Reversal Learning and Associative Memory Impairments in a BACHD Rat Model for Huntington Disease

Chorea and psychiatric symptoms are hallmarks of Huntington disease (HD), a neurodegenerative disorder, genetically characterized by the presence of expanded CAG repeats (>35) in the HUNTINGTIN (HTT) gene. HD patients present psychiatric symptoms prior to the onset of motor symptoms and we recently found a similar emergence of non motor and motor deficits in BACHD rats carrying the human full length mutated HTT (97 CAG-CAA repeats). We evaluated cognitive performance in reversal learning and associative memory tests in different age cohorts of BACHD rats. Male wild type (WT) and transgenic (TG) rats between 2 and 12 months of age were tested. Learning and strategy shifting were assessed in a cross-maze test. Associative memory was evaluated in different fear conditioning paradigms (context, delay and trace). The possible confound of a fear conditioning phenotype by altered sensitivity to a ‘painful’ stimulus was assessed in a flinch-jump test. In the cross maze, 6 months old TG rats showed a mild impairment in reversal learning. In the fear conditioning tasks, 4, 6 and 12 months old TG rats showed a marked reduction in contextual fear conditioning. In addition, TG rats showed impaired delay conditioning (9 months) and trace fear conditioning (3 months). This phenotype was unlikely to be affected by a change in ‘pain’ sensitivity as WT and TG rats showed no difference in their threshold response in the flinch-jump test. Our results suggest that BACHD rats have a profound associative memory deficit and, possibly, a deficit in reversal learning as assessed in a cross maze task. The time course for the emergence of these symptoms (i.e., before the occurrence of motor symptoms) in this rat model for HD appears similar to the time course in patients. These data suggest that BACHD rats may be a useful model for preclinical drug discovery.     

Reduced Motivation in the BACHD Rat Model of Huntington Disease Is Dependent on the Choice of Food Deprivation Strategy

Huntington disease (HD) is an inherited neurodegenerative disease characterized by motor, cognitive, psychiatric and metabolic symptoms. Animal models of HD show phenotypes that can be divided into similar categories, with the metabolic phenotype of certain models being characterized by obesity. Although interesting in terms of modeling metabolic symptoms of HD, the obesity phenotype can be problematic as it might confound the results of certain behavioral tests. This concerns the assessment of cognitive function in particular, as tests for such phenotypes are often based on food depriving the animals and having them perform tasks for food rewards. The BACHD rat is a recently established animal model of HD, and in order to ensure that behavioral characterization of these rats is done in a reliable way, a basic understanding of their physiology is needed. Here, we show that BACHD rats are obese and suffer from discrete developmental deficits. When assessing the motivation to lever push for a food reward, BACHD rats were found to be less motivated than wild type rats, although this phenotype was dependent on the food deprivation strategy. Specifically, the phenotype was present when rats of both genotypes were deprived to 85% of their respective free-feeding body weight, but not when deprivation levels were adjusted in order to match the rats'' apparent hunger levels. The study emphasizes the importance of considering metabolic abnormalities as a confounding factor when performing behavioral characterization of HD animal models.     

Assessment of Motor Function,Sensory Motor Gating and Recognition Memory in a Novel BACHD Transgenic Rat Model for Huntington Disease

Rationale

Huntington disease (HD) is frequently first diagnosed by the appearance of motor symptoms; the diagnosis is subsequently confirmed by the presence of expanded CAG repeats (> 35) in the HUNTINGTIN (HTT) gene. A BACHD rat model for HD carrying the human full length mutated HTT with 97 CAG-CAA repeats has been established recently. Behavioral phenotyping of BACHD rats will help to determine the validity of this model and its potential use in preclinical drug discovery studies.

Objectives

The present study seeks to characterize the progressive emergence of motor, sensorimotor and cognitive deficits in BACHD rats.

Materials and Methods

Wild type and transgenic rats were tested from 1 till 12 months of age. Motor tests were selected to measure spontaneous locomotor activity (open field) and gait coordination. Sensorimotor gating was assessed in acoustic startle response paradigms and recognition memory was evaluated in an object recognition test.

Results

Transgenic rats showed hyperactivity at 1 month and hypoactivity starting at 4 months of age. Motor coordination imbalance in a Rotarod test was present at 2 months and gait abnormalities were seen in a Catwalk test at 12 months. Subtle sensorimotor changes were observed, whereas object recognition was unimpaired in BACHD rats up to 12 months of age.

Conclusion

The current BACHD rat model recapitulates certain symptoms from HD patients, especially the marked motor deficits. A subtle neuropsychological phenotype was found and further studies are needed to fully address the sensorimotor phenotype and the potential use of BACHD rats for drug discovery purposes.     

Cardiac Dysfunction in the BACHD Mouse Model of Huntington’s Disease

While Huntington’s disease (HD) is classified as a neurological disorder, HD patients exhibit a high incidence of cardiovascular events leading to heart failure and death. In this study, we sought to better understand the cardiovascular phenotype of HD using the BACHD mouse model. The age-related decline in cardiovascular function was assessed by echocardiograms, electrocardiograms, histological and microarray analysis. We found that structural and functional differences between WT and BACHD hearts start at 3 months of age and continue throughout life. The aged BACHD mice develop cardiac fibrosis and ultimately apoptosis. The BACHD mice exhibited adaptive physiological changes to chronic isoproterenol treatment; however, the medication exacerbated fibrotic lesions in the heart. Gene expression analysis indicated a strong tilt toward apoptosis in the young mutant heart as well as changes in genes involved in cellular metabolism and proliferation. With age, the number of genes with altered expression increased with the large changes occurring in the cardiovascular disease, cellular metabolism, and cellular transport clusters. The BACHD model of HD exhibits a number of changes in cardiovascular function that start early in the disease progress and may provide an explanation for the higher cardiovascular risk in HD.     

Effect of genotype and day or night time of testing on mice behavior in the light-dark box and the open-field tests

The light-dark box (LDB) and the open-field (OF) tests are widespread experimental models for studying locomotion and anxiety in laboratory rats and mice. The fact that rodents are nocturnal animals and more active at night raises a critical question of whether behavioral experiments carried out in the light phase are methodologically correct. Parameters of behavior of four mouse strains (C57BL/6J, DBA2/J, AKR/J and CBA/LacJ) in the light-dark box and open-field tests in the light and dark phases were compared. No significant influence of the phase of testing on anxiety in LDB and OF tests was revealed. In the OF test CBA mice showed increased locomotor activity, whereas AKR and C57BL/6 mice showed increased defecation in the dark phase. It was concluded that: 1) the phase of testing is not crucial for the expression of anxiety in LDB and OF; 2) the sensitivity to the phase of testing depends on the genotype; 3) the indices of behavior in the genotypes sensitive to the phase of testing (locomotion in the CBA and defecation in the AKR and C57BL/6 mouse strains) are increased in the dark phase.     

Behavioral and neurochemical changes in the zebrafish leopard strain

The zebrafish leopard phenotype (leo) displays abnormal pigmentation and shows increased anxiety‐like behavior. The neurochemical changes associated with this anxious phenotype are not known. Here, we demonstrate that leo show increased anxiety‐like behavior in the light/dark box and in the novel tank test. This anxious phenotype is rescued by acute treatment with a dose of a serotonin reuptake inhibitor, fluoxetine, that is inactive in wild‐type animals. Moreover, leo show decreased tissue levels of serotonin, increased serotonin turnover and slightly increased monoamine oxidase activity. These results suggest that the anxious phenotype observed in leo zebrafish is caused by a decrease in serotonin uptake. This work could open an important avenue in defining the neurochemical underpinning of natural variation in anxiety disorders .     

CB1 receptor signaling regulates social anxiety and memory

The endocannabinoid (eCB) system regulates emotion, stress, memory and cognition through the cannabinoid type 1 (CB₁) receptor. To test the role of CB₁ signaling in social anxiety and memory, we utilized a genetic knockout (KO) and a pharmacological approach. Specifically, we assessed the effects of a constitutive KO of CB₁ receptors (CB₁KOs) and systemic administration of a CB₁ antagonist (AM251; 5 mg/kg) on social anxiety in a social investigation paradigm and social memory in a social discrimination test. Results showed that when compared with wild‐type (WT) and vehicle‐treated animals, CB₁KOs and WT animals that received an acute dose of AM251 displayed anxiety‐like behaviors toward a novel male conspecific. When compared with WT animals, KOs showed both active and passive defensive coping behaviors, i.e. elevated avoidance, freezing and risk‐assessment behaviors, all consistent with an anxiety‐like profile. Animals that received acute doses of AM251 also showed an anxiety‐like profile when compared with vehicle‐treated animals, yet did not show an active coping strategy, i.e. changes in risk‐assessment behaviors. In the social discrimination test, CB₁KOs and animals that received the CB₁ antagonist showed enhanced levels of social memory relative to their respective controls. These results clearly implicate CB₁ receptors in the regulation of social anxiety, memory and arousal. The elevated arousal/anxiety resulting from either total CB₁ deletion or an acute CB₁ blockade may promote enhanced social discrimination/memory. These findings may emphasize the role of the eCB system in anxiety and memory to affect social behavior .     

Avoidance behaviour and anxiety in rats irradiated with a sublethal dose of gamma-rays

The aim of this study was to assess, whether a sublethal dose of gamma-rays will influence the avoidance behaviour and anxiety in rats and whether the response to radiation depends on time of day of its application. Adult male Wistar rats were tested in elevated plus-maze, in hot plate test and in the light/dark box in 4 regular intervals during a day. After two weeks the animals were irradiated with a whole-body dose 6 Gy of gamma-rays. One day after irradiation the animals were repeatedly tested in the same way, as before irradiation. In the plus-maze test an increased level of anxiety was established. The irradiation significantly decreased the locomotor activity of rats, but the extent of exploratory and comfortable behaviour were not altered. After irradiation, an elevated aversion to the thermal stimulus was observed in the hot plate test. The effects of radiation were more pronounced in the light period of the day, than in the dark one. No significant differences in aversion to light were detected after irradiation. The obtained results indicate, that sublethal doses of ionizing radiation can markedly influence the reactivity of animals to adverse stimuli, their motoric activity and emotional status, as well.     

Long-term individual housing in C57BL/6J and DBA/2 mice: assessment of behavioral consequences

The aim of the present study was to investigate the effects of individual housing on mouse behavior. The male mice of the C57BL/6J and DBA/2 strains were separated at the age of 4 weeks and kept in individual housing for 7 weeks until behavioral testing began. Their behavior was compared to the group-housed mice in a battery of tests during the following 7 weeks. The single-housed mice were hyperactive and displayed reduced habituation in the tests assessing activity and exploration. Reduced anxiety was established in the elevated plus-maze, but an opposite effect was observed in the dark-light (DL) and hyponeophagia tests. Immobility in the forced swimming test was reduced by social isolation. The DBA mice displayed higher anxiety-like behavior than the B6 mice in the plus-maze and DL exploration test, but hyponeophagia was reduced in the DBA mice. Moreover, all effects of individual housing on the exploratory and emotional behavior were more evident in the DBA than in the B6 mice. Novel object recognition and fear conditioning (FC) were significantly impaired in the single-housed mice, whereas water-maze (WM) learning was not affected. Marked strain differences were established in all three learning tests. The B6 mice performed better in the object recognition and FC tasks. Initial spatial learning in the WM was faster and memory retention slightly enhanced in the B6 mice. The DBA mice displayed lower preference to the new and enhanced preference to the old platform location than the B6 mice after reversal learning in the WM. We conclude that individual housing has strong strain- and test-specific effects on emotional behavior and impairs memory in certain tasks.     

Evidence for lack of direct causality between pain and affective disturbances in a rat peripheral neuropathy model

Chronic pain is frequently accompanied by the manifestation of emotional disturbances and cognitive deficits. While a causality relation between pain and emotional/cognitive disturbances is generally assumed, several observations suggest a temporal dissociation and independent mechanisms. We therefore studied Sprague‐Dawley rats that presented a natural resistance to pain manifestation in a neuropathy model (spared nerve injury [SNI]) and compared their performance in a battery of behavioral paradigms—anxiety, depression and fear memory—with animals that presented a pain phenotype. Afterward, we performed an extensive volumetric analysis across prefrontal, orbitofrontal and insular cortical areas. The majority of SNI animals manifested mechanical allodynia (low threshold [LT]), but 13% were similar to Sham controls (high threshold [HT]). Readouts of spontaneous hypersensivity (paw flinches) were also significantly reduced in HT and correlated with allodynia. To increase the specificity of our findings, we segregated the SNI animals in those with left (SNI‐L) and right (SNI‐R) lesions and the lack of association between pain and behavior still remains. Left‐lesioned animals, independent of the LT or HT phenotype, presented increased anxiety‐like behaviors and decreased well‐being. In contrast, we found that the insular cortex (agranular division) was significantly smaller in HT than in LT. To conclude, pain and emotional disturbances observed following nerve injury are to some extent segregated phenomena. Also, HT and LT SNI presented differences in insular volumes, an area vastly implicated in pain perception, suggesting a supraspinal involvement in the manifestation of these phenotypes.     

Altered stress responsiveness and hypothalamic‐pituitary‐adrenal axis function in male rat offspring of socially isolated parents

Social isolation in male rats at weaning results in reduced basal levels of the neuroactive steroid 3α,5α‐tetrahydroprogesterone (3α,5α‐TH PROG) in the brain and plasma as well as increased anxiety‐like behavior. We now show that socially isolated female rats also manifest a reduced basal cerebrocortical concentration of 3α,5α‐TH PROG as well as an anxiety‐like profile in the elevated plus‐maze and Vogel conflict tests compared with group‐housed controls. In contrast, despite the fact that they were raised under normal conditions, adult male offspring of male and female rats subjected to social isolation before mating exhibited an increased basal cerebrocortical level of 3α,5α‐TH PROG but no difference in emotional reactivity compared with the offspring of group‐housed parents. These animals also showed an increased basal activity of the hypothalamic‐pituitary‐adrenal axis as well as reduced abundance of corticotropin‐releasing factor in the hypothalamus and of corticotropin‐releasing factor receptor type 1 in the pituitary. Moreover, negative feedback regulation of hypothalamic‐pituitary‐adrenal axis activity by glucocorticoid was enhanced in association with up‐regulation of glucocorticoid receptor expression in the hippocampus. There was also attenuation of corticosterone release induced by foot‐shock stress in the offspring of socially isolated parents. The increase in the brain concentration of 3α,5α‐TH PROG induced by acute stress was also blunted in these animals. Our results thus show that a stressful experience before mating can influence neuroendocrine signaling in the next generation.     

Differential Effects of Dietary Oils on Emotional and Cognitive Behaviors

Several dietary oils have been used preventatively and therapeutically in the setting of neurological disease. However, the mechanisms underlying their influence on brain function and metabolism remain unknown. It was investigated whether 3 types of dietary oils affected emotional behaviors in mice. Wild-type (WT) mice and sialyltransferase ST3Gal IV-knockout (KO) mice, which exhibit increased emotional and cognitive behaviors, were fed diets containing 20% dietary oils from post-weaning to adulthood. Mice were fed pellets made from control feed AIN93G powder containing 18% fish oil, soybean oil, or a mixture of 1-palmitoyl-2-oleoyl-3-palmitoyl glycerol (POP) and 1-stearoyl-2-oleoyl-3-stearoyl glycerol (SOS), plus 2% soybean oil. Once mice reached adulthood, they were subjected to fear conditioning test to measure cognitive anxiety and forced swim test to measure depression. WT mice fed the POP-SOS diet showed a 0.6-fold decrease in percent freezing with contextual fear compared with WT mice fed the control diet. KO mice fed the fish oil diet showed a 1.4-fold increase in percent freezing with contextual fear compared with KO mice fed the control diet. These findings indicate that response to contextual fear was improved in WT mice that consumed POP-SOS but aggravated in KO mice that consumed fish oils. Furthermore, KO mice showed a 0.4-fold decrease in percent freezing in response to tone fear when they were fed POP-SOS diet compared to a control diet. Thus, POP-SOS diet reduced tone fear level of KO mice until the same level of WT mice. Finally, KO mice fed the soybean oil diet showed a 1.7-fold increase in immobility in the forced swim test compared to KO mice fed the control diet. Taken together, oil-rich diets differentially modulate anxiety and depression in normal and anxious mice. Oils rich in saturated fatty acids may alleviate anxiety more strongly than other oils.     

Chronic exposure to an extremely low‐frequency magnetic field induces depression‐like behavior and corticosterone secretion without enhancement of the hypothalamic–pituitary–adrenal axis in mice

An extremely low‐frequency magnetic field (ELF‐MF) is generated by power lines and household electrical devices. Many studies have suggested an association between chronic ELF‐MF exposure and anxiety and/or depression. The mechanism of these effects is assumed to be a stress response induced by ELF‐MF exposure. However, this mechanism remains controversial. In the present study, we investigated whether chronic ELF‐MF exposure (intensity, 3 mT; total exposure, 200 h) affected emotional behavior and corticosterone synthesis in mice. ELF‐MF‐treated mice showed a significant increase in total immobility time in a forced swim test and showed latency to enter the light box in a light–dark transition test, compared with sham‐treated (control) mice. Corticosterone secretion was significantly high in the ELF‐MF‐exposed mice; however, no changes were observed in the amount of the adrenocorticotropic hormone and the expression of genes related to stress response. Quantification of the mRNA levels of adrenal corticosteroid synthesis enzymes revealed a significant reduction in Cyp17a1 mRNA in the ELF‐MF‐exposed mice. Our findings suggest the possibility that high intensity and chronic exposure to ELF‐MF induces an increase in corticosterone secretion, along with depression‐ and/or anxiety‐like behavior, without enhancement of the hypothalamic–pituitary–adrenal axis. Bioelectromagnetics 34:43–51, 2013. © 2012 Wiley Periodicals, Inc.     

Housing mice in the individually ventilated or open cages—Does it matter for behavioral phenotype?

Individually ventilated caging (IVC) systems for rodents are increasingly common in laboratory animal facilities. However, the impact of such substantial change in housing conditions on animal physiology and behavior is still debated. Most importantly, there arise the questions regarding reproducibility and comparison of previous or new phenotypes between the IVC and open cages. The present study was set up for detailed and systematic comparison of behavioral phenotypes in male and female mice of three widely used inbred strains (C57BL/6JRccHsd, DBA/2JRccHsd, 129S2/SvHSd) after being kept in two housing environments (IVC and open cages) for 6 weeks (since 4 weeks of age) before behavioral testing. The tests addressed exploratory, anxiety‐like and stress‐related behavior (light‐dark box, open field, forced swim test, stress‐induced hyperthermia), social approach and species‐specific behavior (nest building, marble burying). In all tests, large and expected strain differences were found. Somewhat surprisingly, the most striking effect of environment was found for basal body temperature and weight loss after one night of single housing in respective cages. In addition, the performance in light‐dark box and open field was affected by environment. Several parameters in different tests showed significant interaction between housing and genetic background. In summary, the IVC housing did not invalidate the well‐known differences between the mouse strains which have been established by previous studies. However, within the strains the results can be influenced by sex and housing system depending on the behavioral tasks applied. The bottom‐line is that the environmental conditions should be described explicitly in all publications.     

A clinical genetic method to identify mechanisms by which pain causes depression and anxiety

Most current methods for assessing pain in animals are based on reflexive measures and require constant interaction between the observer and the animal. Here we explore two new fully automated methods to quantify the impact of pain on the overall behavior of the organism. Both methods take advantage of the animals' natural preference for a dark environment. We used a box divided into two compartments: dark and bright. In the motoric operant task, "AngleTrack", one end of the box was raised so that the animals had to climb uphill to go from the light to the dark compartment. In the thermal operant task, "ThermalTrack", the floor of the dark compartment was heated to a given temperature, while the light compartment remained at 25°C. Rats were individually placed in the light box and their crossing between chambers monitored automatically for 30 minutes. The angle of the box, or the temperature of the dark compartment, was altered to challenge the animals' natural preference. We test the hypothesis that different models of pain (inflammatory or neuropathic) can be differentiated based on performance on these devices. Three groups of rats were tested at five different challenge levels on both tasks: 1) normal, 2) neuropathic injury pain (Spared Nerve Injury), and 3) inflammatory pain (intraplantar injection of Carrageenan). We monitored the position of the animals as well as their rate of switching between compartments. We find significant differences between the three groups and between the challenge levels both in their average position with respect to time, and in their switching rates. This suggests that the angle-track and thermal-track may be useful in assessing automatically the global impact of different types of pain on behavior.     

Enkephalin knockout male mice are resistant to chronic mild stress

Enhanced stress reactivity or sensitivity to chronic stress increases the susceptibility to mood pathologies such as major depression. The opioid peptide enkephalin is an important modulator of the stress response. Previous studies using preproenkephalin knockout (PENK KO) mice showed that these animals exhibit abnormal stress reactivity and show increased anxiety behavior in acute stress situations. However, the consequence of enkephalin deficiency in the reactivity to chronic stress conditions is not known. In this study, we therefore submitted wild‐type (WT) and PENK KO male mice to chronic stress conditions, using the chronic mild stress (CMS) protocol. Subsequently, we studied the CMS effects on the behavioral and hormonal level and also performed gene expression analyses. In WT animals, CMS increased the expression of the enkephalin gene in the paraventricular nucleus (PVN) of the hypothalamus and elevated the corticosterone levels. In addition, WT mice exhibited enhanced anxiety in the zero‐maze test and depression‐related behaviors in the sucrose preference and forced swim tests. Surprisingly, in PENK KO mice, we did not detect anxiety and depression‐related behavioral changes after the CMS procedure, and even measured a decreased hormonal stress response. These results indicate that PENK KO mice are resistant to the CMS effects, suggesting that enkephalin enhances the reactivity to chronic stress.     

Different emotional disturbances in two experimental models of temporal lobe epilepsy in rats

Affective symptoms such as anxiety and depression are frequently observed in patients with epilepsy. The mechanisms of comorbidity of epilepsy and affective disorders, however, remain unclear. Diverse models are traditionally used in epilepsy research, including the status epilepticus (SE) model in rats, which are aimed at generating chronic epileptic animals; however, the implications of different SE models and rat strains in emotional behaviors has not been reported. To address this issue, we examined the emotional sequelae of two SE models of temporal lobe epilepsy (TLE)--the lithium-pilocarpine (LIP) model and the kainic acid (KA) model--in two different rat strains (Wistar and Sprague-Dawley), which differ significantly in the pattern and extent of TLE-associated brain lesions. We found differences between LIP- and KA-treated animals in tests for depression-like and anxiety-like behaviors, as well as differences in plasma corticosterone levels. Whereas only LIP-treated rats displayed increased motivation to consume saccharin, both SE models led to reduced motivation for social contact, with LIP-treated animals being particularly affected. Evaluation of behavior in the open field test indicated very low levels of anxiety in LIP-treated rats and a mild decrease in KA-treated rats compared to controls. After exposure to a battery of behavioral tests, plasma corticosterone levels were increased only in LIP-treated animals. This hyperactivity in the hypothalamus-pituitary-adrenocortical (HPA) axis was highly correlated with performance in the open field test and the social interaction test, suggesting that comorbidity of epilepsy and emotional behaviors might also be related to other factors such as HPA axis function. Our results indicate that altered emotional behaviors are not inherent to the epileptic condition in experimental TLE; instead, they likely reflect alterations in anxiety levels related to model-dependent dysregulation of the HPA axis.