Comparison of SHR, WKY and Wistar rats in different behavioural animal models: effect of dopamine D1 and alpha2 agonists

Spontaneously hypertensive rats (SHR) and its counterpart, the Wistar-Kyoto rats (WKY), are probably the most often used animal model of ADHD. However, SHR as model of ADHD have also been criticised partly because of not differing to outbred rat strains. In the present study, adolescent SHR, WKY and Wistar rats from Charles River were tested in open-field, elevated plus maze and novel object recognition and on gastrointestinal transport to more intensively evaluate the strain characteristics. Non-habituated SHR and Wistar rats were more active than WKY rats but contrary to Wistar rats SHR stay hyperactive in a familiar environment. SHR were more sensitive to the alpha2-adrenoceptor agonist guanfacine and the dopamine D1 agonist A-68930 than WKY and Wistar rats, whereas amphetamine, the D1/D5 agonist ABT431 and the D2 agonist quinpirole, similarly affected open-field activity in all strains. In the elevated plus maze, SHR and Wistar rats showed less anxiety-related behaviour than WKY rats. Guanfacine and amphetamine induced an anxiolytic-like activity in SHR but not in WKY and Wistar rats. SHR showed the highest long-term memory in the novel object recognition. Gastrointestinal transport was similar and comparably affected by guanfacine in all rat strains. The present study shows clear differences in the behaviour of SHR and Wistar rats but also of WKY and Wistar rats. The use of SHR as animal model of ADHD is supported.     

Increased Oxidative Parameters and Decreased Cytokine Levels in an Animal Model of Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is a highly heterogeneous disorder characterized by impairing levels of hyperactivity, impulsivity and inattention. Oxidative and inflammatory parameters have been recognized among its multiple predisposing pathways, and clinical studies indicate that ADHD patients have increased oxidative stress. In this study, we aimed to evaluate oxidative (DCFH oxidation, glutathione levels, glutathione peroxidase, catalase and superoxide dismutase activities) and inflammatory (TNF-α, IL-1β and IL-10) parameters in the most widely accepted animal model of ADHD, the spontaneously hypertensive rats (SHR). Prefrontal cortex, cortex (remaining regions), striatum and hippocampus of adult male SHR and Wistar Kyoto rats were studied. SHR presented increased reactive oxygen species (ROS) production in the cortex, striatum and hippocampus. In SHR, glutathione peroxidase activity was decreased in the prefrontal cortex and hippocampus. TNF-α levels were reduced in the prefrontal cortex, cortex (remaining regions), hippocampus and striatum of SHR. Besides, IL-1β and IL-10 levels were decreased in the cortex of the ADHD model. Results indicate that SHR presented an oxidative profile that is characterized by an increase in ROS production without an effective antioxidant counterbalance. In addition, this strain showed a decrease in cytokine levels, mainly TNF-α, indicating a basal deficit. These results may present a new approach to the cognitive disturbances seen in the SHR.     

The role of temporal generalization in a temporal discrimination task

Two experiments trained rats to discriminate two or three stimulus durations using a temporal discrimination task. A standard peak shift effect was observed when training was administered with short versus long signals in Experiment 1. Both discrimination accuracy scores and the generalization gradients revealed that shorter intervals were discriminated more accurately, which may be due to the scalar property of timing. In Experiment 2, three signals (short, medium, and long) were associated with three different responses, or two of the intervals were associated with one response (short and long or short and medium) and the other interval with a different response. Here, the short/medium versus long discrimination was learned most readily of the three tasks. The results of both experiments indicated a strong contribution of learning of individual durations combined with scalar generalization gradients, but Experiment 2 indicated that categorical encoding of durations may have also been operating.     

奖励性操作式条件反射任务在大鼠学习记忆研究中的应用

目的将奖励性操作式条件反射方法应用于学习记忆研究。方法首次采用奖励性操作式条件反射方法,设置单次操作训练、连续多次操作训练、信号辨识与消退四种检测模式,研究Wistar大鼠和SHR大鼠学习记忆能力。结果奖赏训练中,SHR组鼻触次数显著增多（vs.Wistar＆Donepezil,P〈0.05）;单次操作训练中,三组动物对操作反应的获得无显著差异;连续多次操作学习任务中,SHR组错误操作次数增多（vs.Wistar,P〈0.05）,奖赏获得次数减少（vs.Donepezil,P〈0.001）,反应准确率显著降低（vs.Wistar＆Donepezil,P〈0.05）;信号辨识任务中SHR组错误反应次数显著增多（vs.Wistar＆Donepezil,P〈0.05）,而反应准确率降低,组间差异有显著性（vs.Wistar＆Donepezil,P〈0.05）;消退实验中,三组动物差异无显著性。神经递质检测发现,SHR组大鼠谷氨酸[（1.0639±0.07086）mg/g]、乙酰胆碱[（2.7760±0.2609）μg/g]与五羟色胺[（1.2200±0.1137）μg/g]含量均显著低于Wistar组大鼠（P〈0.05）,多奈哌齐组大鼠乙酰胆碱含量显著增加[（3.9344±0.2747）μg/g]（vs.Wistar,P〈0.05;vs.SHR,P〈0.001）。结论奖励性操作式条件反射方法可作为一种正性增强条件反射检测新方法应用于大鼠学习记忆研究。     

Lifelong hyperarousal in the spontaneously hypertensive rat indicated by operant behavior

Instrumental conditioning techniques were used to obtain objective evidence of differences in behavioral arousal between the spontaneously hypertensive rat (SHR) and the normotensive ancestral Wistar Kyoto (WKY) strain. Subjective emotionality ratings previously indicated that the genetically hypertensive rats were more active and aggressive than their normotensive cousins. In a lengthy series of operant conditioning sessions using a small number of adult female SHR and WKY rats, hyperarousal in the SHR was confirmed by their significantly higher response outputs on either response contingent or time contingent schedules of reinforcement. Conditioned emotionality tests during this series of experiments also suggested hyperarousal and aggressiveness in the SHR, since the fear-conditioned stimulus suppressed bar-pressing in the SHR much less than in the WKY. Further experiments with young prehypertensive SHR rats provided the same evidence of hyperresponsivity in the SHR compared to the WKY strain. Furthermore, these young SHR failed to develop hypertension by the end of the study (14 weeks of age), while their nonconditioned SHR cousins had become clearly hypertensive by the same age. This suggests that factors related to the conditioning methods modified the development of high blood pressure in this animal model of essential hypertension.     

Differential Behavioral and Biochemical Responses to Caffeine in Male and Female Rats from a Validated Model of Attention Deficit and Hyperactivity Disorder

Epidemiological studies suggest sex differences in attention deficit and hyperactivity disorder (ADHD) symptomatology. The potential benefits of caffeine have been reported in the management of ADHD, but its effects were not properly addressed with respect to sex differences. The present study examined the effects of caffeine (0.3 g/L) administered since childhood in the behavior and brain-derived neurotrophic factor (BDNF) and its related proteins in both sexes of a rat model of ADHD (spontaneously hypertensive rats—SHR). Hyperlocomotion, recognition, and spatial memory disturbances were observed in adolescent SHR rats from both sexes. However, females showed lack of habituation and worsened spatial memory. Although caffeine was effective against recognition memory impairment in both sexes, spatial memory was recovered only in female SHR rats. Besides, female SHR rats showed exacerbated hyperlocomotion after caffeine treatment. SHR rats from both sexes presented increases in the BDNF, truncated and phospho-TrkB receptors and also phospho-CREB levels in the hippocampus. Caffeine normalized BDNF in males and truncated TrkB receptor at both sexes. These findings provide insight into the potential of caffeine against fully cognitive impairment displayed by females in the ADHD model. Besides, our data revealed that caffeine intake since childhood attenuated behavioral alterations in the ADHD model associated with changes in BDNF and TrkB receptors in the hippocampus.     

Interval timing by an invertebrate, the bumble bee Bombus impatiens

Sensitivity to temporal information and the ability to adjust behavior to the temporal structure of the environment should be phylogenetically widespread. Some timing abilities, such as sensitivity to circadian cycles, appear in a wide range of invertebrate and vertebrate taxa [1,2]. Interval timing--sensitivity to the duration of time intervals--has, however, only been shown to occur in vertebrates [3,4]. Insect pollinators make a variety of decisions that would appear to require the ability to estimate elapsed durations. We exposed bumble bees to conditions in which proboscis extension was reinforced after a fixed duration had elapsed or after either of two fixed durations had elapsed. Two groups of bees were trained with a short duration (either 6 s or 12 s) and a long duration (36 s) in separate experimental phases (independent timing groups), whereas two other groups were trained with a short duration (either 6 s or 12 s) and long duration (36 s) always intermixed unpredictably (multiple timing groups). On long intervals, independent timing groups waited longer than mixed timing groups to generate the first response and responded maximally near the end of the interval. Multiple timing groups waited the same amount of time on average before generating the first response on both long and short intervals. On individual trials, multiple timing groups appeared to time either the long duration only or both the short and long durations: most trials were characterized by a single burst of responding that began between the short and long duration values or by two bursts of responding with the first burst bracketing the short value and the second burst beginning in anticipation of the long value. These results show that bumble bees learn to time interval durations and can flexibly time multiple durations simultaneously. The results indicate no phylogenetic divide between vertebrates and invertebrates in interval timing ability.     

Dopamine agonists and antagonists can produce an attenuation of response bias in a temporal discrimination task depending on discriminability of target duration

The current study examined the effects of the D2 agonist (quinpirole) and D2 antagonist (eticlopride) on temporal discrimination performance in a conditional discrimination task (Experiment I) and a delayed conditional discrimination task (Experiment II). In both experiments rats discriminated between a scheduled stimulus duration of 3 s versus 9 s. Consistent with previous reports, overall discrimination performance decreased in a dose-dependent manner with both drugs. Changes in response bias (the tendency to choose-short or choose-long irrespective of actual stimulus duration), however, were best characterized in terms of both drugs tending to attenuate the bias effects normally observed during baseline drug-free performance. Specifically, the 'choose-short' bias observed in Experiment I and at a relatively short, 0.1 s, delay in Experiment II became less extreme with increasing doses. In addition, the 'choose-long' bias observed at a relatively long, 6 s, delay in Experiment II also became less extreme with increasing doses. Thus, whether there was an apparent shift from a short response bias to long, or vice versa, was the product of the delay interval between stimulus presentation and choice rather than whether the drug in question was a D2 agonist or antagonist. Such an attenuation of bias may have arisen because of subjects confounding the delay interval with the actual discriminative stimulus duration.     

Choice behavior in spontaneously hypertensive rats: Variable vs. fixed schedules of reinforcement

Spontaneously hypertensive rats (SHR) and Wistar rats were evaluated in the successive-encounters procedure (an operant simulation of natural foraging) with the idea of assessing differences between them in their preference for variable schedules of reinforcement. In this procedure, after satisfying a schedule of reinforcement associated with search time, the subjects could “accept” or “reject” another schedule of reinforcement associated with handling time. Two schedules of reinforcement were available: a fixed interval (FI), and a variable interval (VI) with the same mean value. The results indicated preference for the variable schedule in both strains, as suggested by the observation that the VI was always accepted while the FI was often rejected. The difference in FI acceptability between strains was not statistically significant, a result which is relevant for the current debate of SHR as an adequate animal model of Attention Deficit Hyperactivity Disorder.     

Maintained generalization of delay-specific remembering

According to the discrimination hypothesis (White, 2002), remembering is a delay-specific discrimination made at the time of retrieval. In the present experiment the delay-specific nature of the discrimination was made explicit by making correct choices in a delayed matching-to-sample task performed by pigeons conditional on whether the retention interval was short or long. Retention interval was varied over several durations in a maintained generalization test without reinforcement for correct matching responses. Opposing gradients demonstrated generalization of delay-specific remembering, consistent with the view that the temporal dimension of the retention interval can be treated in the same way as non-temporal dimensions of the sample stimulus.     

Interstimulus interval as a discriminative stimulus: Evidence of the generality of a novel asymmetry in temporal discrimination learning

Three appetitive conditioning experiments with rats examined temporal discrimination learning within Pavlovian conditioning trials. In all experiments, the duration of a feature white noise stimulus signaled whether or not a subsequent 10-s target tone would be reinforced. In Experiment 1, the feature durations were 4 and 1 min. For one group of rats (Group 4+/1−), 4 min of noise signaled that the tone would be reinforced and 1 min of noise signaled that the tone would not be reinforced. A second group (Group 1+/4−) was trained with the reverse contingency. The results showed a clear asymmetry in temporal discrimination learning: rats trained with 4+/1− (Long+/Short−) learned the discrimination readily (responding more in the tone on reinforced than on nonreinforced trials), whereas rats trained with 1+/4− (Short+/Long) did not. In Experiment 2, the feature durations were shortened to 60 and 15 s. Due to strong excitatory conditioning of the 15-s feature, the reverse asymmetry was observed, with the Short+/Long− discrimination learned more readily than the Long+/Short− discrimination. However, Experiment 3 demonstrated that the original Long+/Short− advantage could be recovered while using 60− and 15-s feature durations if the excitatory conditioning of the feature was reduced by including nonreinforced feature trials. The results support previous research involving the timing of intertrial intervals and are consistent with the temporal elements hypothesis which holds that the passage of time is encoded as a series of hypothetical stimulus elements.     

Spontaneously hypertensive and Wistar Kyoto rats are genetically disparate.

Spontaneously hypertensive rats (SHR) are one of the most common animal models used to study essential hypertension in humans. Because SHR and normotensive Wistar Kyoto (WKY) rats were both established from the same parental, normotensive Wistar stock, WKY animals have been used almost exclusively as control animals in studies of SHR. Recently, the suitability of WKY rats as normotensive controls for SHR has been challenged. To establish whether or not SHR and WKY rats share the same immunologic backgrounds, we initially performed a series of skin grafting experiments on these animals. In all cases, grafts of SHR donor skin to WKY recipients and of WKY donor skin to SHR recipients resulted in complete rejection within 7 to 10 days. In addition, grafts of WKY donor skin to other WKY recipients resulted in graft rejection. By contrast, skin grafts between SHRs were always accepted. To further characterize the genetic distinctions between SHR and WKY rats, allelic profiles based on a series of immunologic and biochemical markers were established for each strain. These findings clearly establish that SHR and WKY rats differ at the major histocompatibility complex, in specific blood group antigens, and in a panel of isozymic markers. Moreover, whereas SHRs have the same genetic profiles irrespective of source, some colonies of WKY rats are outbred, as judged by their variant allelic profiles.     

Spatial Memory in Spontaneously Hypertensive Rats (SHR)

The spontaneously hypertensive rat (SHR) is an established animal model of ADHD. It has been suggested that ADHD symptoms arise from deficits in executive functions such as working memory, attentional control and decision making. Both ADHD patients and SHRs show deficits in spatial working memory. However, the data on spatial working memory deficits in SHRs are not consistent. It has been suggested that the reported cognitive deficits of SHRs may be related to the SHRs’ locomotor activity. We have used a holeboard (COGITAT) to study both cognition and activity in order to evaluate the influence of the activity on the cognitive performance of SHRs. In comparison to Wistar-Kyoto (WKY) rats, SHRs did not have any impairment in spatial working memory and reference memory. When the rats’ locomotor activity was taken into account, the SHRs’ working memory and reference memory were significantly better than in WKY rats. The locomotor activity appears to be a confounding factor in spatial memory tasks and should therefore be controlled for in future studies. In the SHR model of ADHD, we were unable to demonstrate an impairment of working memory which has been reported in patients with ADHD.     

The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain

Spontaneously hypertensive rats (SHR) are considered to represent a genetic animal model for attention-deficit hyperactivity disorder (ADHD). In the present studies, we compared the locomotor activity, learning and memory functions of juvenile male SHR, with age- and gender-matched genetic control Wistar-Kyoto rats (WKY). In addition, we investigated potential differences in brain morphology by magnetic resonance imaging (MRI). In other complimentary studies of the central nervous system, we used real-time PCR to examine the levels of several dopaminergic-related genes, including those coding for the five major subtypes of dopamine receptor (D1, D2, D3, D4 and D5), those coding for enzymes responsible for synthesizing tyrosine hydroxylase and dopamine-beta-hydroxylase, and those coding for the dopamine transporter. Our data revealed that SHR were more active than WKY in the open field (OF) test. Also, SHR appeared less attentive, exhibiting inhibition deficit, but in the absence of memory deficits relative to spatial learning. The MRI studies revealed that SHR had a significantly smaller vermis cerebelli and caudate-putamen (CPu), and there was also a significantly lower level of dopamine D4 receptor gene expression and protein synthesis in the prefrontal cortex (PFC) of SHR. However, there were no significant differences between the expression of other dopaminergic-related genes in the midbrain, prefrontal cortex, temporal cortex, striatum, or amygdala of SHR and WKY. The data are similar to the situation seen in ADHD patients, relative to normal volunteers, and it is possible that the hypo-dopaminergic state involves a down regulation of dopamine D4 receptors, rather than a general down-regulation of catecholamine synthesis. In conclusion, the molecular and behavioural data that we obtained provide new information that may be relevant to understanding ADHD in man.     

Myocardial hypertrophy of normotensive Wistar-Kyoto rats

In our studies with spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats, we observed normotensive WKY rats with cardiac hypertrophy determined by a greater left ventricular (LV) mass (LVM)-to-body weight (BW) ratio (LVM/BW) than that of normotensive Wistar rats. Thus we compared the following parameters in SHR, WKY, and Wistar rats: LVM/BW, cell capacitance as index of total surface area of the myocytes, length, width, and cross-sectional area of cardiac myocytes, LV collagen volume fraction, and myocardial stiffness. The LVM/BW of WKY (2.41 +/- 0.03 mg/g, n = 41) was intermediate between SHR (2.82 +/- 0.04 mg/g, n = 47) and Wistar rats (1.98 +/- 0.04 mg/g, n = 28). A positive correlation between blood pressure and LVM was found in SHR, whereas no such relationship was observed in WKY or Wistar rats. Cell capacitance and cross-sectional area were not significantly different in SHR and WKY rats; these values were significantly higher than those of Wistar rats. The cell length was smaller but the width was similar in WKY compared with SHR. Papillary muscles isolated from the LV of WKY and SHR were stiffer than those from Wistar rats. Consistently, a greater level of myocardial fibrosis was detected in WKY and SHR compared with Wistar rats. These findings demonstrate blood pressure-independent cardiac hypertrophy in normotensive WKY rats.     

Gene expression of synaptosomal-associated protein 25 (SNAP-25) in the prefrontal cortex of the spontaneously hypertensive rat (SHR)

Dopamine is believed to play an important role in the etiology of attention-deficit/hyperactivity disorder (ADHD). In our previous study, we showed that gene expression of dopamine D4 receptor decreased in the spontaneously hypertensive rat (SHR) in the prefrontal cortex (PFC). In the present study, we explored the potential causes of dysfunction in the dopamine system in ADHD. It is the first time that neuronal activities in both juvenile SHR and WKY rats have been measured by functional MRI (fMRI). Our results showed that in PFC the Blood Oxygenation Level Dependent (BOLD) signal response in SHR was much higher than WKY under stressful situations. We tested the effects of acute and repeated administration of amphetamine on behavioral changes in SHR combined with the expression of the neuronal activity marker, c-fos, in the PFC. Meanwhile dopamine-related gene expression was measured in the PFC after repeated administration of amphetamine. We found that potential neuronal damage occurred through deficit of D2-like receptor protective functions in the PFC of the SHR. We also measured the expression of synaptosomal-associated protein 25 (SNAP-25) in SHR in PFC. The results showed decreased expression of SNAP-25 mRNA in the PFC of SHR; this defect disappeared after repeated injection of D-AMP.     

The failure of Weber's law in time perception and production

Weber's law--constancy of the coefficient of variation--is an apparently ubiquitous feature of time perception, and forms the foundation of several theories of timing. We sought evidence for Weber's law in temporal production and categorization experiments. The production task required pigeons to switch between keys within a specified temporal window. The categorization task required them to classify a stimulus duration as either short or long. Weber fractions did not descend to a horizontal asymptote, but were U-shaped: they decreased as a function of target duration, and increased again at intermediate and long durations. This pattern conforms neither to Weber's law, nor to its generalized form (Getty, D.J., 1975. Discrimination of short temporal intervals: a comparison of two models. Percept. Psychophys. 18, 1-8). A model of counter failure accommodated the U-shaped pattern.     

Serial conditional discrimination and temporal bisection in rats selectively lesioned in the dentate gyrus

In positive serial conditional discrimination, animals respond during a target stimulus when it is preceded by a feature stimulus, but they do not respond when the same target stimulus is presented alone. Moreover, the feature and target stimuli are separated from each other by an empty interval. The present work aimed to investigate if two durations (4 or 16 s) of the same feature stimulus (light) could modulate the operant responses of rats to different levers (A and B) during a 5-s target stimulus (tone). In the present study, lever A was associated with the 4-s light, and lever B was associated with the 16-s light. A 5-s empty interval was included between the light and the tone. In the same training procedure, the rats were also presented with the 5-s tone without the preceding light stimuli. In these trials, the responses were not reinforced. We evaluated the hippocampal involvement of these behavioral processes by selectively lesioning the dentate gyrus with colchicine. Once trained, the rats were submitted to a test using probe trials without reinforcement. They were presented with intermediate durations of the feature stimulus (light) to obtain a temporal bisection curve recorded during the exposure to the target stimuli. The rats from both groups learned to respond with high rates during tones preceded by light and with low rates during tones presented alone, which indicated acquisition of the serial conditional discrimination. The rats were able to discriminate between the 4- and 16-s lights by correctly choosing lever A or B. In the test, the temporal bisection curves from both experimental groups showed a bisection point at the arithmetic mean between 4 and 16 s. Such processes were not impaired by the dentate gyrus lesion. Thus, our results showed that different durations of a feature stimulus could result in conditional properties. However, this processing did not appear to depend on the dentate gyrus alone.     

Rats' memory for event duration in delayed matching-to-sample with nonspatial comparison response alternatives

Previous research has suggested that using stationary and moving levers as nonspatial response alternatives can significantly enhance the speed of acquiring a temporal discrimination in rats. In Experiment 1, rats were trained to discriminate 2 and 8s of magazine light illumination by responding to either a stationary lever or a moving lever with a cue light illuminated above it. Rats learned to discriminate event durations at a high level of accuracy after 25 sessions of training. During subsequent delay tests, rats exhibited a strong choose-long bias, indicating that they were timing from the onset of the magazine light until the entry of levers into the chamber. This occurred regardless of whether intertrial intervals and delay intervals were dark or illuminated. On test trials in which the sample was omitted, rats responded as if the short sample had been presented. In Experiment 2, the rats received extensive training with dark and illuminated variable delay intervals (1-4 s). However, they continued to exhibit a tendency to time from the onset of the magazine light until entry of the levers into the chamber. Although the use of stationary/uncued and moving/cued levers as response alternatives enhanced the speed of acquisition of the event duration discrimination in rats, additional procedural modifications will be necessary to prevent rats from timing during the delay interval.