Structure of rat behavior in hole-board: I) multivariate analysis of response to anxiety.

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.

Structure of rat behavior in hole-board: II) multivariate analysis of modifications induced by diazepam.

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.

Multivariate analysis of the modifications induced by an environmental acoustic cue on rat exploratory behavior.

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.

Effects of 7-OH-DPAT and U 99194 on the behavioral response to hot plate test, in rats.

Aim of present study was to investigate in male Wistar rats, whether behavioral response to hot plate test application could be influenced by systemic administration of 7-OH-DPAT, a dopaminergic (DA) D3 versus D2 receptor agonist, or U 99194, a DA D3 versus D2 receptor antagonist. Each trial lasted no more than 10 s and the whole experimental session lasted 120 min. Animal behavior was recorded by means of a digital videocamera and later, frame by frame examined using a professional videorecorder. Latency of each behavioral pattern, characterizing the response, was analysed, showing significant changes only with U 99194. A multivariate cluster analysis indicated the presence of three main behavioral clusters (exploratory, primary responses to pain, escape) which, also, resulted significantly modified by both drugs. In addition, diagrams of preferential direction, obtained through multivariate stochastic analysis, evidenced switching probabilities differences among different patterns and clusters. Results demonstrate that the behavioral response to hot plate test application is altered following 7-OH-DPAT or U 99194 administration. These findings are discussed in terms of a) drugs influence on behavioral switching and learning processes; b) a likely activity on DA D3 versus D2 receptors.

The effects of different basal levels of anxiety on the behavioral shift analyzed in the central platform of the elevated plus maze.

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 effects of diazepam on the behavioral structure of the rat's response to pain in the hot-plate test: anxiolysis vs. pain modulation.

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.

Learning influence on the behavioral structure of rat response to pain in hot-plate.

Aim of the research was to study, by means of descriptive and multivariate analyses, whether, and how, learning influences the behavioral structure of rat response to pain. To this purpose, a hot-plate test daily repetition procedure was carried out on male Wistar rats for five days. A 6-day interval without stimulation elapsed before last test was carried out on day 12. After composition of an ethogram, descriptive (number, latency, per cent distribution) and multivariate analyses (cluster, stochastic) were carried out for each scheduled test day. One-way ANOVA and Newman-Keuls post-hoc test for multiple comparisons revealed significant changes for climbing, jumping, front-paw licking and stamping mean latencies. Student's t-test, carried out between days 1 and 12, showed that significant modifications persisted over time only for climbing and jumping. Cluster analysis evidenced three different clusters: exploratory (sniffing, walking), primary noxious evoked elements (front-paw licking, hind-paw licking, stamping), and escape (climbing and jumping), each showing modifications during test repetition. Comparison between days 1 and 12 revealed substantial differences in similarity values of escape patterns whereas, for the response to pain ones, a conservative structure of dendrograms was maintained. Stochastic analysis revealed a progressive increase of transitions toward jumping and a significant reduction of the ones between sniffing and walking. Such modifications persisted also after 6 days without any stimulation. Present study shows that learning provokes a complex and fine temporal evolution of the innermost behavioral structure of rat response to pain aiming at a more efficient escape strategy.

T-pattern analysis of diazepam-induced modifications on the temporal organization of rat behavioral response to anxiety in hole board.

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.

Temporal patterns analysis of rat behavior in hole-board.

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.

Microstructure of rat behavioral response to anxiety in hole-board.

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 behavioral 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.

Multivariate data handling in the study of rat behavior: an integrated approach.

The aim of the present article is to provide a methodological description of various approaches to multivariate data handling in the study of rodent behavior. To this purpose, 42 male Wistar rats were tested in an open field, and their behavior was recorded through a digital video camera for a subsequent analysis by means of a software coder. After a preliminary evaluation of descriptive features such as durations and percent distributions, we carried out different kinds of multivariate approaches represented by stochastic, cluster, adjusted residual, and T-pattern analyses. In the attempt to depict behavior in a straightforward way, the results of each analysis were graphically illustrated through path diagrams, dendrograms, histograms, and tree-shaped T-patterns. Path diagrams showed a clear behavioral convergence toward immobile sniffing; dendrograms highlighted three different dyadic clusters: walking/climbing, immobile-sniffing/immobility, and paw-licking/grooming; adjusted residuals confirmed, for the same patterns, highly significant association values; finally, T-pattern analysis showed a highly recurring temporal sequence of events encompassing walking, climbing, immobile sniffing, and immobility. Such results, drawing attention to specific behavioral patternings, strengthen and extend previous findings on rodent behavior. We suggest that T-pattern analysis, integrated with other multivariate approaches, can provide a more detailed and complete rat behavior representation, very different from classical quantitative approaches.