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Graduate
Category: Physical and Life Sciences Degree Level: Ph.D
Abstract ID# 347
Upstream Dopamine D2 Receptors Modulate
Anabolic/Androgenic Steroid-Induced Aggression
T.R. Morrison, L.A. Ricci, R.H. Melloni Jr. -- Northeastern University -- Behavioral Neuroscience Program -- Boston, MA
Methods
Subjects
Male Syrian hamsters (Mesocricetus auratus) were obtained from Charles River Laboratories (Wilmington,
Massachusetts, USA), individually housed in polycarbonate cages, and maintained at ambient room temperature
on a reverse light/dark cycle (14:10, lights off at 08:00h), with free access to food and water. For aggression testing,
stimulus (intruder) males of equal size and weight to the experimental animals were obtained 1 week before the
behavioral test, group-housed (five animals per cage) in large polycarbonate cages, and maintained as above.
Experimental treatment
From postnatal (P) day 27, Syrian hamsters (n=79) were weighed and received daily subcutaneous injections (0.1–
0.2 ml) of an AAS mixture consisting of 2mg/kg testosterone cypionate, 2mg/kg nandrolone decanoate, and 1 mg/
kg boldenone undecylenate dissolved in sesame oil, for 30 consecutive days during adolescent development (P27–
P56).
On the day following the last injection (P57), AAS- treated hamsters were randomly assigned to one of three
treatment groups (n = 12/group) and tested for offensive aggression after two microinjections into the LAH, with an
interval of 5-10 min between each injection. The drug regimens were as follows: D2 antagonist Eticlopride (.5 mM)
and saline (NaCl; 0.9%) (Etic+NaCl); Eticlopride and AVP (0.09 μM) (Etic+AVP); Eticlopride and Quinpirole (1 nM)
(Etic+Quin); and NaCl followed by NaCl (NaCl+NaCl). The doses of Eticlopride and AVP were selected based on
previous reports of efficacy in suppressing and enhancing aggressive behaviors, respectively (Schwartzer et al.,
2010 and Ferris et al. 1997).
Surgical procedures
One week before aggression testing (P50), animals were anesthetized with Ketamine/Xylazine (80 mg/kg:12mg/kg)
and placed into a stereotaxic device for unilateral implantation of a 26 gauge guide cannula aimed at the LAH. A
small hole was drilled into the skull at the coordinate position necessary to gain access to the LAH, (i.e. 0.2 mm
posterior to Bregma, 1.2mm lateral to the midsagittal suture, 7.0 mm ventral from dura). The cannula was lowered
and anchored to the skull using dental screws and acrylic, and the head wound sutured closed.
Microinjections and Behavioral testing
Two 1μL Hamilton syringes were used for the injection of .5 μL of each drug over the course of 2 min into the LAH
and left in position for an additional minute to allow for drug diffusion. After the last injection, animals were returned
to their home cage for 10 min before behavioral testing. Hamsters were tested for offensive aggression using the
resident-intruder paradigm. The composite aggression score was used as a general measure of offensive
aggression, and is defined as the total number of attacks and bites during the 10 min behavioral test. Residents
were also measured for social interest toward intruders (i.e. total contact time [TCT]), stereotypy (i.e., grooming),
and locomotion (i.e., wall climbing). Following behavioral testing, brains were removed and histologically processed
to locate the site of cannula placement in the LAH.
0
SO
AAS
40
ab*
4
2
0
SO
10
a**
5
0
AAS
SO
Etic+AVP
12
10
8
6
4
2
0
SO
15
ab***
20
15
10
5
0
AAS
6
4
a**
2
0
AAS
Etic+AVP
25
Etic+NaCl
8
5
Etic+AVP
8
ab*
6
4
2
0
Etic+NaCl
3
2
a*
1
0
SO
AAS
Etic+AVP
5
a***
b*
4
3
2
1
0
SO
AAS
SO
Results
4
AAS
SO
AAS
SO
Frequency (Mean + SEM)
6
20
Frequency (Mean + SEM)
8
AAS
10
0
SO
AAS
Etic+Quin
40
ab***
30
12
10
8
6
***
aa***
4
2
0
SO
AAS
Etic+Quin
20
15
10
5
0
10
mSON/NC
0
DA
AVP
GABA
AVP
Eticlopride
DA
DA D2
GABA
GABAA
AVP
AVP
receptor
SO
AAS
ab***
6
4
2
0
AAS
SO
Model
non-AAS
AAS
a*
8
Etic+Quin
5
ab***
4
3
2
1
0
SO
AAS
Dopamine D2 receptor blockade in the presence and absence of AVP and Quinpirole
within the LAH differentially affect aggressive behaviors after adolescent AAS exposure.
Bars denote SEM; a denotes significant difference from respective Saline control, b
denotes significant difference from SO control *p<0.05, **p<0.01,***p<0.001.
20
SO
ab***
25
Etic+Quin
Frequency (Mean + SEM)
20
Etic+Quin
Frequency (Mean + SEM)
30
Frequency (Mean + SEM)
Frequency (Mean + SEM)
Etic+AVP
10
25
Etic+NaCl
Frequency (Mean + SEM)
10
Etic+NaCl
Frequency (Mean + SEM)
a **
12
Frequency (Mean + SEM)
20
Frequency (Mean + SEM)
30
Frequency (Mean + SEM)
40
Frequency (Mean + SEM)
Frequency (Mean + SEM)
Etic+NaCl
Frequency (Mean + SEM)
Anabolic/androgenic steroid (AAS) use by adolescent teens is associated with a higher
incidence of aggression and violence, yet little is known about how these drugs produce
the aggressive phenotype. In previous studies, we have shown that male Syrian hamsters
chronically exposed to AAS during the developmental time period that parallels human
adolescence are highly aggressive in early adulthood (see Melloni & Ricci, 2010). This
increase in aggressive responding occurs in the absence of social learning, and as a
function of various neuroanatomical changes. For example, adolescent AAS exposure in
hamsters increases the density of arginine vasopressin (AVP) afferent fibers in the anterior
hypothalamic region (AH; Grimes et al., 2006) and enhances AVP release during
aggressive encounters with conspecifics (Melloni & Ricci, 2010). In general, AH AVP has
been implicated in the aggressive response across various species. For instance, AVP
delivered directly into the AH of the male hamster has been shown to enhance flank
marking behavior (Ferris et al., 1984) and at lower concentrations, enhance the display of
aggressive behaviors (Ferris et al., 1997).
Similar to AVP, AAS exposure alters dopamine (DA) signaling, and is associated with
increased aggressive behavior. In AAS treated hamsters, the number of inhibitory
dopamine D2 receptors is increased in the lateral anterior hypothalamus (LAH) (Ricci et
al., 2009; Schwartzer et al., 2009). This increase is noteworthy to the AAS-aggression
circuit as data show that heightened DA concentrations correlate with increased
aggressive behavior (Louilot et al., 1986; Ferrari et al., 2003), and D2 agonists and
antagonists affect aggressive responding when delivered systemically (Navarro and
Manzaneque, 1997; Rodriguez-Arias et al., 1999; Dennis et al., 2006) or locally into the
LAH region after AAS exposure (Schwartzer & Melloni, 2010a, 2010b). Further, various AH
efferent regions (e.g., the medial supraoptic nucleus, mSON; and nucleus circularis, NC)
are involved in the aggressive response (Ricci et al., 2009; Schwartzer et al., 2009) and
are known to possess tyrosine hydroxylase containing cell bodies (Ricci et al., 2009).
Recently, we have shown that the DA D2 receptor antagonist Eticlopride inhibits
aggressive responding in adolescent AAS treated hamsters after it is microinjection into
the LAH shortly before an aggressive encounter (Schwartzer & Melloni, 2010a, 2010b).
Considering that the DA system affects aggression, as well as the effects of AAS exposure
on AVP neural signaling, we hypothesized that these two systems interact within the LAH,
and that this interaction is likely involved in the aggressive response induced by AAS
exposure during development. From our previous data, however, it was unclear as to
whether drugs that affect affect DA activity have their anti-aggressive properties as a result
of downstream or upstream modulation of LAH AVP neural signaling. To address this
question, following adolescent AAS exposure, hamsters were administered a dose of
Eticlopride directly into the LAH at a concentration known to inhibit aggression. To
functionally assess whether DA D2 receptors modulate aggression-inducing AVP release,
each dose of Eticlopride was followed by a dose of saline (NaCl), D2 agonist (Quinpirole),
or AVP at a concentration known to enhance aggressive responding. After both drugs were
administered, hamsters were tested for drug-induced alterations to aggressive and social
behaviors.
Aggressive Behavior Breakdown
Attacks
Frequency (Mean + SEM)
Introduction/Abstract
GLU
Under normal (non-AAS)
conditions, AVP and DA
afferents from the mSON
and NC project to the LAH
region. These axonal
projections possess
GABAA receptors and lie
in close apposition to
GABA neurons that
possess DA D2 receptors.
Data from previous
studies suggest that
under these non-AAS
conditions, DA D2
receptors possessed by
inhibitory GABA
interneurons are
unbound, allowing GABA
to be released and inhibit
AVP release in the LAH,
and thus inhibit the of the
aggressive phenotype
observed after AAS
exposure.
AAS
mSON/NC
DA
AVP
GABA
AVP
GLU
Department of Psychology
Following AAS
exposure, and in the
presence of the DA D2
receptor antagonist
Eticlopride, in our
model, we presume
that by indirectly
inhibiting the
endogenous release
of AVP through
microinjection of
Eticolopride,
exogenously
microinjected AVP
activates AVP
sensitive neurons
(GLU), thus facilitating
the display of
aggressive behavior to
levels that are similar
to that of AASexposed NaCl+NaCl
microinjected controls.
For a summary of effects of the drug treatments in animals chronically treated
throughout adolescence with SO or AAS, see Figure 1. Ancillary behavior analysis
(i.e., Wall climbs, total contact time, and grooming behavior) showed no significant
differences between groups with the exception of a significant difference between
AAS and SO animals pre-treated with Quinpirole and Eticlopride (p<0.001), where
AAS animals spent significantly more time in contact with the intruder. AAS animals
administered Eticlopride and AVP or Quinpirole also displayed more grooming
behavior than AAS-animals administered saline (p<0.05 for both). Significant
differences were also found for wall climbs between AAS and SO animals
administered Eticlopride and Quinpirole and between SO animals administered
saline and those microinfused with Eticlopride and Quinpirole (p<0.05 for both).
These differences are not surprising considering the fact that drugs that interact
with the D2 receptor often interfere with motor activity.
Discussion
The aggression circuit is complex and has various neurochemical and
anatomical components. Hypothalamic AVP has long been associated with the
aggressive response, and is known to enhance aggressive responding in various
animals. Considerably less is known about how the DA system works to affect
aggression, and almost nothing is known about how the AVP and DA systems
work together to influence the display of aggressive behavior within the LAH. The
present data fall in line with previously reported data from our lab that show that
blockade of D2 receptor function in the LAH inhibits the aggressive response of
adolescent AAS exposed hamsters. The data also support our hypothesis that
the DA and AVP systems interact at the level of the LAH to influence aggressive
behavior. Lastly, the results of this study guide the notion that the elevated
aggression observed in AAS exposed animals is at least partially regulated by
activation of DA D2 receptors, and suggests that this mechanism lies upstream
of AVP sensitive neurons in the LAH brain region. This notion is further described
with more detail with the Models section in the middle bottom portion of this
poster.
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