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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.

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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.

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

In our laboratories we use T-pattern analysis to study rat behavior in different and well-known experimental assays widely employed as rodent models of anxiety: the open field, the hole board and the elevated plus maze. By using Theme software and T-pattern analysis, we have observed that numerous events, characterizing rodent behavior in each experimental model, occurred sequentially and with significant constraints on the interval lengths separating them. In this chapter, for each test, we highlight some key aspects of our behavioral analyses, with a twofold attempt: first to provide the researcher with useful information concerning the application of T-pattern analysis in the study of rodent behavior and, second, to present and discuss various results of our studies., peer-reviewed

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.

Aim of present paper was to carry out an analysis of rat behavior in hole-board following different multivariate approaches. Thirty male Wistar rats were observed in a hole-board apparatus and their behavior recorded for 10 min through a digital videocamera for a following frame-by-frame analysis. Both descriptive and multivariate analyses were used. Descriptive analysis showed that roughly 85% of the whole behavioral structure encompassed six patterns appearing during the first minute of observation: walking, climbing, rearing, immobile-sniffing, edge-sniff and head-dip. As to multivariate approach, cluster analysis showed three main dyadic associations: [edge-sniff/head-dip], [walking/climbing], [face-grooming/body-grooming]. Path diagram, obtained on the basis of relative frequencies of transitions among behavioral patterns (stochastic analysis), emphasized cluster analysis results. Adjusted residuals confirmed, from a statistical point of view, the strong relationships among specific patterns. Results demonstrate that immobile-sniffing has a crucial role in rat behavioral organization and head-dip is closely related with edge-sniff which is suggested to be a specific sniffing activity of holes edge never properly considered. Present research shows that multivariate approaches, revealing patterning among different behavioral elements in hole-board, could improve test reliability, providing a more useful tool to investigate behavioral effects of anxiolytic drugs.

Nicotine is one of the most addictive drugs of abuse. Tobacco smoking is a major cause of many health problems, and is the first preventable cause of death worldwide. Several findings show that nicotine exerts significant aversive as well as the well-known rewarding motivational effects. Less certain is the anatomical substrate that mediates or enables nicotine aversion. Here, we show that acute nicotine induces anxiogenic-like effects in rats at the doses investigated (0.1, 0.5, and 1.0 mg/kg, i.p.), as measured by the hole-board apparatus and manifested in behaviors such as decreased rearing and head-dipping and increased grooming. No changes in locomotor behavior were observed at any of the nicotine doses given. T-pattern analysis of the behavioral outcomes revealed a drastic reduction and disruption of complex behavioral patterns induced by all three nicotine doses, with the maximum effect for 1 mg/kg. Lesion of the lateral habenula (LHb) induced hyperlocomotion and, strikingly, reversed the nicotine-induced anxiety obtained at 1 mg/kg to an anxiolytic-like effect, as shown by T-pattern analysis. We suggest that the LHb is critically involved in emotional behavior states and in nicotine-induced anxiety, most likely through modulation of monoaminergic nuclei., peer-reviewed

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 the present study was to evaluate, by means of quantitative and multivariate analyses, the effects of diazepam on the behavioral structure of the rat's response to pain in the hot-plate test as well as whether such changes are associated with drug-induced effects on anxiety and/or nociception. To this purpose, ten groups of male Wistar rats were intraperitoneally injected with saline, diazepam (0.25, 0.5 and 2 mg/kg), FG-7142 (1, 4 and 8 mg/kg) or morphine (3, 6 and 12 mg/kg). The mean number and mean latency to first appearance were calculated for each behavioral component. In addition, multivariate cluster and adjusted residual analyses based on the elaboration of transition matrices were performed. Three main behavioral categories were identified: exploratory (walking, sniffing), primary noxious-evoked (hind paw licking, front paw licking, shaking/stamping) and escape (climbing, jumping). Although no significant modifications in the latencies of the primary noxious-evoked components were induced by treatment with diazepam or FG-7142, significant effects were provoked by morphine treatment. Multivariate analyses showed that diazepam-induced anxiolysis redirected the rat's behavior toward a more purposeful and effective escape strategy. In contrast, the high level of anxiety induced by FG-7142 caused the behavioral structure to become disorganized and not purposefully oriented. Changes in the organization of behavioral components were observed in morphine-treated animals and mainly consisted of modifications in the primary noxious-evoked and escape components. The findings suggest that the effects of diazepam on the structure of the rat's response to pain in the hot-plate test are more likely attributable to anxiolysis than pain modulation.

The aim of the present study was to evaluate, by means of a temporal pattern analysis, rat’s behavior in the elevated plus maze test. A specific software called THEME has been used. On the basis of an ethogram encompassing 24 behavioral elements, results demonstrated that 14 components represented the 98% of the displayed activities. 145 different temporal patterns have been detected; length distribution of such patterns showed that three-, four-, and five-elements patterns were more represented than two-elements ones. Finally, a complex temporal pattern encompassing 8 behavioral elements has been discussed. Present article demonstrated the presence of complex temporal patterns characterizing the behavior of rats in the elevated plus maze test.

The aim of the present methodological article was to assess whether a microstructure of hole-exploratory activity can be demonstrated in rats tested in the hole-board, an experimental assay widely used to study anxiety-related behaviors in rodents. Six never properly described hole exploratory behavioral components were identified: active-dip, static-dip, rapid-dip, circular-sniff, point-sniff and central-sniff. Results, assessed by means of descriptive and multivariate approaches, reveal a complex microstructural organization of these behavioral elements. The hypothesis of an important role of the relationships between point-sniff and head dipping activities, in anxiety-related hole exploratory behavior, is proposed. It is suggested that the microstructural analysis can evidence even subtle behavioral changes induced by manipulations of animal anxiety condition.

Rationale: By means of t-pattern analysis, it has been observed that the different events, characterizing rat behavior in hole board (HB), present close interrelationships which occur sequentially and with significant constraints on the interval lengths separating them. Objectives: The aim of present research was to study, by means of descriptive and multivariate t-pattern analyses, the effects of the reference anxiolytic drug diazepam (DZP) on temporal structure of a rat’s anxiety-related behavior in HB. Methods: Fifty-six male Wistar rats were tested for 10 min in HB. Video files, collected for each animal, were coded by means of a software coder, and event log files, generated for each subject, were analyzed by means of a specific software for temporal pattern analysis (t-pattern analysis). Results: Significant diazepam-induced modifications were observed for durations of walking, climbing, edge-sniff, and face grooming. Dose-dependent decreases of t-patterns’ total amount, of their mean occurrences and of their mean length for each group were detected. Also, t-patterns’ mean occurrences, in terms of different composition, were reduced. Percent distributions showed a significant increase of t-patterns including walking for all administered groups, and significant reductions of t-patterns including climbing, immobile sniffing, and edge-sniff. Front-paw licking and face grooming were reduced at the higher DZP dose. Conclusions: Present study demonstrates, for the first time, that the temporal structure of Wistar rats’ behavioral response to anxiety in HB changes following pharmacological manipulation of anxiety condition. Moreover, t-pattern analysis is suggested to represent a useful tool to evaluate and compare different classes of anti-anxiety molecules.

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.