Lydia Gimenez-Llort, Toni Canete, Marc
Guitart-Masip, Alberto Fernandez-Teruel, Adolf
Tobena
Abstract
Strain differences in spontaneous locomotor
activity and the behavioral patterns induced by
dopaminergic agonists in rodents can disclose
differential genetic susceptibilities to
dopaminergic dysfunction (i.e. vulnerability to
psychosis). Psychogenetic selection of
hypoemotional Roman High-Avoidance (RHA) and
hyperemotional Low-Avoidance (RLA) rats leads to
divergence in dopaminergic function as well. The
present study was designed to characterize their
spontaneous activity and their responses to
apomorphine (0.067&endash;3 mg/kg, s.c.) as
compared to those of the standard
Sprague&endash;Dawley (SD) strain.
The Roman strains displayed higher
spontaneous activity than SD rats and RHA
exhibited the higher response to novelty which
agrees with a higher sensitivity to apomorphine
in this strain. The biphasic effect induced by
apomorphine (locomotor inhibition and yawning at
low doses but stimulation of locomotion and
stereotyped behavior at higher ones) was
reproduced in the standard SD strain. Low doses
were less effective inducing locomotor
inhibition in RHAwhereas these animals were much
more sensitive to high dose-induced stereotyped
behavior. In contrast, RLA was characterized as
a high-yawning strain and low doses of
apomorphine also induced a striking motor
inhibition suggesting functional enhancement of
dopamine receptors mediating these
behaviors.
The detailed and distinctive behavioral
profiles described in this work suggest between
strain differences both at the presynaptic and
postsynaptic dopaminergic function and may serve
as paths to better specify functional mechanisms
in future studies of risk of developing
dopaminergic dysfunctions.
The Swiss sublines of Roman high-avoidance
(RHA/ Verh) and low-avoidance (RLA/Verh) rats,
psychogenetically selected for rapid (RHA/Verh)
vs. extremely poor (RLA/Verh) two-way avoidance
acquisition in the shuttle box [3,8]
differ in many behavioral and neural traits
indicating that the RLA/Verh line is
hyperemotional and hyperreactive to a variety of
stressful situations (for review, see Ref.
[35]). These two lines consistently
differ in dopamine (DA) function as well.
Compared to RLA/Verh rats, RHA/Verh rats show:
(i) higher stereotypy response to high doses of
DA agonists apomorphine [11] and
amphetamine [9]; (ii) higher levels of
exploratory behavior in tests of novelty seeking
[13&endash;16]; (iii) higher preference
for alcohol [10,16,19,31] as well as
saccharin and quinine solutions [27,31];
(iv) higher impulsivity during acquisition of a
DRL-20 task [46]; (v) less sensitivity
to aversive effects of lateral hypothalamic
stimulation [25]; (vi) stronger
mesolimbic dopaminergic responses to drugs of
abuse (e.g. cocaine, morphine and alcohol,
[18,19,23]); (vii) higher density of DA
D1 receptors in the nucleus accumbens
[4]; (viii) enhanced mesocortical DA
release evoked by stressors [20]; and
(ix) enhanced sensitisation to amphetamine
[5]. All these evidences indicate a DA
hyperfunctionality in RHA/Verh rats and suggests
this line may be an animal model of
spontaneous/genetic vulnerability to
psychosis.
Apomorphine-induced responses are regularly
used as a model of acute psychosis with studies
ranging from screening compounds for potential
antipsychotic activity to testing the
sensitivity of central DA system in vivo. Acute
systemic administration of apomorphine induces a
biphasic behavioral response. Inhibition of
locomotor activity and appearance of yawning are
observed at low doses of apomorphine whereas
high doses produce locomotor stimulation and
perioral stereotypes [7,26]. The
distinctive features of the motility patterns
including yawning, locomotion and stereotypes
induced by a range of doses of apomorphine
depend on differential activation of D1, D2 and
D3 DA receptors located at pre- or postsynaptic
levels and of discrete postsynaptic DA-receptors
subpopulations as well (i.e. Refs.
[36,40,44]). The variability of
behavioral responses to apomorphine in different
rat strains has also been the basis for
selective breeding for their low or high
sensitivity to this drug in an attempt to model
certain characteristics of dopaminergic
dysfunction. This is the case of the
wellcharacterized apomorphine susceptible
(Apo-SUS) and non-susceptible (Apo-nonSUS) rats
generated by Ellenbroek and Cools (see their
review, Ref. [12]). In other cases,
differences in the DA systems of selectively
breeded rat lines emerged as a result of
divergences in other neural/behavioral
phenotypes (i.e. High (SwHi) and low (SwLo)
swim-test activity rats of West et al.
[45], spontaneously hypertensive rats by
van den Buuse and de Jong [41] and the
RHA and RLA rats as well [13]).
Interestingly, extreme performances in the
acquisition of active avoidance in the
shuttle-box used as criterion for selective
breeding of Roman rats have been suggested to be
causally related to divergences in dopaminergic
tone [23,29,34].
In the present study we wanted to extend
previous findings with the Roman rat strains
(mainly obtained with outbred animals) with a
wider characterization of spontaneous activity
and behavioral responses to apomorphine in our
colony of inbred RHA/Verh and RLA/Verh strains.
Spontaneous locomotor activity served as a
measure of basal dopaminergic function and the
behavioral patterns induced with a wide range of
doses of apomorphine served as measures of acute
DA challenges. For the first time, we compared
the Roman dopaminergic profiles with those in a
well-known standard strain of rats commonly used
in pharmacological research such as
Sprague&endash;Dawley (SD). The comparison of
Roman strains with this external control strain
is a necessary contrast to provide an external
standardized reference with genetic
heterogeneity. In sum, we expected that the
characterization of spontaneous locomotor
activity and the differential motility patterns
induced by apomorphine (yawning, locomotion and
stereotyped behavior) in these three strains
would improve the phenotypic description of
their responsiveness to dopaminergic behavioral
stimulation. A prerequisite that may be relevant
to investigate the basis of the genetic
vulnerability to psychosis and their underlying
neuronal mechanisms.
Discussion In the present study the
comparative analysis of the spontaneous
locomotor activity and apomorphine-induced motor
activity patterns (yawning, locomotion and
stereotypes) in the RHA/Verh-I, RLA/Verh-I and
SD strains of rats revealed significant
phenotypical differences that may serve as paths
to better specify functional mechanisms in
future studies of genetic vulnerability of
developing dopaminergic dysfunctions.
3.1. Spontaneous locomotor activity Intense
locomotor activity was recorded in all the
strains during the first 10 min interval of the
test with RHA being the most active rats.
Between-strain differences were found in the
habituation curves and the RHA rats also showed
the highest activity levels during the majority
of the intervals with differences vs. their
genetic counterpart, RLAs, persisting during the
initial 20&endash;30 min of the test. Globally,
both Roman strains showed higher total
spontaneous locomotor activity than the standard
SD strain.
The exposure to a novel environment elicits
intense exploratory behavior susceptible of
between-strain differences which seem to be
related to neurochemical differences as well
[38,43]. This is the case for RHA and
RLA rats which diverge not only in dopaminergic
function at mesolimbic areas such as the nucleus
accumbens, but also mesostriatal and/or
mesocortical dopaminergic pathways
[18,20,23] known to participate
regulating spontaneous locomotor activity
[22,24], although other neuronal
pathways may be involved (see Ref.
[28]). The higher total spontaneous
locomotor activity shown by both Roman strains
as compared to SD rats is described here for the
first time. We have observed it also after
repeated testing (unpublished results)
suggesting important intrinsic strain
differences between SD and the Swiss sublines
which still need to be explored. It also points
out the relevance of genetic counterparts as
control strains.
The present results do provide new evidences
for RHA as a novelty-seeker strain since
increased exploratory behavior reported in
standard tests assessing this psychological
trait [13&endash;16] can be observed
here as a sustained phenomena. Thus, when
compared to RLA rats, RHA show enhanced
locomotor activity for 20&endash;30 min until
time elapses and the novelty recedes, and during
most of the intervals when they are compared to
SD rats.
3.2. Motility patterns induced by
apomorphine In the second experiment, the Roman
strains administered with vehicle where again
more active than SD rats whereas the analysis of
the different motility patterns induced by
apomorphine (yawning, locomotion and stereotypy)
showed distinctive behavioral gradients among
the three strains. The biphasic effect
(locomotor inhibition and yawning at low doses
but stimulation of locomotion and stereotyped
behavior at higher ones) was reproduced in the
standard SD strain. However, a shift to the
right was observed in RHA rats as low doses were
less effective inducing locomotor inhibition and
the stimulatory ones elicited intense
stereotyped behavior associated with restricted
locomotion. In RLA, low doses produced a strong
motor inhibition (a 6-fold reduction) and also
entitled RLA as a high yawning strain.
The present study provides evidence of
higher basal locomotor activity levels in Roman
rats as compared to the SD strain. This
difference was seen in a spontaneous activity
test, after administration of a vehicle, and in
unpublished results during longer habituation
periods and repeated testing. Taken together,
these findings suggest a genetic variation in
dopaminergic function that should be explored
further.
A behavioral gradient in the locomotor
response and concomitant stereotypic activity
induced by apomorphine was observed between the
three strains. The high novelty responder strain
RHA in experiment 1 was also the one showing
higher sensitivity to stimulatory doses of
apomorphine in agreement with literature
suggesting that response to novelty may be a
good predictor of sensitivity to
psychostimulants based on functional
neuroanatomical differences (i.e. Refs.
[1,30]). There was a shift to the right,
with the first effective dose of apomorphine in
the other strains (0.067 mg/kg) being unable to
induce a locomotor inhibitory effect in RHA rats
and high doses of apomorphine eliciting an
enhanced intensity of stereotypic feature
(mouthing and gnawing) involving restricted
locomotion. Similar differential sensitivities
of DA receptors function were also described in
the outbred RHA and RLA rats by Durcan et al.
[11] with significant strain differences
found in the stereotypy resulting from a high
dose of apomorphine (2 mg/kg) and the response
of an F1 hybrid cross of the outbred RHA and RLA
falling between the two strains. Although there
was no evidence of any strain variation in the
behavioral effects of the low dose of
apomorphine (0.05 mg/kg), the authors reported
that the drug did, however, lead to a depression
of activity relative to controls.
The strong inhibitory effect elicited by low
doses of apomorphine in RLA and their high
yawning response was also surprising. Such
increased yawning activity had only been
previously reported in HY (High Yawning) rats
genetically selected for this purpose by
Urba`-Holmgren et al. [39].
Interestingly, HY and RLA rats do also share
some other behavioral patterns such as higher
grooming behavior, higher emotivity and
reactivity to stress with higher HPA axis
activation than their genetic counterparts. The
biphasic behavioral effect of apomorphine has
been studied for at least 30 years. Recent
reviews [2,36] document that the
distinctive features of the motility patterns
including yawning, locomotion and stereotypes
depend on differential activation of D1, D2 and
D3 DA receptors located at pre- or postsynaptic
levels and of discrete postsynaptic DA-receptor
subpopulations as well (also Refs.
[6,7,26,33,37,40,42,44]). Decreased
locomotion and yawning can be mediated by
postsynaptic DA receptors located in neuronal
populations distinct from those involved in
locomotor stimulation [33,40].
According to this, the distinct features in
the low-dose of apomorphine-induced phenotype in
RHA, RLA and SD rats may reflect strain
differences at those postsynaptic DA receptors
mediating locomotor inhibition and yawning
though differences at the presynaptic junction
such as in autoreceptor affinities or in the
induced inhibitory tone could also contribute to
explain the results. On the other hand,
locomotion and stereotypes induced by high doses
of apomorphine are, in fact, competing behaviors
that seem to depend on different DA neuronal
populations and neuroanatomical substrates. The
predominant response to a moderate dose of
apomorphine has been used by Surman and
Havemann-Reinecke [37] to categorize
male rats into two groups, which the authors
attributed to differences in postsynaptic DA
receptor mechanisms. A Fmesolimbic active_ group
showed intense sniffing behavior with increased
locomotor activity and a Fnigrostriatal active_
group mainly exhibited licking and gnawing
behavior with less increase in the locomotor
activity. This categorization of the DA function
was used to explain strain differences in the
behavioral response of SwLo and SwHi rats
[45].
Molecular studies in inbred RHA and RLA rats
are still lacking but because of their
behavioral similitude with outbred rats we
expect they would also exhibit similar D1/D2
receptor densities to those described in
outbreds by Corda et al. [4]. Then, a
role for increased stimulation on D1 DA
receptors which are known to differ between RHA
and RLA rats [4] could be related to
this potentiation of apomorphine-induced
stereotyped behavior [40] since this
receptor subtype seems to be related with
increases in oral stereotypic movements (i.e.
Ref. [6]). More recently, Germeyer et
al. [17] demonstrated in Wistar rats
that the individual apomorphine-induced
behavioral phenotype (different motility
patterns regarding 464 L. Gime´nez-Llort et
al. / Physiology & Behavior 86 (2005)
458&endash; 466 locomotor activities and oral
stereotypes) might be due to characteristic DA
D2 receptor polymorphisms, an aspect that cannot
be excluded here because it was not considered
when lack of differences between the two Roman
strains were described at the D2 receptor level
[4].
In conclusion, the present results
confirm and extend previous results obtained
with the outbred RHA/Verh and RLA/Verh lines
after treatment with apomorphine [11].
We also show that the inbred Roman rat strains
display higher total spontaneous locomotor
activity than the widely used SD strain.
However, it is the RHA strain which is the one
exhibiting the higher response to novelty, a
finding which appears to be congruent with a
higher sensitivity to apomorphine in that
strain. The differential motility patterns
induced by apomorphine (yawnings, locomotion and
stereotypy) showed differential dose-related
gradients among strains. The apomorphine-induced
locomotor inhibition at low doses was less
effective in RHA, whereas these animals were
much more sensitive to high doses of
apomorphineinduced stereotyped behavior.
The higher severity of stereotypy in RHA, as
compared to both RLA and SD rats, clearly
suggests a differential postsynaptic stimulatory
DA receptor function among these strains, which
in turn may lead to differential postsynaptic
dopaminergic vulnerability. In RHA rats, there
was also a lack of effectiveness of very low
doses of apomorphine to inhibit locomotor
activity. This result would suggest possible
differences among these strains at the
presynaptic junction although they are more
likely to depend upon postsynaptic differences
at D2/D3 dopaminergic neuronal subpopulations
mediating inhibition [44].
Oppositely, low doses of apomorphine induced
a strong motor inhibition (a 6-fold reduction)
in the RLA rats which may indicate functional
enhancement of presynaptic autoreceptors or of
those inhibitory DA receptors postsynaptically
located. Low doses of apomorphine also
characterized, for the first time, RLA as a high
yawning strain. This finding also points to
possible differences at postsynaptic DA
receptors (vs. the other two strains) in those
neuroanatomical regions involved in that
behavior [2].
These detailed and distinctive behavioral
profiles may serve as good leads to specify
functional mechanisms and perhaps pave the way
to use the Roman strains in future studies of
risk of developing dopaminergic dysfunctions.
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