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Aim of this article is to illustrate the application of a multivariate approach known as t-pattern analysis in the study of rat behavior in elevated plus maze. By means of this multivariate approach, significant relationships among behavioral events in the course of time can be described. Both quantitative and t-pattern analyses were utilized to analyze data obtained from fifteen male Wistar rats following a trial 1 - trial 2 protocol. In trial 2, in comparison with the initial exposure, mean occurrences of behavioral elements performed in protected zones of the maze showed a significant increase counterbalanced by a significant decrease of mean occurrences of behavioral elements in unprotected zones. Multivariate t-pattern analysis, in trial 1, revealed the presence of 134 t-patterns of different composition. In trial 2, the temporal structure of behavior become more simple, being present only 32 different t-patterns. Behavioral strings and stripes (i.e. graphical representation of each t-pattern onset) of all t-patterns were presented both for trial 1 and trial 2 as well. Finally, percent distributions in the three zones of the maze show a clear-cut increase of t-patterns in closed arm and a significant reduction in the remaining zones. Results show that previous experience deeply modifies the temporal structure of rat behavior in the elevated plus maze. In addition, this article, by highlighting several conceptual, methodological and illustrative aspects on the utilization of t-pattern analysis, could represent a useful background to employ such a refined approach in the study of rat behavior in elevated plus maze.

The aim of present research is to study the temporal structure of the behavior in two strains of rats with different basal level of emotionality. To this purpose, the temporal profile of the behavior in Wistar rat and in the spontaneously more anxious DA/Han strain was analyzed in the Elevated Plus Maze. Both quantitative and multivariate t-pattern analyses were carried out. In comparison with Wistar, DA/Han subjects showed a significant reduction of the permanence in open arm and a significant increase of the time spent in the central platform of the maze. Mean frequencies of each behavioral element showed significant modifications both in open and in closed arm. Multivariate t-pattern analyses demonstrated a very different temporal profile of behavior in the two strains: Wistar rats presented 197 t-patterns of different composition, whereas DA/Han rats only 26; as to the mean number of t-patterns, Wistar presented a value of 698.90, whereas DA/Han only 92.80. Similar clear-cut differences were detected for the mean number of t-patterns in the two arms and for the time course of such t-patterns. Present study has evidenced that the temporal organization of the bahavior in Elevated Plus Maze is differently structured in two strains of rats with different basal level of emotionality.

Aim of the research was to evaluate, by means of quantitative and multivariate temporal pattern analyses, the behavior of Wistar rat in elevated plus maze (EPM) test. On the basis of an ethogram encompassing 24 behavioral elements, quantitative results showed that 130.14 ± 8.01 behavioral elements occurred in central platform and in closed arms (protected zones), whereas 88.62 ± 6.04 occurred in open arms (unprotected zones). Percent distribution was characterized by a prevalence of sniffing, walking and vertical exploration. Analysis of minute-by-minute duration evidenced a decrease for time spent in open arms and central platform and an increase for time spent in closed arms. As to multivariate t-pattern analysis, 126 different temporal patterns were detected. Behavioral stripes, summarizing distribution of such t-patterns along time, showed that several t-patterns were not homogeneously distributed along the test observational period: t-patterns encompassing behavioral events occurring prevalently in central platform-open arms were observed during the first minutes, whereas t-patterns structured on the basis of events occurring mainly in central platform-closed arms were detected during the last minutes. Therefore, during the observation in elevated plus maze, rat's behavior undergoes significant rearrangements of its temporal features. Present research demonstrates, for the first time, the existence of complex and significantly timed behavioral sequences in the activity of Wistar rats tested in elevated plus maze. Application of t-pattern analysis can provide useful tools to characterize the behavioral dynamics of anxiety-related rodent behavior and differentiate the effect of various anxioselective substances.

Tobacco smoking is a serious health problem worldwide and a leading cause of mortality. Nicotine, the addictive component of tobacco, affects a range of emotional responses, including anxiety-related behaviors. Although perceived by smokers to be anxiolytic, evidence suggests that smoking increases anxiety and that mood fluctuates with nicotine intake. Thus, nicotine addiction may depend on easing the psychobiological distress caused by its abuse. The lateral habenula (LHb) has been implicated as a neural substrate for acute nicotine-induced anxiety, but its role in anxiety-like behaviors associated with chronic nicotine exposure has not been explored. Here, we assessed the effect of chronic nicotine exposure and its subsequent overnight withdrawal on anxiety-like behavior using both quantitative and multivariate T-pattern analysis in rats tested using the hole-board apparatus. Additionally, we explored the role of the LHb by comparing the behavioral effects of short-term nicotine withdrawal in chronically treated LHb-lesioned rats. Quantitative analysis revealed increased anxiety-like behavior in chronically treated overnight nicotine-deprived rats, as manifested in reduced general and focused exploratory behaviors, which was eased in animals that received nicotine. Quantitative analysis failed to reveal a role of the LHb in overnight nicotine deprivation-induced anxiety. Conversely, T-pattern analysis of behavioral outcomes revealed that chronic nicotine-treated rats still show anxiety-like behavior following nicotine challenge. Moreover, it demonstrated that the LHb lesion induced a stronger anxiolytic-like response to the acute challenge of nicotine in chronically nicotine-exposed animals, implicating the LHb in the anxiogenic effect of chronic nicotine exposure. These data further highlight the LHb as a promising target for smoking cessation therapies and support the importance of T-pattern analysis for behavioral analysis.

Little is known about the structural characteristics of the behavior of rats with enhanced anxiety level. To fill this gap, a study was undertaken where effects of an anxiogenic drug were examined on behavioral structure of rats tested in hole board.This study investigates effects of increased anxiety level on the structure of the behavior of rats tested in hole board METHODS: Different doses of FG7142 (1, 4, 8 mg/kg IP), a potent anxiety-inducing drug, were administered to three groups of male Wistar rats. A further group was administered saline. Experiments were recorded through a digital camera. Quantitative and multivariate approaches were applied.Percent distributions and durations showed increases of immobile sniffing, rearing, head dip, and edge sniff and a significant reduction of grooming activities and of walking. In addition, a decrease of head dip/edge sniff ratio was detected. Transition matrices evidenced that FG7142 provoked evident modifications of behavioral structure mainly of general exploration of environment and focused exploration of the hole. Finally, adjusted residuals showed a reduced effectiveness of FG7142 on transitions from head dip to edge sniff; on the contrary, transitions from edge sniff to head dip underwent evident dose-dependent changes.Present study provides a useful tool to analyze behavioral responses to different anxiety conditions. Accordingly, it is demonstrated that a condition of increased anxiety deeply modifies the structure of male Wistar rat's behavior in hole board. In addition, our results suggest that evaluation of head dip/edge sniff ratio can be considered a reliable index to appraise effects of pharmacological manipulation of anxiety and related behavioral elements.

We have analyzed the temporal patterns of behaviour of male rats of the Wistar and DA/Han strains on the central platform of the elevated plus maze. The ethogram encompassed 10 behavioural elements. Durations, frequencies and latencies showed quantitative differences as to walking and sniffing activities. Wistar rats displayed significantly lower latency and significantly higher durations and frequencies of walking activities. DA/Han rats showed a significant increase of sniffing duration. In addition, DA/Han rats showed a significantly higher amount of time spent in the central platform. Multivariate T-pattern analysis revealed differences in the temporal organization of behaviour of the two rat strains. DA/Han rats showed (a) higher behavioural complexity and variability and (b) a significantly higher mean number of T-patterns than Wistar rats. Taken together, T-pattern analysis of behaviour in the centre of the elevated plus maze can noticeably improve the detection of subtle features of anxiety related behaviour. We suggest that T-pattern analysis could be used as sensitive tool to test the action of anxiolytic and anxiogenic manipulations., peer-reviewed

Rationale: The largest amount of researches on the hot-plate test was carried out using quantitative assessments. However, the evaluation of the relationships among the different elements that compose the behavioral response to pain requires different approaches. Although previous studies have provided clear information on the behavioral structure of the response, no data are available on its temporal structure. Objectives: The objective of this study was to investigate the temporal structure of the behavioral response to pain in Wistar rat tested in hot-plate and how this structure was influenced by morphine-induced analgesia. Methods: The behavior of four groups of subjects tested in hot-plate, one administered saline and three with different doses (3, 6, 12mg/kg) of morphine IP, was analyzed by means of quantitative and t-pattern analyses. The latter is a multivariate technique able to detect the existence of statistically significant temporal relationships among the behavioral events in time. Results: A clear-cut influence of morphine on quantitative parameters of the response to the noxious stimulation was observed. T-pattern analysis evidenced profound structural changes of behavior. Twenty-four different t-patterns were identified following saline, whereas a dose-dependent reduction was observed following morphine. Such a reduction was accompanied by a decrease of the total amount of t-patterns detected. Conclusions: Morphine, by reducing the effects of the noxious stimulation, orients animal behavior prevalently toward exploratory t-patterns. In addition, it is suggested that the temporal structure of the response is very quickly organized and adapted to environmental noxious cues.

Markers of histaminergic dysregulation were found in several neuropsychiatric disorders characterized by repetitive behaviours, thoughts and stereotypies. We analysed the effect of acute histamine depletion by means of i. c.v. injections of alpha-fluoromethylhistidine, a blocker of histidine decarboxylase, on the temporal organization of motor sequences of CD1 mice behaviour in the open-field test. An ethogram encompassing 9 behavioural components was employed. Durations and frequencies were only slightly affected by treatments. However, as revealed by multivariate t-pattern analysis, histamine depletion was associated with a striking increase in the number of behavioural patterns. We found 42 patterns of different composition occurring, on average, 520.90±50.23 times per mouse in the histamine depleted (HD) group, whereas controls showed 12 different patterns occurring on average 223.30±20.64 times. Exploratory and grooming behaviours clustered separately, and the increased pattern complexity involved exclusively exploratory patterns. To test the hypothesis of a histamine-dopamine interplay on behavioural pattern phenotype, non-sedative doses of the D2/D3 antagonist sulpiride (12.5-25-50mg/kg) were additionally administered to different groups of HD mice. Sulpiride counterbalanced the enhancement of exploratory patterns of different composition, but it did not affect the mean number of patterns at none of the doses used. Our results provide new insights on the role of histamine on repetitive behavioural sequences of freely moving mice. Histamine deficiency is correlated with a general enhancement of pattern complexity. This study supports a putative involvement of histamine in the pathophysiology of tics and related disorders.

Aim of present research was to assess, by means of descriptive and multivariate approaches, in Wistar rats tested in hole-board apparatus, whether a microstructure of hole exploratory behavior may be identified in the behavioral response to anxiety. Two groups of 15 subjects were tested: one received 1 ml of saline IP and one was IP injected with diazepam at 0.5 mg/kg dissolved in 1 ml of saline. Following saline, six not yet reported hole-exploratory components were identified: active-dip, static-dip, rapid-dip, circular-sniff, point-sniff and central-sniff. Diazepam provoked significant changes of their per cent distribution. Results of multivariate analyses revealed a complex microstructure of hole-exploratory elements characterized by behavioral flows between sniffing and dipping patterns. Diazepam significantly modified several patternings, mainly those involving point-sniff and static dip. Hence, the hypothesis of an important role of such behavioural elements and of their patternings, in anxiety-related hole-exploratory behavior, can be proposed. It is suggested that modifications of point-sniff and static-dip patternings could evidence even subtle changes of animal anxiety condition.

The aim of the present research was to study the effects of different basal levels of anxiety on the behavioral shift studied in the central platform of the elevated plus maze. To this purpose, quantitative and multivariate analyses, the latter based on transition matrix elaboration, were carried out on Wistar and on DA/Han rats the latter belonging to a strain characterized by different reactivity to anxiogenic stimuli. Wistar rats spent 74.11 ± 5.11 s in the central platform, whereas DA/Han significantly more: 127.08 ± 9.87. Per cent distributions evidenced a clear-cut difference in walking activities (46.25% in Wistar, 28.4% in DA/Han rats) and in the sniffing activities (45.82% in Wistar, 62.54% in DA/Han). Mean frequencies of each behavioral element showed in DA/Han strain a value significantly lower than in Wistar for central-platform entry, open arm-entry and closed-arm entry and a significant higher value for central- platform sniffing, open-arm sniffing and corner-rearing. Moreover, the ratio open-arm entry/open-arm sniffing and closed-arm entry/closed-arm-sniffing showed significant higher values in the Wistar strain. Finally, by means of hierarchical clustering analysis, strong differences between the two strains were observed in the behavioral architecture: a cascade-shaped dendrogram, branching from Walking activities, indicates that Wistar rat behavior is oriented to cross the central platform so to rapidly reach an arm; on the contrary, the dendrogram of DA/Han rats displays a behavior heavily oriented toward the permanence in the central platform. The results show that different basal levels of anxiety provoke significant differences in the behavioral shift studied in the central platform of the elevated plus maze. Such differences, evidenced by means of transition matrices elaboration, might represent the behavioral expression of anxiety-induced modifications of decision making process underlying behavioral shift activities.

The aim of present research was to analyze the temporal structure of rodent's anxiety-related behavior in hole-board apparatus (HB). Fifteen male Wistar rats were tested for 10 min. Video files, collected for each subject, were coded by means of a software coder and event log files generated for each subject. To assess temporal relationships among behavioral events, log files were processed by means of a t-pattern analysis. 14 two-element t-patterns, four t-patterns encompassing 3 events and 2 t-patterns encompassing 4 and 5 events respectively were revealed. It was demonstrated that rat behavior in HB was mainly structured on the basis of the temporal patterning among exploratory events; these ones were the most structured t-patterns detected and appeared mainly during the first 5 min of exploration, while grooming t-patterns were present prevalently after the fifth minute. Specific t-pattern parameters, such as overall occurrences and mean duration of each given t-pattern in each subject, were also studied. Present research: (a) reports for the first time that some behavioral events occur sequentially and with significant constraints on the interval lengths separating them; (b) presents the temporal flows of some behavioral elements through multimodal behavioral vectors; (c) could also be used to improve HB test reliability and its ability to detect even small induced behavioral changes.

In our previous study we suggested that multivariate analysis could improve hole-board test reliability providing a more useful tool to determine behavioral effects of anxiolytic drugs. To support this hypothesis, a multivariate analysis of rat behavior in hole-board, following administration of the reference anxiolytic drug diazepam, was carried out. Four groups, each composed of thirty male Wistar rats, were used: one saline and three diazepam injected (0.25, 0.5 and 2 mg/kg IP). Rat behavior was recorded for 10 min through a digital videocamera. Descriptive and multivariate analyses were carried out. In all groups, more than 80% of whole behavioral structure encompassed walking, climbing, rearing, immobile-sniffing, edge-sniff and head-dip. Moreover, modifications observed of a specific index, represented by edge-sniff/head-dip ratio, were correlated to diazepam-induced modifications of anxiety level. Cluster analysis showed that diazepam at 0.5 and 2 mg/kg induced important changes for [edge-sniff/head-dip] cluster. In addition, in all diazepam groups a [walking/climbing] cluster appeared. Path diagrams showed close relationships among different patterns both in saline and diazepam injected animals. Also, significant changes were detected following diazepam for transitions encompassing both general exploratory patterns (walking, climbing) and the specific ones (head-dip and edge-sniff). Adjusted residuals confirmed in all groups patterns relationships and, where present, significant behavioral associations. Results demonstrate that an anxiolytic activity can be revealed by head-dip/edge-sniff association weakening and by the addressing of behavioral structure toward general exploratory activity. Improvement of hole-board test reliability in behavioral study of anxiety, following multivariate analysis, is emphasized.

The aim of the present paper is to study by means of a multivariate analysis the modifications induced by an environmental acoustic cue on the structure of rat exploratory behavior. Adult male Wistar rats were observed during the exploration of a soundproof observation box. Each rat was acoustically stimulated after 150 s from the beginning of the experimental session, lasting 300 s, and recorded through a digital videocamera. A frame by frame analysis was thereafter carried out using a professional video-recording system. Thirteen behavioral patterns were selected: immobility, immobile-sniffing, walking, rearing, climbing, chewing, paw-licking, face-grooming, body-grooming, head-turning, tuning, oriented-sniffing, focusing. Both descriptive and multivariate analyses (cluster, stochastic, adjusted residuals) were carried out. Through descriptive statistical analysis, latencies and per cent distribution of each pattern were studied. A multivariate cluster analysis revealed the presence of three main behavioral clusters, an additional one being identified following acoustic stimulation. Multivariate stochastic analysis showed that all the patterns converged on immobile-sniffing which could represent a key component in behavioral switching processes related to environmental exploration. Moreover, through adjusted residuals, the degree of relationship among different patterns was shown according to statistic Z-distribution. Our data assign new ethological meanings to different behavioral patterns. Notably, head-turning is suggested to be considered as a generic directional search and tuning as a subtle activity of stimulus localization.