mise à jour du
8 mai 2006
Physiol Behav

Two distinctive apomorphine-induced phenotypes
in the Roman high- and low-avoidance rats
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.


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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HY (High Yawning) rats