Sex-dependent behavioral effects of chronic nicotine during adolescence evaluated in young adult rats tested in Hole-Board.

As one of the leading causes of death and serious illnesses, tobacco smoking remains a significant issue in modern societies. Many individuals smoke during adolescence, a trend that has been exacerbated by the prevalence of vaping among young people. In this context, studying the behavioral effects induced by nicotine administration in male and female rats, during the adolescent period, assumes great importance because it can help to better understand the dynamics underlying tobacco use in the two sexes. For this purpose, we employed 4 groups of rats, 2 male and 2 female groups, chronically treated with saline or nicotine 3 mg/kg i.p. for 30 days, spanning from postnatal day 30 to postnatal day 60. Utilizing quantitative analyses and T-pattern detection and analysis, our findings revealed a complex and multifaceted behavioral reorganization in adolescent rats subjected to chronic nicotine administration. Specifically, we observed an increase of anxiety in males and a reduction in females. The distinctive structural changes, induced by chronic nicotine in both sexes, have significant implications, from a translational perspective, for studies on nicotine dependence disorders.

A quantitative and T-pattern analysis of anxiety-like behavior in male GAERS, NEC, and Wistar rats bred under the same conditions, against a commercially available Wistar control group in the hole board and elevated plus maze tests.

AIM: The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred polygenic model of childhood absence epilepsy (CAE), which, as their non-epileptic control (NEC) rats, are derived from Wistar rats. While the validity of GAERS in reproducing absence seizures is well established, its use as a model for CAE psychiatric comorbidities has been subject to conflicting findings. Differences in colonies, experimental procedures, and the use of diverse controls from different breeders may account for these disparities. Therefore, in this study, we compared GAERS, NEC, and Wistar bred in the same animal facility with commercially available Wistar (Cm Wistar) as a third control. METHODS: We performed hole board (HB) and elevated plus maze (EPM) tests that were analyzed with standard quantitative and T-pattern analysis in male, age-matched Cm Wistar and GAERS, NEC, and Wistar, bred under the same conditions, to rule out the influence of different housing factors and provide extra information on the structure of anxiety-like behavior of GAERS rats. RESULTS: Quantitative analysis showed that GAERS and NEC had similar low anxiety-like behavior when compared to Cm Wistar but not to Wistar rats, although a higher hole-focused exploration was revealed in NEC. T-pattern analysis showed that GAERS, NEC, and Wistar had a similar anxiety status, whereas GAERS and NEC exhibited major differences with Cm Wistar but not Wistar rats. EPM results indicated that GAERS and NEC also have similar low anxiety compared to Cm Wistar and/or Wistar rats. Nevertheless, the analysis of the T-pattern containing open-arm entry showed GAERS and Wistar to be less anxious than NEC and Cm Wistar rats. CONCLUSION: To summarize, comorbid anxiety may not be present in male GAERS rats. This study also highlighted the importance of including a control Wistar group bred under the same conditions when evaluating their behavior, as using Wistar rats from commercial breeders can lead to misleading results.

The hole-board apparatus in the study of anxiety.

Anxiety disorders pose a significant challenge in contemporary society, and their impact in terms of social and economic burden is overwhelming. Behavioral research conducted on animal subjects is crucial for comprehending these disorders and, from a translational standpoint, for introducing innovative therapeutic approaches. In this context, the Hole-Board apparatus has emerged as a widely utilized test for studying anxiety-related behaviors in rodents. Although a substantial body of literature underscores the utility and reliability of the Hole-Board in anxiety research, recent decades have witnessed a range of studies that have led to uncertainties and misinterpretations regarding the validity of this behavioral assay. The objective of this review is twofold: firstly, to underscore the utility and reliability of the Hole-Board assay, and concurrently, to examine the underlying factors contributing to potential misconceptions surrounding its utilization in the study of anxiety and anxiety-related behaviors. We will present results from both conventional quantitative analyses and multivariate approaches, while referencing a comprehensive collection of studies conducted using the Hole-Board.

Structural analyses in the study of behavior: From rodents to non-human primates.

The term "structure" indicates a set of components that, in relation to each other, shape an organic complex. Such a complex takes on essential connotations of functionally unitary entity resulting from the mutual relationships of its constituent elements. In a broader sense, we can use the word "structure" to define the set of relationships among the elements of an emergent system that is not determined by the mere algebraic sum of these elements, but by the interdependence relationships of these components from which the function of the entire structure itself derives. The behavior of an integrated living being can be described in structural terms via an ethogram, defined as an itemized list of behavioral units. Akin to an architectural structure, a behavioral structure arises from the reciprocal relationships that the individual units of behavior establish. Like an architectural structure, the function of the resulting behaving complex emerges from the relationships of the parts. Hence, studying behavior in its wholeness necessitates not only the identification of its constitutive units in their autarchic individuality, but also, and importantly, some understanding of their relationships. This paper aimed to critically review different methods to study behavior in structural terms. First, we emphasized the utilization of T-pattern analysis, i.e., one of the most effective and reliable tools to provide structural information on behavior. Second, we discussed the application of other methodological approaches that are based on the analysis of transition matrices, such as hierarchical clustering, stochastic analyses, and adjusted residuals. Unlike T-pattern analysis, these methods allow researchers to explore behavioral structure beyond its temporal characteristics and through other relational constraints. After an overview of how these methods are used in the study of animal behavior, from rodents to non-human primates, we discussed the specificities, advantages and challenges of each approach. This paper could represent a useful background for all scientists who intend to study behavior both quantitatively and structurally, that is in terms of the reciprocal relationships that the various units of a given behavioral repertoire normally weave together.

Cannabinoid 1/2 Receptor Activation Induces Strain-Dependent Behavioral and Neurochemical Changes in Genetic Absence Epilepsy Rats From Strasbourg and Non-epileptic Control Rats.

Childhood absence epilepsy (CAE) is characterized by absence seizures, which are episodes of lack of consciousness accompanied by electrographic spike-wave discharges. About 60% of children and adolescents with absence seizures are affected by major neuropsychological comorbidities, including anxiety. Endocannabinoids and monoamines are likely involved in the pathophysiology of these CAE psychiatric comorbidities. Here, we show that the synthetic cannabinoid receptor type 1/2 (CB1/2R) agonist WIN 55,212-2 (2 mg/kg) has a strain-dependent effect on anxiety-like and motor behavior when assess in the hole board test and cerebral monoaminergic levels in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and their non-epileptic control (NEC) rat strain. Using quantitative and Temporal pattern (T-pattern) analyses, we found that WIN 55,212-2 did not affect the emotional status of GAERS, but it was anxiolytic in NEC. Conversely, WIN 55,212-2 had a sedative effect in GAERS but was ineffective in NEC. Moreover, vehicle-treated GAERS more motivated to explore by implementing more complex and articulated strategies. These behavioral changes correlate with the reduction of 5-HT in the hippocampus and substantia nigra (SN) and noradrenaline (NA) in the entopeduncular nucleus (EPN) in vehicle-treated GAERS compared to NEC rats, which could contribute to their low anxiety status and hypermotility, respectively. On the other hand, the increased level of NA in the EPN and 5-HT in the SN is consistent with an activation of the basal ganglia output-mediated motor suppression observed in WIN 55,212-2-treated GAERS rats. These data support the view of a strain-dependent alteration of the endocannabinoid system in absence epilepsy by adding evidence of a lower emotional responsiveness and a basal ganglia hypersensitivity to cannabinoids in GAERS compared to NEC rats.

The effect of cannabinoid receptor agonist WIN 55,212-2 on anxiety-like behavior and locomotion in a genetic model of absence seizures in the elevated plus-maze.

GAERS and NEC rats were treated with cannabinoid 1/2 receptor agonist WIN 55,212-2 2 mg/kg and tested on the Elevated Plus-Maze.

Nicotine modulation of the lateral habenula/ventral tegmental area circuit dynamics: An electrophysiological study in rats.

Nicotine, the addictive component of tobacco, has bivalent rewarding and aversive properties. Recently, the lateral habenula (LHb), a structure that controls ventral tegmental area (VTA) dopamine (DA) function, has attracted attention as it is potentially involved in the aversive properties of drugs of abuse. Hitherto, the LHb-modulation of nicotine-induced VTA neuronal activity in vivo is unknown. Using standard single-extracellular recording in anesthetized rats, we observed that intravenous administration of nicotine hydrogen tartrate (25-800 µg/kg i.v.) caused a dose-dependent increase in the basal firing rate of the LHb neurons of nicotine-naïve rats. This effect underwent complete desensitization in chronic nicotine (6 mg/kg/day for 14 days)-treated animals. As previously reported, acute nicotine induced an increase in the VTA DA neuronal firing rate. Interestingly, only neurons located medially (mVTA) but not laterally (latVTA) within the VTA were responsive to acute nicotine. This pattern of activation was reversed by chronic nicotine exposure which produced the selective increase of latVTA neuronal activity. Acute lesion of the LHb, similarly to chronic nicotine treatment, reversed the pattern of DA cell activation induced by acute nicotine increasing latVTA but not mVTA neuronal activity. Our evidence indicates that LHb plays an important role in mediating the effects of acute and chronic nicotine within the VTA by activating distinct subregional responses of DA neurons. The LHb/VTA modulation might be part of the neural substrate of nicotine aversive properties. By silencing the LHb chronic nicotine could shift the balance of motivational states toward the reward.

Effects of Different Anxiety Levels on the Behavioral Patternings Investigated through T-pattern Analysis in Wistar Rats Tested in the Hole-Board Apparatus.

The Hole-Board is an ethologically based tool for investigating the anxiety-related behavior of rats following manipulation of the central anxiety level. The present paper aims to assess behavioral patterning following pharmacological manipulation of emotional assets in Wistar rats tested in this experimental apparatus. For this purpose, the behavior of three groups of rats injected with saline, diazepam or FG7142 was evaluated using conventional quantitative and multivariate T-pattern analyses. The results demonstrate that quantitative analyses of individual components of the behavior, disjointed from the comprehensive behavioral structure, are of narrow utility in the understanding of the subject's emotional condition. Among the components of the behavioral repertoire in rodents tested in the Hole-Board, Edge-Sniff and Head-Dip represent the most significant ones to rate anxiety level. They are characterized by a strong bivariate relationship and are also firmly part of the behavioral architecture, as revealed by the T-pattern analysis (TPA), a multivariate technique able to detect significant relationships among behavioral events over time. Edge-Sniff → Head-Dip sequences, in particular, are greatly influenced by the level of anxiety: barely detectable in control animals, they completely disappear in subjects with a reduced level of anxiety and are present in almost 25% of the total of T-patterns detected in subjects whose anxiety level increased.

Detection of a temporal structure in the rat behavioural response to an aversive stimulation in the emotional object recognition (EOR) task.

Aim of the research was to investigate whether a temporal structure could be detected in the behavioural response to an aversive stimulation. A fear-related memory task was used in rats, placed in a modified version of the Novel Object Recognition task known as Emotional Object Recognition task, i.e. a behavioural assay that orbits around the declarative memory for an aversive experience. To this purpose, twelve male Wistar rats, divided in two groups (Control and Aversive memory), observed after 4 h (OR4h) and after 24 h (OR24h) from the delivery of an aversive stimulation, associated to a specific object, were used. Data were evaluated both in terms of conventional quantitative approaches and by means of T-pattern analysis, namely a multivariate technique able to unveil the temporal structure of behaviour and the relationships amongst the behavioural items in time. Results evidenced several changes between groups and over time as well. Mean occurrences and mean durations showed significant differences between OR4h and OR24h sessions and between Control and Aversive memory groups for behavioural items of Exploration, Object-related aversion and Immobility. T-pattern analysis revealed important changes of behavioural variability, complexity and repetitiveness, (i.e., the three main qualitative features of T-patterns) in the Aversive memory group. These outcomes highlight a simpler and linear behavioural profile, focused only on specific sequences of particularly repetitive events. Overall, the present study demonstrates a) the presence of a temporal organization of fear-related behavioural events and b) the influence of learning on the modifications observed over time.

Lateral Habenula 5-HT2C Receptor Function Is Altered by Acute and Chronic Nicotine Exposures.

Serotonin (5-HT) is important in some nicotine actions in the CNS. Among all the 5-HT receptors (5-HTRs), the 5-HT2CR has emerged as a promising drug target for smoking cessation. The 5-HT2CRs within the lateral habenula (LHb) may be crucial for nicotine addiction. Here we showed that after acute nicotine tartrate (2 mg/kg, i.p.) exposure, the 5-HT2CR agonist Ro 60-0175 (5-640 µg/kg, i.v.) increased the electrical activity of 42% of the LHb recorded neurons in vivo in rats. Conversely, after chronic nicotine treatment (6 mg/kg/day, i.p., for 14 days), Ro 60-0175 was incapable of affecting the LHb neuronal discharge. Moreover, acute nicotine exposure increased the 5-HT2CR-immunoreactive (IR) area while decreasing the number of 5-HT2CR-IR neurons in the LHb. On the other hand, chronic nicotine increased both the 5-HT2CR-IR area and 5-HT2CR-IR LHb neurons in the LHb. Western blot analysis confirmed these findings and further revealed an increase of 5-HT2CR expression in the medial prefrontal cortex after chronic nicotine exposure not detected by the immunohistochemistry. Altogether, these data show that acute and chronic nicotine exposure differentially affect the central 5-HT2CR function mainly in the LHb and this may be relevant in nicotine addiction and its treatment.

Brain histamine and oleoylethanolamide restore behavioral deficits induced by chronic social defeat stress in mice.

The physiological mechanisms underlying the complex interplay between life stressors and metabolic factors is receiving growing interest and is being analyzed as one of the many factors contributing to depressive illness. The brain histaminergic system modulates neuronal activity extensively and we demonstrated that its integrity is necessary for peripheral signals such as the bioactive lipid mediator oleoylethanolamide (OEA) to exert its central actions. Here, we investigated the role of brain histamine and its interaction with OEA in response to chronic social defeat stress (CSDS), a preclinical protocol widely used to study physio-pathological mechanisms underlying symptoms observed in depression. Both histidine decarboxylase null (HDC-/-) and HDC+/+ mice were subjected to CSDS for 21 days and treated with either OEA or vehicle daily, starting 10 days after CSDS initiation, until sacrifice. Undisturbed mice served as controls. To test the hypothesis of a histamine-OEA interplay on behavioral responses affected by chronic stress, tests encompassing the social, ethological and memory domains were used. CSDS caused cognitive and social behavior impairments in both genotypes, however, only stressed HDC+/+ mice responded to the beneficial effects of OEA. To detect subtle behavioral features, an advanced multivariate approach known as T-pattern analysis was used. It revealed unexpected differences of the organization of behavioral sequences during mice social interaction between the two genotypes. These data confirm the centrality of the neurotransmitter histamine as a modulator of complex behavioral responses and directly implicate OEA as a protective agent against social stress consequences in a histamine dependent fashion.

The impact of chronic daily nicotine exposure and its overnight withdrawal on the structure of anxiety-related behaviors in rats: Role of the lateral habenula.

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.

Possible Contribution of T-pattern Detection and Analysis to the Study of the Behavioral Correlates of Afferent Inhibition.

A pivotal tenet in modern behavioral sciences is that the study of behavior, in its most intimate structure, necessarily deals with time and, for this reason, behavioral dynamics are not intuitively perceivable and/or detectable (Eibl-Eibesfeldt, 1970). In reality, the possibility to describe a given behavior in terms of its structural/temporal features makes available new and detailed information otherwise unavailable. The aim of the present paper is to discuss the possible application of T-pattern detection and analysis, i.e., a multivariate approach specifically developed to describe the temporal structure of behavior, to the study of an important and still scantly investigated issue, namely the detection of behavioral correlates of the neurophysiological phenomenon known as afferent inhibition.

Is female-male mounting functional? An analysis of the temporal patterns of sexual behaviors in Japanese macaques.

In certain populations of Japanese macaques, adult females mount adult males in the context of heterosexual consortships (i.e., temporary but exclusive sexual associations between a male and a female). Previous research suggested that, in this primate species, female-male mounting (FMM) may be a behavioral adaptation. This functional hypothesis holds that FMM is a (special) courtship behaviour, or a (super) sexual solicitation, that serves the function of focusing the male's attention, preventing him from moving away, and expediting male-female mounting, in the context of high female competition for male mates. In this study, we aimed to test some of the proposed functional features of FMM in Japanese macaques by comparing the temporal structure of mating behavioral sequences, including various well-known sexual solicitations, exhibited during heterosexual consortships with and without FMM. To identify and compare recurring series of behavioral events within and across sequences, we used a temporal analysis known as "T-pattern detection and analysis". Our results (partly) supported the "FMM as a (super) sexual solicitation" hypotheses, and supported the "FMM as a sexual adaptation" hypothesis. The utilization of TPA allows for the detection of hidden features of primates' behaviors otherwise undetectable by using conventional quantitative approaches, such as the calculation of frequencies or durations of isolated behavioral components, disjointed from the comprehensive behavioral architecture. This study fits into the scheme of a broader investigation of the functionality of non-conceptive mounting patterns observed in Japanese macaques and a reconstruction of their evolutionary history.

Inferring functional patterns of tool use behavior from the temporal structure of object play sequences in a non-human primate species.

Inferring functional components of behavioral sequences is a crucial but challenging task. A systematic comparison of their temporal structure is a good starting point, based on the postulate that more functional traits are less structurally variable. We studied stone handling behavior (SH) in Balinese long-tailed macaques, a versatile form of stone-directed play. We tested the hypothesis that stones are used by male monkeys to stimulate their genitals in a sexual context (i.e., "sex toy" hypothesis). Specifically, two SH actions (i.e., "tap-on-groin" (TOG) and "rub-on-groin" (ROG), respectively the repetitive tapping and rubbing of a stone onto the genital area) gained functional properties as self-directed tool-assisted masturbation. Owing to the structural organization of playful activities, we predicted that SH sequences without TOG/ROG would exhibit higher levels of variability, repeatability and exaggeration than SH sequences with TOG/ROG. We also predicted that TOG/ROG would occur more often and last longer in SH sequences in which penile erection - a sexually-motivated physiological response in primates - was observed than in SH sequences in which penile erection was not observed. To identify and compare recurring series of SH patterns otherwise undetectable by using conventional quantitative approaches across SH sequences containing TOG/ROG or not, we used a temporal analysis known as "T-pattern detection and analysis" (TPA). Our predictions about variability, exaggeration and temporal association between TOG/ROG in males and penile erection were supported. As expected, SH sequences without TOG/ROG were, on average, more repeatable than SH sequences with TOG/ROG, but the difference was not statistically significant. Overall, the "sex toy" hypothesis was partly supported, and our results suggested that TOG and ROG are two forms of tool-assisted genital stimulation, possibly derived from the playful handling of stones. These findings are consistent with the view that tool use may evolve in stages from initially non-functional object manipulation, such as object play.

Recurring sequences of multimodal non-verbal and verbal communication during a human psycho-social stress test: A temporal pattern analysis.

BACKGROUND: The Trier Social Stress Test (TSST) is a widely used protocol to study human psycho-social stress responses. Quantitative reports of non-verbal behaviors have been carried out by means of the Ethological Coding System for Interviews (ECSI). However, no data have described whether and how non-verbal and verbal behaviors take part in the composition of multimodal sequences of communication during the test. METHOD: Five non-verbal ECSI categories and four verbal behaviors related with communication were included in the Ethogram. A focal sampling was employed to ensure a high temporal resolution of the behavioral annotation. T-Pattern Analysis was employed to detect statistically-grounded behavioral sequences. RESULTS: As a first step, frequency, overall duration and mean time length were reported for each component of the Ethogram. Besides, T-Pattern Analysis revealed that communication during TSST is organized according to a complex temporal patterning. We found 51 different sequences (T-patterns): 8 T-patterns included exclusively non-verbal behaviors; 17 T-patterns included verbal behaviors and 26 T-patterns encompassed mixed non-verbal and verbal behaviors. T-patterns were discussed depending on their putative functional meaning since non-verbal behaviors almost did not overlap within patterns. CONCLUSIONS: The implementation of an Ethogram including non-verbal and verbal components highlights the multimodal human communication in TSST. T-Pattern Analysis unveils the real-time interplay among these components. In this study results are discussed according to Jakobson's six constitutive factors of communication.

Early alterations of the behavioural structure of mice affected by Duchenne muscular dystrophy and tested in open-field.

Present study has been carried out to assess whether early alterations of the behavioural structure may be detected in mice affected by Duchenne muscular dystrophy (DMD). To this purpose, both quantitative and T-pattern analysis (TPA) were used to analyse the behaviour of two groups of male, two months old mice, 18 MDX and 18 normal as control, tested in an open-field apparatus. T-pattern analysis is a multivariate technique able to reveal hidden structural features of behaviour and, in particular, its temporal characteristics. As to quantitative analyses, mean durations evidenced a significant increase of Walking, Modified Climbing and Rearing and a significant reduction of Immobile-Sniffing, Paw Licking and Immobility in MDX animals. A similar outcome was present in mean occurrences where the only difference was a significant result in Climbing rather than Immobile Sniffing. In addition, mean occurrences, evaluated for all the behavioural components, showed a significant increase for MDX mice. As to TPA, control mice performed 78 different T-patterns occurring 9500 times, whereas in MDX group 47 different T-patterns occurring 7082 times. Overall, MDX mice showed T-patterns of significantly shorter length. Finally, percent distribution of T-patterns encompassing each component of the behavioural repertoire showed significant differences between Control and MDX groups in all the behavioural components, except Climbing. Results suggest that the combined use of quantitative and temporal pattern analyses offers a useful approach to deeply investigate from a behavioural point of view pre-symptomatic stages of DMD in humans and related animal models as well.