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The preeminent role of corticotropin-releasing factor
(CRF) in mediating the neuroendocrine, immune, autonomic, and behavioral
responses to stress has been clearly established. Therefore it has
been proposed that a CRF receptor antagonist may demonstrate anxiolytic
and/or antidepressant like activity. We tested the small molecule
CRF1 receptor antagonist R121919 in three classic behavioral models,
Open Field (OF), Elevated Plus Maze (EPM), and Restraint-Stress
Defensive Withdrawal (DW). Forty male Sprague-Dawley rats
(200-300g) were used in each study. Four dose concentrations,
1mg/kg, 3.16 mg/kg, 10mg/kg, 31.6mg/kg, and a vehicle of acetic
acid were delivered during each experiment (n=8). Chosen variables
of import in OF were time spent in the center and exploratory activity
as expressed in distance moved. Variables of import in EPM
were time spent in the open versus closed arms and corticosterone
levels directly subsequent to testing. In DW, significant variables
were time spent in the tube (hidden zone) versus the field and distance
moved. The trials were scored via Ethovision (a computerized
tracking system) and the data analyzed by one and two-way analyses
of variance (ANOVA) as appropriate. Our OF results indicate
that R121919 effectively increased exploratory behavior, but showed
no efficacy on time spent in the center. There was no behavioral
effect of R121919 in the EPM experiment. However, R121919 did dose
dependently reduce corticosterone levels following exposure to the
EPM. In DW, R121919 significantly attenuated the decrease in exploration
seen following restraint stress.
Corticotropin-releasing factor (CRF), an endogenous
peptide, is the primary mediator of the neuroendocrine response
to environmental stressors. An abundance of clinical and preclinical
data has also shown that CRF mediates the behavioral, autonomic,
and immune responses to stress. CRF is synthesized in the paraventricular
nucleus of the hypothalamus (PVN), is secreted into the median eminence,
then travels into the pituitary gland to activate CRF1 receptors.
This then promotes the release of adrenocorticotropin hormone (ACTH)
into the blood stream. ACTH in turn stimulates the secretion of
glucocorticoids (GCs) from the adrenal cortex. GCs help mobilize
energy needed to respond to stressors and they later shut off the
stress response by inhibiting further HPA axis activity. Studies
have shown that a large number of patients with depression and anxiety
disorders have HPA axis dysfunction and/or chronic hypersecretion
of CRF. Accordingly, it is believed that CRF1 antagonists may represent
novel treatments for depression and anxiety related disorders. This
study examines the effects of a novel, small-molecule CRF1 antagonist,
R121919, as tested in the Open Field (OF), Restraint-stress Defensive
Withdrawal (DW), and Elevated Plus Maze (EPM) behavior paradigms.
Forty male Sprague Dawley rats (200-300g) served as
research subjects. We utilized an acetic acid vehicle, and doses
consisted of 1, 3.16, 10, and 31.6mg/kg of the CRF1 receptor antagonist
R121919. Open Field (OF) Two arenas were adjacently situated in
the testing room. Lighting was maintained at approx. 600 lux. Each
cage of animals was injected 60 min. prior to experimentation. Elevated
Plus Maze (EPM) A plus maze, elevated 73cm above ground, was placed
into the testing room.
On testing days we injected pairs of animals approx.
60 min. prior to placement into the arena. At testing, each animal
was positioned at the junction of the open and closed arms, held
for 3 seconds, and then released into the maze for 10 minutes. Animals
that fell off the open arm were replaced immediately. At trial expiration
each rat was promptly tail nicked and 100ul of blood was collected
for a corticosterone assay. Restraint-Stress Defensive Withdrawal
(DW) On Day 1 of DW the animals received no drug. Each was guided
into a black tube which was then placed near and parallel to one
wall of the open field. Trial duration was 15min. This process was
repeated days 2 and 3 to eradicate environment novelty. On Day 4,
the standard 5 doses were delivered on a variable concentration
schedule. Sixty min. after dosage, pairs were restrained for 10min.
in a plastic restraint cone. Directly after release they were placed
into DW arenas for 15min.
R121919 showed some efficacy in the OF experiment.
The drug dose dependently increased exploratory behavior expressed
as distance moved [Figure
5, Figure 6]
This locomotion was not exaggerated as in the presence of stimulants.
R121919 did not increase time in the center zone, typically interpreted
as the most aversive portion of the field [Figure
4] Surprisingly, R121919 showed no anxiolytic effect on behavior
in the EPM paradigm [Figure
7, Figure 9].
However, our results show a dose-dependent decrease in levels of
corticosterone, indicating that our drug can block stress-induced
changes in corticosterone [Figure
8] R121919 showed behavioral efficacy in the combined restraint-stress
DW challenge [Figure
3], similar to our previous findings using only 1 day of DW
[Figure 1, Figure
2].
This experiment highlights the fact that several different behavioral
assays are needed in order to establish the anxiolytic properties
of novel psychotherapeutics.
Funded by the STC Program and the Center for Behavioral Neuroscience
of the National Science Foundation under Agreement No. IBN-9876754
The aim of my project was to test the effects of a
psychotherapeutic drug called R121919 in several animal models of
behavior. The treatment is an antagonist (blocks the binding action)
for the CRF1 receptor, and if its action is successful, R121919
will have anxiety relieving and antidepressents effects in the central
nervous system (CNS) of patients with anxiety and affective disorders
(i.e. panic attacks, PTSD, major depression). A vehicle (no drug)
and four dose concentrations were issued during experiments. Our
subjects were forty male rats. We tested the drug in the arenas
of Open Field (large box), Elevated Plus Maze (cross with one walled,
one open arm), and Restraint-Stress Defensive Withdrawal (restrain
animal, put inside a tube then into a large box. If the rats entered
the center of OF versus the walls, they were less anxious. If they
entered the open versus closed arms of EPM they were less anxious.
If our animals spent more time in the field verus the tube in DW
their anxiety was reduced. We also examined distance moved as a
sign of exploration. R121919 showed some efficacy in the OF test.
Center time wasn't increased, but there was more exploration with
higher drug doses. It showed no behavioral effect in EPM, but there
was promising hormonal change. R121919 worked effectively in DW
as the rats spent less time in the tube with increasing drug dose,
and there was an expected hormonal change. It is important to test
new drugs in several behavioral situations to prove they work well.
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