Fig. 2.1.
Social play behavior in periadolescent (aged between 28 and 35 days) rats. SERT−/− rats played significantly less compared to SERT+/+ rats.
Social aggression has also been studied in the SERT−/− rat, using the resident-intruder test. As was found for social play, the initiation to attack an intruder was delayed in SERT−/− rats. Because the inhibitory control in the five-choice serial reaction time task is better in SERT−/− rats (19), reduced impulsivity could well contribute to the increased latency to initiate an attack. Aggression-unrelated social behavior was not found to be affected in SERT−/− rats.
Given that social play serves to train adult social interactions, the finding that reduced social play can be extended to adult aggressive behavior indicates that the reduced social approach has a strong genetic basis that becomes overt at an early age, as is the situation in autism. In the studies described, the SERT−/− rats are less able to interact socially with the other rats when compared with the wild type rats, suggesting that they suffer from changes in the function of brain circuits that are part of the “social brain.” Which brain circuits are involved in what way remains to be determined.
1.1.4 Repetitive Behavior and Resistance to Change
SERT−/− rats have been tested in the Phenotyper cage, an automatic behavioral observation system. In this cage, SERT−/− rats were found to display a preservative-like behavior: the frequency of immobility and mobility were increased, and SERT−/− rats displayed an increased speed by which the rats move towards their shelter (Nonkes, Homberg, unpublished findings). Wild type rats explored their environment more smoothly and habituated over time, whereas the knockout rats remained restless (Nonkes, Homberg, unpublished findings). In the open field test, self-grooming behavior in a new environment has been monitored. SERT−/− rats self-groomed significantly more than SERT+/+ rats (Nonkes, Homberg, unpublished findings), which might imply that the SERT−/− rat is unable to stop a certain behavioral pattern. This observation could also relate to the increased novelty-induced anxiety in the SERT−/− rat (see below), given the thought that self-grooming under stressful circumstances serves as displacement behavior.
To assess resistance to change, cognitive flexibility was measured in a visuospatial reversal learning paradigm. Unexpectedly, reversal learning was unchanged in SERT−/− rats, suggesting that SERT−/− rats are flexible and able to adapt to changing environmental conditions. Given that 5-HT plays a crucial role in cue-driven reversal learning, rather than spatial reversal learning (36), it remains to be tested how SERT−/− rats perform during a cue-based reversal learning test.
Together, the data show that genetic variation in the SERT gene strongly affects the social behavior, which nicely elaborates findings in SERT−/− mice (37) and in monkeys with the 5-HTTLPR s allele (35). However, resistance to change does not seem to be affected by SERT deficiency. To some extent, repetitive tendencies, as are seen in autism, may be affected by the serotonergic system, although it is debatable whether the locomotor pattern on the SERT−/− rats is representative for repetitive behavior in autism. That is, repetitive tendencies may reflect habitual acts that are dependent on aberrant learning processes. Given that the SERT-reducing s allele of the human 5-HTTLPR is associated with reduced social interactions in autism, and that the SERT-increasing l allele is associated with repetitive tendencies (38), it can be argued that SERT−/− rodents are specifically useful in modeling the s allele associated social domain of autism.
Depression
Major depression is one of the most common psychiatric disorders and is characterized by a variety of symptoms. A depressed person shows a lowered mood, loss of interest in normal activities and diminished ability to experience pleasure, which is called anhedonia. Also appetite, sleep and sex life can be negatively affected. Furthermore, depression is strongly characterized by increased anxiety, and stress-responsiveness. Finally, depression is also accompanied by reduced social interaction. As described above, this is strongly reduced in SERT−/− rats.
While autism is mainly caused by genetic factors, both genetic and environmental factors, like stressful events, are involved in the onset of depression. The 5-HT system plays a major role. In depression, it has been found that the plasma tryptophan levels are decreased (39, 40), and also the 5-HIAA level in CSF is lower (41, 42). Because the 5-HT homeostasis is strongly disturbed in the SERT−/− rat, this knockout rat is an excellent model to study the role of 5-HT in depression.
We performed several experiments to test depression-like behavior, as well as acute stress responses. To test depression-related behavior, two experiments were performed: The Forced Swim Test to assess learned helplessness, and the Sucrose Preference Test to measure anhedonia. SERT−/− rats displayed increased immobility in the Forced Swim Test when compared with the SERT+/+ rats (43). In addition, in a two-bottle paradigm with increasing concentrations of sucrose (2-10%), SERT−/− rats consumed less sucrose when compared with the SERT+/+ rats. These results indicate a higher depression-like state in SERT−/− rats. The forced swim test data are suggestive for an enhanced negatively reinforced conditioning, while the sucrose consumption test data imply that the SERT−/− rats are less responsive to positive natural stimuli.
Acute stress responses were measured in the Open Field Test and the Elevated Plus Maze Test. In the open field, the SERT−/− rats spent significantly less time in the central part (43), and on the elevated plus maze they spent less time on the open arms compared to SERT+/+ rats. In addition, anxiety-like behavior was detected in the SERT−/− rats in the Phenotyper cages. While locomotor activity between SERT−/− rats and wild type animals did not differ, the knockout rats avoided the center of the cage and showed thigmotaxis. Furthermore, the SERT−/− rats spent more time in the shelter of the Phenotyper cages, normally used for sleeping, than SERT+/+ rats (Homberg, unpublished observations), which could be related either to an increased anxiety or changes in sleeping behavior.
Together, these results indicate an enhanced responsivity to acute stress in SERT−/− rats, which is likely to serve the basis for the increased negatively reinforced behavior in the forced swim test.
Schizophrenia
Schizophrenic individuals are characterized by positive symptoms (e.g., psychosis and underlying sensorimotor gating deficits), negative symptoms (e.g., anhedonia), and cognitive deficits (poor attention, impaired working memory, and impaired executive functioning (e.g., prioritizing tasks, inhibition and decision making)). We tested the SERT−/− rats in these three domains (for anhedonia see the section on depression).
To assess sensorimotor gating, the Prepulse Inhibition Test was used. The automatic filtering of sensory information, measured in this test, is impaired in schizophrenic people (44). We found that the basal startle response, and PPI (3, 5 and 10 dB tones before the 120 db tone) were similar in SERT−/− and SERT+/+ rats. Attention was measured in the Startle Habituation Test. In this test, the reduction of startle over time is measured. In medicated and unmedicated patients with schizophrenia, startle habituation is diminished (45, 46). Both, SERT−/− and SERT+/+ rats displayed a reduction in startle magnitude over time, but no genotype differences were found (47). Overall, despite that SERT−/− rats show a slightly impaired working memory as well as anhedonia (see section on depression), these data imply that the SERT−/− rat is not a strong model to address the psychopathology underlying schizophrenia. Yet, impaired latent inhibition, reflecting altered processing of sensory information, and attentional set shifting, a type of cognitive flexibility impaired in schizophrenic patients, remain to be studied in SERT−/− rats. Given the role of 5-HT in cognitive functions (36), phenotypes in these domains are possible in SERT−/− rats.
Drug Addiction
Drug addiction is an important health problem and a major socioeconomic issue that affects millions of people (48). An often abused drug is the psychostimulant cocaine, which affects the central nervous system by blocking the monoamine transporters. While individual differences in vulnerability to psychostimulants have been largely attributed to dopaminergic neurotransmission, the role of 5-HT is not fully understood. It is possible that cocaine addiction and the 5-HTTLPR are somehow linked, but this has been rarely explored and findings are inconsistent (49, 50). Given that the intravenous drug self-administration model is originally developed in rats, the SERT−/− rats are very useful to determine the role of the 5-HT system in cocaine self-administration behavior, and eventually to obtain insights into the effect of the 5-HTTLPR on susceptibility to cocaine dependence.
To assess the role of the SERT in different aspects of cocaine’s effects; that is its psychomotor effects, its incentive salience, and its reinforcing and motivation effects, the SERT−/− rats have been tested for cocaine-induced locomotor activity, cocaine-induced conditioned place preference, and intravenous cocaine self-administration (51). It was found that cocaine-induced (20 mg/kg) locomotor activity was strongly increased in SERT−/− rats. Similarly, cocaine-induced conditioned place preference was enhanced in SERT−/− rats. Finally, SERT−/− rats displayed an increased acquisition of intravenous cocaine self-administration under a fixed ratio 1 schedule, an increased motivation to self-administer cocaine under a progressive ratio schedule, and a delayed extinction of self-administration behavior. These data point out that SERT−/− rats are supersensitive to cocaine. Given that cocaine-induced 5-HT release is reduced in SERT−/− rats (Olivier, Homberg, unpublished findings), and that 5-HT exerts an inhibitory control over DA release, it is possible that the balance between 5-HT and DA is shifted to DA in the SERT−/− rats, which may at least explain an increased cocaine sensitivity. Other factors could also be involved. For instance, improved conditioning could contribute to the increased acquisition of cocaine self-administration behavior. Further, compensatory changes may play a role. Thus, 5-HT1A receptors are desensitized in SERT−/− rats, and it was found that 5-HT1A receptor ligands differentially affect the cocaine-induced locomotor activity in SERT−/− and SERT+/+ rats (51).
1.1.5 Relevance
Considering the central role of 5-HT in several central processes and major psychiatric conditions, and the rich background of the rat as a model of psychiatric disorders, it is clear that the SERT−/− rat will play an important role in the understanding of the psychopathology underlying psychiatric disorders. It is important to find an answer to the question of how results can be extrapolated to humans. At first sight, a knockout rat appears to be an inadequate resemblance of the human condition. In humans, genes can be modified by mutations, but are usually not completely absent or non-functional. Moreover, besides genetic factors, environmental factors play an important role in the development of human psychiatric disorders, although the genetic component is more prevalent in neurodevelopmental psychiatric disorders when compared with other psychiatric disorders. Knockout animals have a life-long disturbance of a certain process and it is likely that compensatory changes take place to deal with such a disturbance. As a consequence, knockout rodents may not directly give insight into gene function, but rather can be useful to model genetic factors in human psychiatric disorders. As compensatory changes will take place in association with human polymorphisms, insights into these changes are not only of fundamental importance, but may also lead to novel targets in the design of improved therapies in the treatment of psychiatric conditions. For instance, as a consequence of the high extracellular level of 5-HT, the 5-HT1A receptor is desensitized in SERT−/− rats (51). This is in agreement with the finding that the 5-HT1A receptor is down-regulated in humans carrying the s/s genotype of the 5-HTTLPR (52).
As implied, the SERT−/− rat could resemble the widely studied human serotonin transporter polymorphism (5-HTTLPR). In more detail, the short (s) allelic variant of this polymorphism involves a 44-base pair deletion (53), resulting in a 40% decrease in serotonin transporter expression and function. This s allele, appearing in 19% of Caucasians, is associated with several profound endophenotypes and may increase the risk of psychiatric disorders. Thus, the s allele is strongly linked to neuroticism, as suggested by the psychologist Hans Eysenck, which is characterized by brooding, being anxious, having a low self-esteem and reacting strongly to criticism. This may, in combination with environmental factors and the other genetic factors, pave the path for depression, (54, 55), autism (38), and to a lesser extent, schizophrenia (56). Given that neuroticism-like endophenotypes can be modeled in SERT−/− rats, future studies will point out their relevance in the dissection of interacting environmental and genetic factors contributing to the development of psychopathology.
1.2 The Dopamine D1 Receptor Mutant Rat
The dopamine D1 receptor (DRD1) mutant Wistar rat bears a mutation, not necessarily leading to a total knockout of the dopamine receptor D1. In this rat line N-ethyl-N-nitrosourea induced a T > A transversion at position 1,215 of the DRD1 gene, which encodes a G-protein coupled receptor (GPCR). This mutation leads to an amino acid exchange in the second transmembrane domain of the receptor. The non-polar amino acid isoleucine is replaced by the polar amino acid serine, which might lead to a conformational change of the receptor. Autoradiography with [3H]SCH23390 revealed a 50% decrease in binding to the dopamine D1 receptor (Homberg, unpublished findings). However, the mechanism underlying this down-regulation is not yet clear. There are different possibilities: (1) The D1 receptor cannot leave the endoplasmatic reticulum and is degraded. (2) The receptor cannot enter the cellular membrane and is degraded. (3) The receptor can enter the cell membrane, but there is reduced ligand binding due to changes in receptor conformation. Functionally, there is the additional option that there is altered G-protein coupling to the receptor. In localization studies the mutated D1 receptor was cloned into canine kidney cells. It was found that the receptor is transported to the cell membrane (Deen, unpublished findings), implying that reduced ligand binding explains the reduced [3H]SCH23390 binding. Yet, the question remains whether there is altered G-protein coupling.
The DRD1−/− rat is now used at our laboratory in several behavioral tasks and experiments to reveal the function of the dopamine D1 receptor and its implication in dopamine-related disorders. Anatomically, there are no gross abnormalities, but the body weight of homozygous mutant males is significantly reduced (25%) as compared to the mutant heterozygous and wild type males, with no differences between the latter two groups. The same effect is seen in the homozygous D1 receptor knockout mice (57–59). It is possible that the mutant rats are less motivated to eat, given that the dopaminergic system plays an important role in motivation and reward.
The dopaminergic system is involved in memory, locomotion, cognition, attention, sensorimotor gating, and to a smaller extent in hormonal regulation. Drugs targeting dopamine receptors have been the focus of much research over the past 30 years, mainly because of their role in treating pathological conditions such as Parkinson’s disease, schizophrenia, Tourette’s syndrome, and hyperprolactinemia. Missense mutations in the dopamine D1 receptor can not only alter ligand-induced signaling (60) but could increase the risk for a neurodevelopmental disorder. There are associations between a haplotype of the DRD1 gene with alcohol dependence. People with the rs686*T-rs4532*G allele seem to be more susceptible to alcohol and have more difficulties in withdrawal (61). Another DRD1 haplotype is associated with the risk for developing autism spectrum disorders (ASDs), especially in male persons. In families with two or more males affected by ASD, there was an over-transmission of the alleles rs265981-C and rs4532-A. Quantitative Transmission Disequilibrium Test (QTDT) analyses showed associations of the rs265981-C, rs4532-A and rs686-T alleles (also called the C-A-T haplotype) with severe problems in social interaction, greater difficulties with nonverbal communication and increased stereotypies when compared with individuals with other haplotypes (62). Furthermore, the A-48G polymorphism of the dopamine D1 receptor may influence prefrontal executive cognition in healthy adults as investigated in a group of Han Chinese adults undergoing the Wisconsin Card Sorting Test (WCST) (63). Executive functioning is also impaired in schizophrenia and might increase the risk to develop the disorder. These findings suggest that a mutant rat for the dopamine D1 receptor has heuristic value to model drug addiction and schizophrenia. The preliminary findings on disease-related symptoms are discussed below.
1.2.1 Preliminary Findings
Locomotor Control
The dopamine D1 receptor is well known to mediate locomotor responses (64, 65) raising the expectation to find phenotypes at this level in DRD1−/− rats. Indeed, already with handling the DRD1−/− rats seem to be different from the DRD1+/+ rats. The mutant rats show slower responses in escaping when they are picked up, and they have difficulties to relax when fixed by the neck skin (Müller, unpublished observations). In the open field, male homozygous DRD1−/− rats show a significant decrease in locomotor activity, but no differences in locomotor activity were seen in female DRD1−/− rats in their home cage (Olivier, Balemans, unpublished findings). In addition, in the Morris Water Maze we found that DRD1−/− males were far less active (there was less swimming and they had difficulties to climb on the platform) when compared with wild type rats (Müller, Homberg, unpublished observations). Collectively, these data strongly suggest that locomotor activity is affected in DRD1−/− rats, which is of relevance in the interpretation of other behavioral tasks.
Schizophrenia
In rodents, sensorimotor gating can be measured with the Prepulse Inhibition (PPI) Test. Previous studies in mice demonstrate a contribution of dopamine D1-family receptors in modulating PPI (66). In that light, we measured PPI in the DRD1−/− rats and found a modest reduction in PPI. However, no changes were observed in the startle response, either acutely, nor during habituation (Müller, unpublished findings).
To elaborate schizophrenia-related measurements in the DRD1−/− rats, we aim to study their spatial learning abilities in the Morris Water Maze. In dopamine D1 receptor knockout mice, it was shown that the spatial learning abilities are decreased (67). Further, as schizophrenics typically display perseveration, inefficient sorting, and non-perseverative errors in the Wisconsin Card Sorting Test (WCST), and because this is associated with reduced dopamine D1 receptor expression in the prefrontal cortex of schizophrenic patients (68), it is of high relevance to address attentional set shifting in the DRD1−/− rats.
Finally, our future experiments will include the observation of self-grooming behavior to establish whether DRD1−/− rats differ in the display of stereotypies, as they are seen in schizophrenia (69). Self-grooming constitute the most widely accepted behavioral index of D1-like receptor function, as it has been shown that dopamine D1 receptor agonists increase self-grooming (70).
Drug Addiction
Dopamine is essential for the reward system, which ensures that an animal eats enough and reproduces and thus survives. A lot of addictive substances are able to manipulate the dopaminergic system by increasing the extracellular concentration of the neurotransmitter (71). It was hypothesized that the DRD1−/− rats would have lower or no responses to drugs and agonists acting on the dopamine D1 receptor, as the dopamine D1 receptor is the major indirect target of dopamine-releasing psychoactive drugs. However, in a Free choice two-bottle paradigm, with water and ethanol simultaneously freely available, male DRD1−/− rats did not significantly differ from male DRD1+/+ rats (van der Kant, Olivier, unpublished findings). Only at a constant level of 10% ethanol (the highest concentration used), DRD1−/− rats showed a higher preference for the alcohol than the wild type rats. Normally, rats stop drinking high concentrations of ethanol, to prevent intoxication (72). The DRD1−/− rats seem to be impaired in this preventive inhibition. On the other hand, it could be that the ethanol has less effect in the brains of the DRD1−/− rats due to the absence of a functional D1 receptor.
Cocaine-induced locomotor activity was measured in the open field test. It was found that this response was strongly reduced in DRD1−/− rats when compared with the wildtype controls, suggesting that the mutant rats display reduced cocaine sensitivity. To further address this issue, cocaine-induced mRNA expression of the genes encoding Arc, a marker of neuronal activity, and FGF-2, a neurotrophic factor, were measured (73, 74). Gene expression of both proteins is under tonic regulation of the dopamine D1 receptor (75–77). In concordance with the reduced locomotor response to cocaine, mRNA expression levels were increased in wild type rats after a cocaine challenge, but unchanged in DRD1−/− rats (Fumagalli, Riva, unpublished findings). These results are not only indicative for a strong reduction in cocaine sensitivity, but also show that dopamine D1 receptor function is reduced in DRD1 mutant rats.
Related posts:
Estrogen-Deficient Mouse Models in the Study of Brain Injury and Disease
Mutant and Transgenic Zebrafish in Modeling Neurobehavioral Disorders
Investigating Rett Syndrome Through Genetic Mouse Models: Presymptomatic, Clearly Symptomatic Phases, and Innovative Therapeutic Approaches
Mutant and Transgenic Tools in Modeling Schizophrenia
The Utility of Genetically Modified Animals in Modeling OCD-Spectrum Disorders
GABAA Receptor α1 Subunit (Gabra1) Knockout Mice: Review and New Results
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
