Changes in the mesocorticolimbic pathway after low dose reserpine-treatment in Wistar and Spontaneously Hypertensive Rats (SHR): Implications for cognitive deficits in a progressive animal model for Parkinson's disease.

Reserpine (RES) is an irreversible inhibitor of VMAT2 used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low dose of reserpine was proposed as a model capable of emulating progressive neurochemical, motor and non-motor impairments in PD. Conversely, compared to Wistar rats, Spontaneously Hypertensive Rats (SHR) are resistant to motor changes induced by repeated treatment with a low dose of RES. However, such resistance has not yet been investigated for RES-induced non-motor impairments. We aimed to assess whether SHR would have differential susceptibility to the object recognition deficit induced by repeated low-dose reserpine treatment. We submitted male Wistar and SHR rats to repeated RES treatment (15 s.c. injections of 0.1 mg/kg, every other day) and assessed object memory acquisition and retrieval 48 h after the 6th RES injection (immediately before the appearance of motor impairments). Only RES Wistar rats displayed memory impairment after reserpine treatment. On the other hand, untreated SHR rats displayed object recognition memory deficit, but RES treatment restored such deficits. We also performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last RES injection. In a different set of animals submitted to the same treatment, we quantified DA, 5-HT and products of lipid peroxidation in the prefrontal cortex (PFC) and hippocampus (HPC). SHR presented increased constitutive levels of DA in the PFC and reduced immunoreactivity to TH in the medial PFC and dorsal HPC. Corroborating the behavioral findings, RES treatment restored those constitutive alterations in SHR. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potentially relevant targets to the study of susceptibility to diseases related to dopaminergic alterations.

Changes in the mesocorticolimbic pathway after low dose reserpine-treatment in Wistar and Spontaneously Hypertensive Rat (SHR): implications for cognitive deficits in a progressive animal model for Parkinson’s Disease

Reserpine (RES) is an irreversible inhibitor of VMAT2 used to study Parkinson’s disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low dose of reserpine was proposed as a model capable of emulating progressive neurochemical, motor and non-motor impairments in PD. Conversely, compared to Wistar rats, Spontaneously Hypertensive Rats (SHR) are resistant to motor changes induced by repeated treatment with a low dose of RES. However, such resistance has not yet been investigated for RES-induced non-motor impairments. We aimed to assess whether SHR would have differential susceptibility to the object recognition deficit induced by repeated low-dose reserpine treatment. We submitted male Wistar and SHR rats to repeated RES treatment (15 s.c. injections of 0.1 mg/kg, every other day) and assessed object memory acquisition and retrieval 48 h after the 6th RES injection (immediately before the appearance of motor impairments). Only RES Wistar rats displayed memory impairment after reserpine treatment. On the other hand, untreated SHR rats displayed object recognition memory deficit, but RES treatment restored such deficits. We also performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last RES injection. In a different set of animals submitted to the same treatment, we quantified DA, 5-HT and products of lipid peroxidation in the prefrontal cortex (PFC) and hippocampus (HPC). SHR presented increased constitutive levels of DA in the PFC and reduced immunoreactivity to TH in the medial PFC and dorsal HPC. Corroborating the behavioural findings, RES treatment restored those constitutive alterations in SHR. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potentially relevant targets to the study of susceptibility to diseases related to dopaminergic alterations.

Variants in SNCA Gene Are Associated with Parkinson's Disease Risk and Cognitive Symptoms in a Brazilian Sample.

Genetic susceptibility contributes to the etiology of sporadic Parkinson's Disease (PD) and worldwide studies have found positive associations of polymorphisms in the alpha-synuclein gene (SNCA) with the risk for PD. However, little is known about the influence of variants of SNCA in individual traits or phenotypical aspects of PD. Further, there is a lack of studies with Latin-American samples. We evaluated the association between SNCA single nucleotide polymorphisms (single nucleotide polymorphisms, SNPs - rs2583988, rs356219, rs2736990, and rs11931074) and PD risk in a Brazilians sample. In addition, we investigated their potential interactions with environmental factors and specific clinical outcomes (motor and cognitive impairments, depression, and anxiety). A total of 105 PD patients and 101 controls participated in the study. Single locus analysis showed that the risk allele of all SNPs were more frequent in PD patients (p < 0.05), and the associations of SNPs rs2583988, rs356219, and rs2736990 with increased PD risk were confirmed. Further, the G-rs356219 and C-rs2736990 alleles were associated with early onset PD. T-rs2583988, G-rs356219 and C-2736990 alleles were significantly more frequent in PD patients with cognitive impairments than controls in this condition. In addition, in a logistic regression model, we found an association of cognitive impairment with PD, and the practice of cognitive activity and smoking habits had a protective effect. This study shows for the first time an association of SNCA polymorphism and PD in a South-American sample. In addition, we found an interaction between SNP rs356219 and a specific clinical outcome, i.e., the increased risk for cognitive impairment in PD patients.

Spontaneously Hypertensive Rats (SHR) Are Resistant to a Reserpine-Induced Progressive Model of Parkinson's Disease: Differences in Motor Behavior, Tyrosine Hydroxylase and α-Synuclein Expression.

Reserpine is an irreversible inhibitor of vesicular monoamine transporter-2 (VMAT2) used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low-dose of reserpine was proposed as a progressive model of PD. Rats under this treatment show progressive catalepsy behavior, oral movements and spontaneous motor activity decrement. In parallel, compared to Wistar rats, spontaneously hypertensive rats (SHR) are resistant to acute reserpine-induced oral dyskinesia. We aimed to assess whether SHR would present differential susceptibility to repeated reserpine-induced deficits in the progressive model of PD. Male Wistar and SHR rats were administered 15 subcutaneously (s.c.) injections of reserpine (0.1 mg/kg) or vehicle, every other day and motor activity was assessed by the catalepsy, oral movements and open field tests. Only reserpine-treated Wistar rats presented increased latency to step down in the catalepsy test and impaired spontaneous activity in the open field. On the other hand, there was an increase in oral movements in both reserpine-treated strains, although with reduced magnitude and latency to instauration in SHR. After a 15-day withdrawn period, both strains recovered from motor impairment, but SHR animals expressed reduced latencies to reach control levels. Finally, we performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last injection or 15 days after withdrawn. Reserpine-treated animals presented a reduction in TH and an increase in α-syn immunoreactivity in the substantia nigra and dorsal striatum (dSTR), which were both recovered after 15 days of withdraw. Furthermore, SHR rats were resistant to reserpine-induced TH decrement in the substantia nigra, and presented reduced immunoreactivity to α-syn in the dSTR relative to Wistar rats, irrespective of treatment. This effect was accompanied by increase of malondaldhyde (MDA) in the striatum of reserpine-treated Wistar rats, while SHR presented reduced MDA in both control and reserpine conditions relative to Wistar strain. In conclusion, the current results show that SHR are resilient to motor and neurochemical impairments induced by the repeated low-dose reserpine protocol. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potential relevant targets to the study of susceptibility to PD.

Diazepam effects on aversive memory retrieval and extinction: Role of anxiety levels.

Benzodiazepines (BDZs) are anxiolytic drugs that impair memory acquisition. Previous studies using the plus-maze discriminative avoidance task (PMDAT, which assesses memory and anxiety concomitantly) indicated that the effects of BDZs on anxiety and acquisition are related to each other. The possible influence of the anxiolytic action of BDZs on their effects on memory retrieval and extinction are poorly understood. This is relevant considering the relationship between aversive memories and anxiety disorders. We designed a modified protocol of PMDAT that evaluates anxiety during retrieval and extinction of the task. Male Wistar rats were trained in the PMDAT (plus-maze with two open and two enclosed arms) using a standard or a modified protocol. In the standard protocol, the aversive stimuli were presented in one of the enclosed arms during training, and the animal had free access to the whole apparatus. In the modified protocol, the open arms were blocked with glass walls. Twenty-four hours after training, the animals subjected to each of the protocols were treated with saline or 2.0mg/kg of diazepam (DZP) 30min before the test. There was a third session in the maze (retest) 24h after the test. During the test, DZP impaired and improved retrieval in rats that had been trained in the standard and the modified protocol when compared to the respective saline-treated groups. In addition, treatment with DZP prior to the test induced anxiolysis, but only in the animals that were not pre-exposed to the open arms of the apparatus (modified protocol). In these animals, DZP impaired extinction, which was evaluated during retest session. The impairing effect of DZP on extinction seems to be related to its anxiolytic action during the test (extinction learning). Further, we suggest that aversive memory retrieval depends on both the treatment and the arousal elicited by exposure to the apparatus.

Cognitive, motor and tyrosine hydroxylase temporal impairment in a model of parkinsonism induced by reserpine.

Studies have suggested that cognitive deficits can precede motor alterations in Parkinson's disease (PD). However, in general, classic animal models are based on severe motor impairment after one single administration of neurotoxins, and thereby do not express the progressive nature of the pathology. A previous study showed that the repeated administration with a low dose (0.1mg/kg) of the monoamine depleting agent reserpine induces a gradual appearance of motor signs of pharmacological parkinsonism in rats. Here, we showed this repeated treatment with reserpine induced a memory impairment (evaluated by the novel object recognition task) before the gradual appearance of the motor signs. Additionally, these alterations were accompanied by decreased tyrosine hydroxylase (TH) striatal levels and reduced number of TH+ cells in substantia nigra pars compacta (SNpc). After 30 days without treatment, reserpine-treated animals showed normal levels of striatal TH, partial recovery of TH+ cells in SNpc, recovery of motor function, but not reversal of the memory impairment. Furthermore, the motor alterations were statistically correlated with decreased TH levels (GD, CA1, PFC and DS) and number of TH+ cells (SNpc and VTA) in the brain. Thus, we extended previous results showing that the gradual appearance of motor impairment induced by repeated treatment with a low dose of reserpine is preceded by short-term memory impairment, as well as accompanied by neurochemical alterations compatible with the pathology of PD.

Repeated treatment with a low dose of reserpine as a progressive model of Parkinson's disease.

Animal models are widely used to study alterations caused by Parkinson's disease (PD). However, in general, pharmacological models do not express the progressive nature of the disease, being characterized by immediate severe motor impairment after a single dose of the drug. Reserpine administration in rodents has been suggested as a pharmacological model of PD based on the effects of this monoamine-depleting agent on motor activity. Here, we describe that repeated administration with a low dose (0.1 mg/kg) of reserpine in rats induces a gradual appearance of motor signs, evaluated by catalepsy behavior. Furthermore, these motor signs are accompanied by increased levels of striatal lipid peroxidation. However, treatment with reserpine failed to induce memory impairments (evaluated by novel object recognition and discriminative avoidance tasks) and alterations in hippocampal lipid peroxidation. Thus, repeated treatment with low doses of reserpine progressively induces alterations in motor function and an increase in striatal oxidative stress, indicating a possible application of this model in the study of the neuroprogressive nature of the motor signs in PD.

Antidepressants differentially modify the extinction of an aversive memory task in female rats.

Treatment of major depression, posttraumatic stress disorder and other psychopathologies with antidepressants can be associated with improvement of the cognitive deficits related to these disorders. Although the mechanisms of these effects are not completely elucidated, alterations in the extinction of aversive memories are believed to play a role in these psychopathologies. We have recently verified that female rats present low levels of extinction when submitted to the plus-maze discriminative avoidance task. In the present study, female rats were treated long term with clinically used antidepressants (fluoxetine, nortriptyline or mirtazapine) and subjected to the plus-maze discriminative avoidance task to evaluate learning, memory, extinction and anxiety-related behaviors as well as behavioral despair in the forced swimming test. All groups learned the task and exhibited retrieval. Chronic treatment with fluoxetine (but not with the other antidepressants tested) increased extinction of the discriminative task. In the forced swimming test, the animals treated with fluoxetine and mirtazapine showed decreased immobility duration. In conclusion, fluoxetine potentiated extinction, while both fluoxetine and mirtazapine were effective in ameliorating depressive-like behavior in the forced swimming test, suggesting a possible dissociation between the effects on mood and the extinction of aversive memories in female rats.

The influence of sex and estrous cycle on QTL for emotionality and ethanol consumption.

The inbred rat strains Lewis (LEW) and Spontaneously Hypertensive Rats (SHR) differ with respect to several emotionality- and ethanol intake-related behaviors, one of which (inner locomotion in the open field; OF) is strongly influenced by a locus (Anxrr16) on chromosome (Chr) 4. We aimed to further investigate the influence of Chr 4 on these behaviors and to evaluate the role of the estrous cycle in QTL expression. LEW females and SHR males were intercrossed to produce F1 and F2 rats (96-97/sex), which were then tested in the OF, light-dark box (LDB), forced swimming test (FST), and an ethanol consumption procedure (ECP). In addition, another group of 96 F2 females were tested in the OF and LDB according to their estrous cycle phase. All animals were genotyped for microsatellite markers located on Chr 4 and two QTL analyses were performed. A factor analysis of the F2 population produced five factors reflecting different behavioral dimensions. QTL analysis revealed five significant loci in males, some of which with pleiotropic effects on behaviors measured in the OF, LDB, and ECP. The second QTL analysis revealed two significant loci in females in diestrous-proestrous and one in females in estrous-metestrous that influence behaviors in the OF and LDB. Results revealed that Anxrr16 and four other new QTL influence emotionality- and ethanol-related behaviors in male rats, whereas Anxrr16 attained suggestive levels only in females in diestrous-proestrous, which raises the need for taking into account factors related to the sex and estrous cycle in behavioral QTL analysis.

Absence of repeated-trial tolerance to the anxiolytic-like effects of chlordiazepoxide in the rat triple test.

The triple test, recently developed to assess anxiety-related behaviors in rodents, combines three widely used behavioral tests: the open field (OF), elevated plus maze (EPM) and light/dark box (LDB). The EPM and LDB, individually, are normally sensitive to the anxiolytic effects of benzodiazepines only in the first trial, due to the phenomenon of one-trial tolerance, which limits their use in longitudinal studies. The main objective of the present investigation was to verify whether the anxiolytic-like effects of chlordiazepoxide (CDZ), previously observed in naive animals submitted to the triple test, would persist after repeated testing. To this end, three experiments were carried out where male Wistar rats received CDZ (10mg/kg) 30min before the triple test for 2, 3 or 20 consecutive days. Except for the first day of drug treatment following a previous test experience in an undrugged state, CDZ had enduring anxiolytic-like effects under all schedules, promoting an increase in the exploration of the EPM open arms (and in some cases of the white compartment of the LDB), without affecting the number of closed-arm entries. The finding that rats did not develop tolerance to CDZ even with chronic treatment and repeated exposures to the triple test suggests that this new device is a promising tool to be used in longitudinal studies involving pharmacological manipulations of anxiety-related behaviors.

Sex differences in aversive memory in rats: possible role of extinction and reactive emotional factors.

Studies usually show better spatial learning in males and stronger emotional memory in females. Spatial memory differences could relate to diverse strategies, while dissimilar stress reactions could cause emotional memory differences. We compared male and female rats in two emotional (classical emotional conditioning and aversive discrimination memory) and two emotionally "neutral" tasks: (1) plus-maze discriminative avoidance, containing two open and two enclosed arms, one of which presenting aversive stimuli (light/noise). No differences were found in learning, retrieving, or basal emotional levels, while only male rats presented extinction of the task; (2) contextual fear conditioning--a cage was paired to mild foot shocks. Upon reexposure, freezing behavior was decreased in females; (3) spontaneous alternation--the animals were expected to alternate among the arms of a four-arm maze. No differences between genders were found and (4) open-field habituation was addressed in an arena which the rats were allowed to explore for 10 min. Habituation was similar between genders. Differences were found only in tasks with strong emotional contexts, where different fear responses and stress effects could be determinant. The lack of extinction of discriminative avoidance by females points out to stronger consolidation and/or impaired extinction of aversive memories.

Drug reinforcement in a rat model of attention deficit/hyperactivity disorder--the Spontaneously Hypertensive Rat (SHR).

The co-occurrence of drug addiction in adults with attention deficit hyperactivity disorder (ADHD) is very common, but its etiology remains largely unknown. Therefore, animal models to study this kind of psychiatric comorbidity are needed. The Spontaneously Hypertensive Rat (SHR) strain shows neurochemical and behavioral characteristics which make it a suitable model of ADHD. Compared with their normotensive controls (Wistar-Kyoto) and with some other rat strains, SHR rats drink more ethanol and are more sensitive to its anxiolytic/stimulant effects. They also show increased sensitivity to psychostimulants, opioids and cannabinoids. Furthermore, chronic treatment with methylphenidate, the first-choice drug to treat ADHD, during adolescence, changes the ethanol intake and the behavioral effects of cocaine in adult SHR rats. Regarding sex differences, females are more sensitive to psychostimulants and drink more ethanol than males, an important condition because in adulthood, more females suffer from ADHD than males. Taken together, the reviewed findings indicate that the SHR strain is a promising tool for studies on drug addiction and, possibly, its relationship with ADHD.

Association of a locus on rat chromosome 4 with anxiety-related behaviors in two selectively bred rat lines

The Floripa H and L rat lines, selected for high and low locomotion in the central aversive area of an open field, a widely used emotionality test, were proposed as a model for studying the genetic basis of anxiety. The present study aimed to verify if the QTL Ofil1, mapped to rat chromosome 4 and previously identified as being related to emotionality in another population of rats, contributes to the behavioral variability observed in the Floripa rat lines. To this purpose, rats of five generations of selective breeding were genotyped for two polymorphic markers, D4RAT59 and D4MGH27, flanking Ofil1. Changes in genotype and allele frequencies throughout generations were evaluated in both H and L lines, in order to assess if the bidirectional selection based on behavioral scores induced divergent changes in the genotype of this genome region. There were significant changes in genotype frequencies for both molecular markers, however, only the genotype variations of the D4RAT59 marker were significantly correlated with the variations in the selected phenotype. This result suggests that the region of the genome near D4RAT59 contains one or more genes contributing to the interindividual variation in central locomotion in the open field test.

Integrating the open field, elevated plus maze and light/dark box to assess different types of emotional behaviors in one single trial.

Current anxiety tests do not provide, individually, a pure and complete picture of an animal's emotional profile. Therefore, many authors test their experimental hypotheses using a series of anxiety-related tests, which are thought to reflect different facets of emotionality. The objective of this study was to investigate the potential usefulness of integrating three widely used behavioral tests, the open field (OF), elevated plus maze (EPM), and light/dark box (LDB), to assess a wider range of emotional and non-emotional behaviors within one single trial. A protocol was developed where rats could freely explore an OF that was physically connected to an EPM and a LDB during 15min. Classical anxiety- and locomotion-related behaviors from each test were measured. Lewis and spontaneously hypertensive rats (SHR) inbred strains, known to present genetic differences in each of the individual tests, differed for all anxiety-related behaviors from the combined apparatus. Factor analyses revealed that similar anxiety- and locomotion-related factors were produced by the three tests applied either separately or in combination. Under both conditions, each test produced its own anxiety-related factor. Two benzodiazepines, chlordiazepoxide (at 5 and 10mg/kg) and midazolam (at 0.75mg/kg), facilitated the approach towards the EPM open arms, whereas pentylenetetrazole (10mg/kg) specifically inhibited exploration of the three aversive areas (OF center, EPM open arms, LDB light compartment). Together, these results suggest that the new integrated apparatus may contribute to the study of anxiety, by providing a rapid, comprehensive and reliable method of assessing emotionality-related behaviors and its underlying components.

Chronic methylphenidate treatment during adolescence increases anxiety-related behaviors and ethanol drinking in adult spontaneously hypertensive rats.

Methylphenidate (MPD) is the most widely used drug in the treatment of attention-deficit hyperactivity disorder (ADHD), the symptoms of which can persist into adolescence and adulthood. The cooccurrence of other psychiatric disorders, such as anxiety and alcoholism, in adults with ADHD is very common, but its etiology remains largely unknown. This study examined the effects of chronic MPD treatment during adolescence on emotional and consummatory behaviors in adult spontaneously hypertensive rats (SHRs), often proposed as an animal model of ADHD. Adolescent SHRs of both sexes were given chronic treatment with MPD (2 mg/kg intraperitoneally; twice daily for 16 days) or vehicle and were subsequently tested in adulthood. The tests used were the open-field and the elevated plus-maze, thought to measure locomotor and emotionality-related behaviors, and a protocol of ethanol consumption. MPD elicited anxious-like behavior in the open-field (but not in the elevated plus-maze) regardless of sex, and enhanced ethanol intake in females. These findings suggest that MPD treatment during adolescence induces persistent changes on emotionality and ethanol consumption in SHRs, but these effects depend on the sex and behavioral test used. Potential clinical implications of these findings are discussed.

Evaluation of two genetic animal models in behavioral tests of anxiety and depression.

Anxiety- and depression-related disorders often appear associated and may be affected by common genetic factors. The inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) and the outbred rat lines Floripa H and L, which were selectively bred for high and low locomotion in the central area of the open field (OF) test, respectively, have been proposed as experimental tools to study anxiety. The main goal of the present study was to characterize the behavior of these animals in two models of anxiety, elevated plus-maze (EPM) and OF, in two models of depression, forced swim test (FST) and tail suspension test (TST) and in their home-cages. Emotionality-related differences between LEW and SHR rats and between Floripa H and L rats were found in the EPM, OF and FST. Those lines showing low anxiety-like profiles in the EPM and OF (SHR and Floripa H) also showed low immobility in the FST. The TST failed to unveil any line differences. Factor analysis involving all tests revealed three independent factors with one of them associating anxiety-related measures from the OF and EPM to immobility in the FST. When observed in their home-cages, LEW and SHR rats showed no differences in general activity, but when acutely treated with imipramine (15mg/kg), only LEW rats were sensitive to its antidepressant effects. These results suggest the existence of a genetic link between two tests used in the screening of anxiolytic drugs and one test of antidepressant activity. Moreover, the LEW and SHR rat strains were shown to be an interesting model to study the comorbidity between anxiety- and depression-related disorders.

Genetic selection of two new rat lines displaying different levels of anxiety-related behaviors.

We have recently identified, using two inbred rat strains (Lewis and SHR), the first two loci for an emotionality-related behavior in rats (locomotion in the central area of the open field test). Aiming to develop an alternative genetic model that will help to corroborate the existence of these loci and that could serve as a tool in the study of anxiety, we have started breeding two new rat lines. From a highly heterogeneous population, we have selectively bred rats for four generations with high and low scores for central locomotion in the open field. At the fourth generation, the new outbred lines differed from each other for the selected trait by as much as threefold. At each generation, all rats were also tested in the elevated plus maze and the black and white box, with the two lines differing significantly also in these two models of anxiety. These results suggest that behavioral measures from different tests of emotionality/anxiety are, at least in part, genetically related. They also indicate that this newly developed genetic model (herein named Floripa H and L rat lines) may be useful in the study of anxiety, as well as serving as a tool to test the existence/significance of the two aforementioned loci putatively associated with emotionality.