Lydia Giménez-Liort , Toni
Cafíete, Marc Guitart-Masip,
Alberto Fernández-Teruel, Adolf
Tobefla
Medical Psychology Unit,
Department of Psychiatry and Forensic Medicine,
Institute of Neuroscience,
Autonomous University of Barcelona,
Spain.
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 (0.067-3 mg/kg, s.c.) as compared to
those of the standard Sprague-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 RHA whereas 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 (RLAIVerh) rats,
psychogenetically selected for rapid (RHAIVerh)
vs. extremely poor (RLAIVerh) 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
[13-16]; (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]; (y) 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
Dl 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 Dl, 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 RHAIVerh and RLAIVerh 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-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.
3. 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 RHAIVerh-I, RLAIVerh-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 20-30 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 [13-16] can be observed here as a
sustained phenomena. Thus, when compared to RLA
rats, RHA show enhanced locomotor activity for
20-30 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 Urbà-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 Dl, 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 'mesolimbic active' group
showed intense sniffmg behavior with increased
locomotor activity and a 'nigrostriatal 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 Dl/D2
receptor densities to those described in
outbreds by Corda et al. [4]. Then, a
role for increased stimulation on Dl 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 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 RHAIVerh and RLAIVerh 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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