated rats displayed more anxiety-related behaviors than did the vehicle-treated rats. These N/

OFQ-treated rats displayed longer latencies to enter the open field (Fig. 2a; F(3,24) = 4.009, p<0.05) than did

the vehicle-treated rats. The N/OFQ-treated rats displayed a decrease in the number of entries into the open

field (Fig. 2b; F(3,24)= 3.005, p<0.05) as compared to the vehicle-treated rats' entries. The N/OFQ-treated rats





did not display a decrease in total time spent in the open field (Fig. 2c; F(3,24)= 2.075, p>0.05) as compared to

the time spent by the vehicle-treated rats; however, there were behavioral trends in the same direction as the

other measures, specifically the rats in the 1.0 nmole group spent less time in the open field.



The N/OFQ-treated rats displayed fewer entries into the inner zone (Fig. 2e; F(3,24)= 3.321, p<0.05) than did

the vehicle-treated rats. The N/OFQ-treated rats did not display significant differences in latency to enter the

inner zone (Fig. 2d; F(3,24)= 2.593, p>0.05) or total time in the inner zone (Fig. 2f; F(3,24)= 2.669, p>0.05)

when compared with the latencies and times of the vehicle-treated rats. However, the results for latency to

enter inner zone and total time in inner zone approached significance (p = 0.07). Furthermore the behavioral

trends for these measures were consistent with other measures: the inmole group displayed increased

anxiety-related behaviors.


Latency to Enter Open Field


3 *i |


20


. R _ ... _ . .. . -... . . .


0.0 0.01 0.1 1.0 AC
NIOFO dose rnmoles)
2b
Number of Entries into Open Field
6-








0 00 01 01 10 AC
NOF;Q dose (nmoles)
2c Total Time Spent In Open Field


0.0 0.01 01 1.0 AC
NOF/Q dose Inmoles)


Latency to Enter Inner Zone









0.0 &001 01 1.0 AC
NIOFQ dose ( romols)

Number of Entries into Inner Zone


O00 001 0 1 10 AC
NOFlQ dose (namoies
2f Total Time in Inner Zone


10






o,


0.0 0 dos01 01 1.0
N1OFQ dose rnnotet)


Figure 2. Anxiety-related behaviors following intra-BNST injections of N/OFQ. Administration of N/

OFQ (a) increased latency to enter the open field, (b) decreased number of entries into the open

field, and (d) increased latency to enter the inner zone. However, N/OFQ did not significantly alter

(c) the total time spent in the open field,, (e) the number of entries into the inner zone, or (f) the


200-

100-

0


T
i20fr


100
B1v


- 69-J6


VsQ 16


i M 6 _V_






total time spent in the inner zone. Values expressed are group means � SEM (n =6-8 rats per group).

AC = anatomical controls.



The N/OFQ-treated rats displayed elevated circulating CORT levels (Fig. 3; F(3,24)= 3.696, p<0.05) as compared

to the vehicle-treated rats.





Levels of Circulating Corticosterone





B00-




00 001 01 10 AC



Figure 3. Concentrations of circulating CORT following intra-BNST injections of N/OFQ. N/

OFQ administration increased levels of circulating CORT in a relatively dose-dependent manner.

Values expressed are group means � SEM (n =6-8 rats per group). AC = anatomical controls.



There were no significant differences between groups in thymus weights (Fig. 4a; F(4,29)= 0.5992, p>0.05),

spleen weights (Fig. 4b; F(4,29)= 0.7459, p>0.05), or adrenal weights (Fig. 4c; F(3,24)= 1.276, p>0.05).

Additionally, there were no significant differences between the anatomical control group and the vehicle-

treated group on any measure of latency (Fig. 2a and 2d, t(10) = 1.297, p > 0.05 for open field latency and t

(10) =.7077, p > 0.05 for inner zone latency), entries (Fig. 2b & 2e; t(10) = 1.077, p > 0.05 for open field

entries and t(10) = 1.357, p > 0.05 for inner zone entries), time (Fig. 2c & 2f; t(10) = 0.1273, p > 0.05 for

open field time and t(10) = 0.8485, p > 0.05 for inner zone time) or CORT (Fig. 3, t(10) = 1.246, p > 0.05).



DISCUSSION


N/OFQ injections into the BNST cause increases in anxiety-related behaviors and circulating CORT in rats.

However, injections into the lateral ventricle appear to produce more potent results, with statistically

significant effects at doses as low as 0.001nmoles16. This difference suggests that the BNST may not be a

primary site of action for the anxiogenic behavioral effects of N/OFQ. Another possible explanation is that

ICV injections diffuse to multiple sites, potentially producing an additive or synergistic effect with multiple

limbic, cortical, and brainstem structures. For example, we know that injections into the amygdala also

produce increases in anxiety-related behaviors23. Therefore, it may be that ICV injections are having an effect at

the BNST and the amygdala, as well as other potential sites such as the septum, the cortex, the hypothalamus,

and numerous brainstem nuclei.










4a Thymus weights


3 -






o.0 0.01 0o1 '1. AC 0.0 o.01 .1 1'0 AC
NOFfR dose (nmIles) NOFIQ dose (nmoles)

4c Adrenal Weights









10.





0.0 0. 01 .1 1.0 AC 0. 0. 1 1.0 AC
Left AdrenalB Right Adrenarh
NOF/Q domes (nmoles)



Figure 4. Analysis of organ masses. Thymus (a), spleen (b), and adrenal (c) weights were

approximately equal across groups, suggesting that there were no group differences in health or

chronic stress exposure that may have affected the behavioral or hormonal responses of the rats.

Values expressed are group means � SEM (n =6-8 rats per group). AC = anatomical controls.



The results of this study are consistent with previous findings that have shown that N/OFQ is anxiogenic15 and that

it causes increases in circulating CORT following ICV14,15 and intra-limbic injections17. This experiment extends

the behavioral findings adding further support that there is limbic involvement in the anxiogenic effects of N/

OFQ. Additionally, we have shown that injections of N/OFQ into the BNST produce further increases in

circulating CORT above those caused by exposure to stressful situations (handling and open field exposure).

Previous work was done with unstressed injections into the BNST, whereby the rats were allowed to recover from

the stress of handling prior to receiving the injection of N/OFQ17. These rats displayed elevations in circulating

CORT above resting baselines. In the current study, we have shown that handling and exposure to the open field

is stressful (CORT is elevated in the vehicle-treated rats) and that N/OFQ produces further elevations in CORT

under these conditions.



Organ masses were measured in order to determine if the animals had been exposed to chronic stress. The


4b Spleen weights





thymus and spleen decrease in size and the adrenals increase in size after exposure to chronic physiological

and psychological stressors. Because there were no differences in size, we can conclude that there were

no differences in the health and/or amounts of chronic stress exposure that may have contributed to the

anxiogenic and HPA axis-activating actions of N/OFQ after administration into the BNST. We are currently

examining the role of the N/OFQ-NOP system in emotional responses by examining gene expression in

various cortical, limbic and brainstem following single and repeated stress exposure.






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