Effects of a rat model of gestational hypothyroidism on forebrain dopaminergic, GABAergic, and serotonergic systems and related behaviors.

We investigated the effects of maternal hypothyroidism on forebrain dopaminergic, GABAergic, and serotonergic systems and related behavior in adult rat offspring. Experimental gestational hypothyroidism (EGH) was induced by administering 0.02% methimazole (MMI) to pregnant rats from gestational day 9 to delivery. Neurotransmitter-related protein and gene expression were evaluated in offspring forebrain at postnatal day (P) 120. Exploratory behavior, contextual fear conditioning, locomotion, and 30-day reserpine Parkinson induction were assessed from P75-P120. Protein and gene expression assessments of medial prefrontal cortex showed group differences in dopaminergic, GABAergic, and serotonergic receptors, catabolic enzymes, and transporters. Striatum of MMI offspring showed an isolated decrease in the dopaminergic enzyme, tyrosine hydroxylase. MMI exposure increased GABA and dopamine receptor expression in amygdala. MMI offspring also had decreased state anxiety and poor contextual fear conditioning. We found that baseline locomotion was not changed, but reserpine treatment significantly reduced locomotion only in MMI offspring. Our results indicated that restriction of maternal thyroid hormones reduced dopaminergic, GABAergic, and serotoninergic forebrain components in offspring. Tyrosine hydroxylase deficiency in the striatum may underlie enhanced reserpine induction of Parkinson-like movement in these same offspring. Deficits across different neurotransmitter systems in medial prefrontal cortex and amygdala may underlie decreased state anxiety-like behavior and reduced fear conditioning in offspring, but no changes in trait anxiety-like behavior occurred with maternal MMI exposure. These findings strongly support the hypothesis that adequate delivery of maternal thyroid hormones to the fetus is crucial to the development of the central nervous system critical for emotion and motor regulation.

Excitotoxic lesion of the medial prefrontal cortex in Wistar rats: Effects on trait and state anxiety.

The neural substrate of anxiety response (state anxiety) to a threatening situation is well defined. However, a lot less is known about brain structures implicated in the individual's predisposition to anxiety (trait anxiety). Scientific evidences lead us to suppose that the medial prefrontal cortex (mPFC) is involved in both trait and state anxiety. Thus, the aim of this study was to investigate the involvement of mPFC in trait anxiety and to further evaluate its participation in state anxiety. Sixty six adult, Wistar, male rats were first tested in the free-exploratory paradigm (FEP) and were categorized according to their levels of trait anxiety (high, medium and low). Three to six days after this exposure, all animals were submitted to stereotaxic brain surgery. Half the animals from each anxiety category was allocated to the mPFC-lesioned group and the other half to the Sham-lesioned group. After seven to nine days, all animals were again tested in FEP. Eight to 10 days later, the animals were tested in the Hole Board test, a model of state anxiety. The mPFC lesion decreased levels of trait anxiety of highly anxious rats, whereas it reduced the state anxiety of all animals, regardless the level of trait anxiety. These data extend evidence of the participation of the mPFC in state anxiety and it demonstrate the involvement of this brain structure in trait anxiety, a personality trait supposed to be a predisposing factor for anxiety disorders.

Trait vs. state anxiety in different threatening situations / Ansiedade traço versus ansiedade estado em diferentes situações de ameaça

Abstract Objective Anxiety as a uni- or multidimensional construct has been under discussion. The unidimensional approach assumes that there is a general trait anxiety, which predisposes the individuals to increases in state anxiety in various threatening situations. In this case, there should be a correlation between state and trait anxiety in any situation of threat. Therefore, the aim of this study was to investigate the correlation between trait and state anxiety in participants exposed to two different anxiogenic situations: interpersonal threat (Video-Monitored Stroop Test - VMST) and physical threat (third molar extraction - TME). Methods Participants with various levels of trait anxiety (general trait: State-Trait Anxiety Inventory - STAI, Hospital Anxiety and Depression Scale; specific trait: Social Phobia Inventory, Dental Anxiety Scale) had their anxious state evaluated (STAI, self-evaluation of tension level, heart rate, electromyogram activity) before, during and after the VMST or the TME. Results In VMST, trait anxiety correlated to state anxiety (psychological parameters) in all test phases. However, in TME, the only trait measurement that correlated to state anxiety (psychological parameters) was the Dental Anxiety Scale. Conclusion Trait anxiety correlates positively to state anxiety in situations of interpersonal threat, but not of physical threat.

Resumo Objetivo A ansiedade como um construto uni ou multidimensional tem estado em discussão. A abordagem unidimensional presume que há uma ansiedade-traço geral, a qual predispõe o indivíduo a aumentar a ansiedade-estado em situações de ameaça. Neste caso, deveria existir uma correlação entre estado e traço ansioso em diferentes situações ameaçadoras. Portanto, o objetivo deste estudo foi avaliar a correlação entre ansiedade-traço e ansiedade-estado em participantes que foram expostos a duas situações ansiogênicas diferentes: ameaça interpessoal (Teste de Stroop Monitorado por Vídeo - TSMV) e ameaça física (exodontia do terceiro molar - ETM). Métodos Participantes com vários níveis de ansiedade-traço (traço geral: Inventário de Ansiedade Traço-Estado - IDATE; Escala Hospitalar de Ansiedade e Depressão; traço específico: Inventário de Fobia Social, Escala de Ansiedade Dental de Corah) tiveram seus estados ansiosos avaliados (IDATE, escala analógica de tensão, frequência cardíaca, eletromiografia) antes, durante e depois do TSMV ou da ETM. Resultados No TSMV, a ansiedade-traço correlacionou-se com a ansiedade-estado (parâmetros psicológicos) em todas as fases do teste. Entretanto, na ETM, a única medida de traço que se correlacionou com a ansiedade-estado (parâmetros psicológicos) foi a Escala de Ansiedade Dental de Corah. Conclusão A ansiedade-traço correlaciona-se positivamente com a ansiedade-estado em situações de ameaça interpessoal, mas não de ameaça física.

Trait vs. state anxiety in different threatening situations.

OBJECTIVE: Anxiety as a uni- or multidimensional construct has been under discussion. The unidimensional approach assumes that there is a general trait anxiety, which predisposes the individuals to increases in state anxiety in various threatening situations. In this case, there should be a correlation between state and trait anxiety in any situation of threat. Therefore, the aim of this study was to investigate the correlation between trait and state anxiety in participants exposed to two different anxiogenic situations: interpersonal threat (Video-Monitored Stroop Test - VMST) and physical threat (third molar extraction - TME). METHODS: Participants with various levels of trait anxiety (general trait: State-Trait Anxiety Inventory - STAI, Hospital Anxiety and Depression Scale; specific trait: Social Phobia Inventory, Dental Anxiety Scale) had their anxious state evaluated (STAI, self-evaluation of tension level, heart rate, electromyogram activity) before, during and after the VMST or the TME. RESULTS: In VMST, trait anxiety correlated to state anxiety (psychological parameters) in all test phases. However, in TME, the only trait measurement that correlated to state anxiety (psychological parameters) was the Dental Anxiety Scale. CONCLUSION: Trait anxiety correlates positively to state anxiety in situations of interpersonal threat, but not of physical threat.

Effect of Lemongrass Aroma on Experimental Anxiety in Humans.

OBJECTIVES: The objective of this study was to evaluate the potential anxiolytic effect of lemongrass (Cymbopogon citratus) aroma in healthy volunteers submitted to an anxiogenic situation. DESIGN: Forty male volunteers were allocated to four different groups for the inhalation of lemongrass essential oil (test aroma: three or six drops), tea tree essential oil (control aroma: three drops), or distilled water (nonaromatic control: three drops). Immediately after inhalation, each volunteer was submitted to an experimental model of anxiety, the video-monitored version of the Stroop Color-Word Test (SCWT). OUTCOME MEASURES: Psychologic parameters (state anxiety, subjective tension, tranquilization, and sedation) and physiologic parameters (heart rate and gastrocnemius electromyogram activity) were evaluated before the inhalation period and before, during, and after the SCWT. RESULTS: Individuals exposed to the test aroma (three and six drops), unlike the control groups, presented a reduction in state anxiety and subjective tension, immediately after treatment administration. In addition, although they presented an anxious response to the task, they completely recovered from it in 5 min, unlike the control groups. Physiologic alterations along the test were not prevented by any treatment, in the same way as has previously been observed for diazepam. CONCLUSIONS: Although more investigations are necessary to clarify the clinical relevance of lemongrass essential oil as an anxiety treatment, this work shows that very brief exposure to this aroma has some perceived anxiolytic effects.

Pharmacological validation of the free-exploratory paradigm in male Wistar rats: A proposed test of trait anxiety.

The free-exploratory paradigm (FEP) has been proposed as a model of trait anxiety for both mice and rats. However, its pharmacological validation has only been carried out for the mice. Thus, the aim of the present study was to pharmacologically validate FEP for Wistar rats, by testing the effects of clinically established anxiolytic and anxiogenic drugs, in four different experiments. In all experiments, male Wistar rats were first tested in FEP to be categorized according to their levels of trait anxiety (high, medium and low). Then, only medium trait anxiety rats were selected to be tested again in FEP, two weeks later, after being pharmacologically treated, according to each experiment as follows: Experiment I: 0.5mg/kg of diazepam (DZP) or vehicle; Experiment II: 20mg/kg of pentylenetetrazole (PTZ) or vehicle; Experiment III: 5mg/kg of fluoxetine (FLX5) or vehicle: and Experiment IV: 0.5mg/kg of fluoxetine (FLX0.5) or vehicle. As a group, the results showed that PTZ and FLX5 increased levels of trait anxiety and reduced locomotor activity, whereas DZP and FLX0.5 decreased levels of trait anxiety, without impairing locomotor activity. These results demonstrate that FEP for rats is able to predict clinical anxiolytic and anxiogenic activities of different drugs, including fluoxetine, which is believed to present a dual effect on anxiety. Therefore, this paradigm can be proposed as an effective method for testing potential trait anxiety-reducing drugs, in rats.

Environmental enrichment for adult rats: effects on trait and state anxiety.

Experimental evidence indicates that enriched environment (EE) induces neurobiological and behavioural alterations. EE in early life improves learning and memory and reduces trait and state anxiety. However, the effect of EE established in adulthood has rarely been investigated. Thus, the aim of this study was to evaluate the possibility of modifying the levels of trait and/or state anxiety of adult rats exposed to EE. Seventy adult Wistar male rats were first tested in the free-exploratory paradigm (FEP) and were categorized according to their levels of trait anxiety (high, medium and low). Subsequently, half of the animals from each category returned to their home cages (standard condition: SC) and the other half was transferred to an enriched environment (enriched condition: EC). After three weeks, all animals were again tested in FEP. Seven to 10 days later, fifty of the seventy animals were tested on the elevated plus-maze test (EPM). In FEP, EE reduced locomotor activity in the second exposition independently of the anxiety category and, it decreased the levels of trait anxiety of highly anxious rats. No effect of EE was observed on EPM. In conclusion, EE established in adulthood was able to reduce high trait anxiety, a major risk factor for anxiety disorders.

The influence of trait anxiety on the elevation of arterial pressure induced by L-NAME in rats.

Due to the high prevalence of anxiety disorders and hypertension comorbidity in the general population, the establishment of anxiety as a risk factor for elevated blood pressure, or the reverse, is of great relevance. In this context, animal models can be of great scientific value, as they permit the control of several variables. Bearing this in mind, the influence of anxiety, not as a state, but as a personality trait (trait anxiety), on blood pressure elevation and vice versa were investigated for the first time in rats, using the free-exploratory paradigm (FEP). Sixty adult male Wistar rats were evaluated on FEP and categorized according to their levels of anxiety. From this sample, 24 animals with high (n=12) and low (n=12) trait anxiety were allocated to two treatment groups: (1) l-NAME (N(G)-nitro-l-arginine methyl ester, 20mg/kg, p.o., for 7 days to increase blood pressure; n=6/anxiety category); (2) CTRL (tap water, p.o., for 7 days; n=6/anxiety category). During treatment, measurements of systolic blood pressure (SBP) were taken daily. After treatment, the animals were again tested on FEP. SBP and trait anxiety levels were compared pre- and post-treatment. Additionally, correlations between trait anxiety levels and SBP increases (l-NAME group) were analyzed. The results showed that l-NAME was able to induce significant SBP elevation, but only for the high-anxious animals, while SBP elevation did not significantly interfere with anxiety levels. A significant correlation between anxiety levels and SBP peaks in response to l-NAME was also shown. No differences were observed between the levels of anxiety before and after treatment. These findings suggest that individuals with high trait anxiety are more susceptible to increases in blood pressure, but that high blood pressure does not affect the levels of trait anxiety.

The free-exploratory paradigm as a model of trait anxiety in female rats: test-retest reliability.

The free-exploratory paradigm has been proposed as a model of trait anxiety and as such has been proven stable over time, which is a sine qua non condition for any model proposing to evaluate a personality trait. However this stability has only been shown for male rats. Considering that anxiety disorders are most prevalent in women, it's crucial that females are tested in animal models. With this in mind, the stability over time of female rats in the free-exploratory paradigm was evaluated using a test-retest procedure. The behaviour of drug-naive, adult, female, Wistar rats was measured in the free-exploratory apparatus on two occasions two months apart. The following parameters were evaluated: percentage of distance travelled in the novel compartment; number of attempts to enter the novel compartment; percentage of time spent in the novel compartment; and percentage of time rearing in the novel compartment. Subsequently, the intraclass correlation coefficient (ICC) and the kappa index (κ) were calculated for each of these parameters. The "percentage of time spent in the novel compartment" (ICC=0.727; κ=0.457), the "percentage of distance travelled in the novel compartment" (ICC=0.680; κ=0.370), and the "percentage of time rearing in the novel compartment" (ICC=0.648; κ=0.309) were found to be stable over time. Analysis of these parameters indicated fair to substantial reliability over time in two-month inter-trial interval. Therefore, our results support the idea of the free-exploratory paradigm as an animal model of trait anxiety for female rats.

Effect of sweet orange aroma on experimental anxiety in humans.

OBJECTIVES: The objective of this study was to evaluate the potential anxiolytic effect of sweet orange (Citrus sinensis) aroma in healthy volunteers submitted to an anxiogenic situation. DESIGN: Forty (40) male volunteers were allocated to five different groups for the inhalation of sweet orange essential oil (test aroma: 2.5, 5, or 10 drops), tea tree essential oil (control aroma: 2.5 drops), or water (nonaromatic control: 2.5 drops). Immediately after inhalation, each volunteer was submitted to a model of anxiety, the video-monitored version of the Stroop Color-Word Test (SCWT). OUTCOME MEASURES: Psychologic parameters (state-anxiety, subjective tension, tranquilization, and sedation) and physiologic parameters (heart rate and gastrocnemius electromyogram) were evaluated before the inhalation period and before, during, and after the SCWT. RESULTS: Unlike the control groups, the individuals exposed to the test aroma (2.5 and 10 drops) presented a lack of significant alterations (p>0.05) in state-anxiety, subjective tension and tranquillity levels throughout the anxiogenic situation, revealing an anxiolytic activity of sweet orange essential oil. Physiologic alterations along the test were not prevented in any treatment group, as has previously been observed for diazepam. CONCLUSIONS: Although more studies are needed to find out the clinical relevance of aromatherapy for anxiety disorders, the present results indicate an acute anxiolytic activity of sweet orange aroma, giving some scientific support to its use as a tranquilizer by aromatherapists.

Automation of the free-exploratory paradigm.

The free-exploratory paradigm (FEP) is currently the only proposed animal model of trait anxiety, making it highly valuable to behavioural neuroscience. However, FEP has not yet been automated, so its results depend on human scoring, which can be quite imprecise. The aim of this study was, therefore, to validate an automated version of FEP, using a commercially available video-tracking system (ANY-maze(©) - Stoelting Co., USA). To achieve this, two experiments were performed. The first one evaluated the reliability of the video-based automation of FEP, and the second, assessed whether the zeolites, used as a bedding material in the first experiment to facilitate video-tracking, influenced the animals' behaviour in FEP. In experiment I, 15 drug-naive, adult, male rats were tested in FEP, while their behaviour was simultaneously evaluated by ANY-maze(©) and two human observers. Subsequently, the intraclass correlation coefficient (ICC) was calculated for the automated and manual results of the parameters "percentage of time in the novel side" (%TNS) and "total units visited" (TUV). The analysis resulted in high, significant values of ICC (%TNS: 0.9962 and TUV: 0.9453). In experiment II, 18 drug-naive, adult, male rats were allocated to two different groups: (1) tested in FEP with zeolites; and (2) tested in FEP with sawdust. The data obtained were analysed using the Student's t-test, which revealed no significant differences between the groups for the parameters %TNS and TUV. In conclusion, the data presented here show that automation of FEP, using a video-based tracking system, is not only possible, but also highly reliable.

The free-exploratory paradigm as a model of trait anxiety in rats: test-retest reliability.

The free exploratory paradigm has been proposed as a model of trait anxiety. By definition, trait anxiety does not vary from moment to moment, therefore any model used to evaluate it needs to give the same results in sequential trials. With this in mind, the test-retest reliability of the free-exploratory paradigm in rats was evaluated. The behaviour of drug-naive, adult, male, Wistar rats was measured in the free-exploratory apparatus on two occasions, either one week apart (Experiment I) or four weeks apart (Experiment II). The following parameters were evaluated: percentage of time spent in the novel compartment of the free exploratory box; percentage of novel compartment units visited; percentage of time rearing in the novel compartment; and attempts to enter the novel compartment. Subsequently, the intraclass correlation coefficient (ICC) and the kappa index (kappa) were calculated for each of these parameters. The "percentage of time spent in the novel compartment" was found to be stable over time. Analysis of this parameter in Experiment I showed high values of ICC (0.805) and kappa (0.716), indicating good reliability with a one-week inter-trial interval. In Experiment II, although the same parameter's kappa index (0.255) failed to show a reasonable test/retest concordance, the ICC value (0.760) still demonstrated a good test/retest correlation, suggesting that with a four-week inter-trial interval, the reliability may diminish, but still exists. Therefore, our results bring further support to the free-exploratory paradigm as an animal model of trait anxiety.

Trait and state anxiety in animal models: Is there correlation?

It is believed that subjects with high trait anxiety levels tend to present state anxiety reactions with greater intensity than individuals with low trait anxiety levels. In order to verify if this premise is valid for animal models of anxiety, the present work investigated the possible correlation between two behavioral tests: the elevated plus-maze, a classic model of state-anxiety, and the free-exploratory paradigm, which has been proposed as a model of trait anxiety. The behavior of 46 drug-naive, adult, Wistar, male rats was measured in these two models on two occasions, 1 week apart. Subsequently, the intraclass correlation coefficient (ICC) was calculated for the parameters "percentage of time in the novel side" (%TNS; free-exploratory paradigm), "percentage of time in the open arms" (%TOA; elevated plus-maze) and "percentage of entries into the open arms" (%EOA; elevated plus-maze). These parameters were also used to classify the animals into groups presenting high, medium or low levels of anxiety in both tests, so that the concordance between the models could be evaluated through the kappa test. The analysis resulted in low ICC (%TNSx%TOA: -0.127; %TNSx%EOA: 0.040) and low kappa index (%TNSx%TOA: -0.017; %TNSx%EOA: -0.044), suggesting a poor correspondence between the free-exploratory paradigm and the elevated plus-maze. In conclusion, the data presented here indicate that the premise of correlation between trait and state anxiety is not necessarily true for animal models of anxiety and, therefore, care must be exercised when using state anxiety models in order to determine animals' anxiety profile.