ß-Hexachlorocyclohexane triggers neuroinflammatory activity, epigenetic histone post-translational modifications and cognitive dysfunction.

Persistent organic pollutants (POPs), which encompass pesticides and industrial chemicals widely utilized across the globe, pose a covert threat to human health. ß-hexachlorocyclohexane (ß-HCH) is an organochlorine pesticide with striking stability, still illegally dumped in many countries, and recognized as responsible for several pathogenetic mechanisms. This study represents a pioneering exploration into the neurotoxic effects induced by the exposure to ß-HCH specifically targeting neuronal cells (N2a), microglia (BV-2), and C57BL/6 mice. As shown by western blot and qPCR analyses, the administration of ß-HCH triggered a modulation of NF-κB, a key factor influencing both inflammation and pro-inflammatory cytokines expression. We demonstrated by proteomic and western blot techniques epigenetic modifications in H3 histone induced by ß-HCH. Histone acetylation of H3K9 and H3K27 increased in N2a, and in the prefrontal cortex of C57BL/6 mice administered with ß-HCH, whereas it decreased in BV-2 cells and in the hippocampus. We also observed a severe detrimental effect on recognition memory and spatial navigation by the Novel Object Recognition Test (NORT) and the Object Place Recognition Task (OPRT) behavioural tests. Cognitive impairment was linked to decreased expression of the genes BDNF and SNAP-25, which are mediators involved in synaptic function and activity. The obtained results expand our understanding of the harmful impact produced by ß-HCH exposure by highlighting its implication in the pathogenesis of neurological diseases. These findings will support intervention programs to limit the risk induced by exposure to POPs. Regulatory agencies should block further illicit use, causing environmental hazards and endangering human and animal health.

Nasal obstruction during the growth period modulates the Wnt/ß-catenin pathway and brain-derived neurotrophic factor production in association with tyrosine kinase receptor B mRNA reduction in mouse hippocampus.

There is accumulating evidence that nasal obstruction induces high-level brain dysfunction, including memory and learning deficits. We previously demonstrated that unilateral nasal obstruction (UNO) during the growth period increases the expression of brain-derived neurotrophic factor (BDNF). The expression of BDNF is regulated by the Wnt/ß-Catenin pathway, which is linked to neuronal differentiation, proliferation, and maintenance. However, little is known about whether Wnt3a protein expression could be an index for modulations analyses in the Wnt/ß-Catenin pathway caused by UNO during the growth period. This study aimed to investigate the effects of UNO during the growth period on the Wnt/ß-Catenin pathway in the hippocampus using combined behavioural, biochemical, and histological approaches. Male BALB/C mice were randomly divided into the control (CONT; n = 6) and experimental (UNO; n = 6) groups. Blood oxygen saturation (SpO2 ) levels were measured, and a passive avoidance test was performed in mice aged 15 weeks. Brain tissues were subjected to immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blot analysis. Compared with control mice, UNO mice had lower SpO2 levels and exhibited memory/learning impairments during behavioural testing. Moreover, Wnt3a protein, BDNF mRNA, and tyrosine kinase receptor B (TrkB) mRNA expression levels were significantly lower in the hippocampus in the UNO group than in the CONT group. Our findings suggested that UNO during the growth period appeared to modulate the hippocampal Wnt/ß-catenin pathway and BDNF production in association with TrkB mRNA reduction, thereby resulting in memory and learning impairments.

The onset of piscivory in a freshwater fish species: analysis of behavioural and physiological traits.

The onset of piscivory in fish, resulting in a shift from zooplankton or invertebrate to fish prey, was studied on pikeperch (Sander lucioperca) larvae displaying and not displaying piscivorous behaviour at different ages (23, 30, 37, 44 and 52 days post-hatching) using behavioural (attack, capture and swimming activity), morphological (allometry) and digestive enzymatic (trypsin, α-amylase and pepsin) analyses. The shift from zooplanktonic food items (Artemia nauplii) to a piscivorous diet did not occur at the same time for all individuals within the same cohort. Predation tests, conducted under controlled conditions (20°C; ad libitum feeding), showed that some larvae attacked fish prey as early as the age of 3 weeks [11.0 ± 1.3 mm total length (TL)], whereas others did not start until the age of 6 weeks (16.6 ± 1.9 mm TL). Piscivorous individuals were bigger, with larger heads, longer tails, higher acid protease and lower alkaline protease activities, than non-piscivorous conspecifics. In conclusion, high interindividual variability in morphological and digestive system developments linked to the development of predatory abilities could induce cannibalism in fish.

Sweeteners modulate bioactivity of endothelial progenitor cells but not induce detrimental effects both on inflammation and behavioural changes.

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p < .05). This reduction was more strongly significant when a double dose of glucose, aspartame, rebaudioside A and cyclamate (p < .005) in comparison to fructose and sucrose (p < .05) was administered. During inflammation carrageenan-induced, all sweeteners produced a significant increase of EPCs compared to the control group (p < .05). Consumption of glucose and sugar substitutes affect mouse EPC number according to the absence or presence of an inflammatory status, but does not induce detrimental effects on inflammation and behavioural changes.

Behavioural phenotypes in mice after prenatal and early postnatal exposure to intermediate frequency magnetic fields.

Electromagnetic fields are ubiquitous in the environment. Human exposure to intermediate frequency (IF) fields is increasing due to applications like electronic article surveillance systems, wireless power transfer, and induction heating cooking hobs. However, there are limited data on possible health effects of exposure to IF magnetic fields (MF). In the present study, we set out to assess cognitive and behavioural effects of IF MF in mice exposed during prenatal and early postnatal periods. Pregnant female mice were exposed continuously to 7.5kHz MFs at 12 and 120µT, from mating until weaning of pups. Sham exposed pregnant mice were used as a control group. A behavioural teratology study was conducted on the male offspring at two months of age to detect possible effects on the developing nervous system. Body weight development did not differ between the exposure groups. The exposure did not alter spontaneous motor activity when exploring a novel cage or anxiety in novelty-suppressed feeding or marble burying tests. Improved performance in the Rotarod task was observed in the 12µT group, while the 120µT exposure group swam more slowly than the sham exposed group in the Morris swim navigation task. However, indices of learning and memory (path length and escape latency during task acquisition and search bias during the probe test) did not differ between the exposure groups. Furthermore, the passive avoidance task did not indicate any impairment of long-term memory over a 48h interval in the exposed groups. In a post-mortem histopathological analysis, there was no evidence for an effect of IF MF exposure on astroglial reactivity or hippocampal neurogenesis. The results suggest that the IF MF used did not have detrimental effects on spatial learning and memory or histological markers of tissue reaction. The two statistically significant findings that were observed (improved performance in the Rotarod task in the 12µT group and decreased swimming speed in the 120µT group) are likely to be chance findings, as they do not form an internally consistent, dose-dependent pattern indicative of specific developmental effects.

Effects of waterborne exposure to the antidepressant fluoxetine on swimming, shoaling and anxiety behaviours of the mosquitofish Gambusia holbrooki.

Chemical pollution from pharmaceuticals is increasingly recognized as a major hazard to the aquatic biota. Among the wide variety of pharmaceuticals, fluoxetine (FLX) is one of the most widely prescribed antidepressants, and therefore, it is frequently identified in the aquatic environment. As FLX is designed to alter human behaviour and many physiological pathways are conserved across vertebrates, this drug may affect the behaviour of fish living in FLX-polluted environments. Here, we exposed groups of female mosquitofish Gambusia holbrooki to waterborne FLX for 14 days, under semi-static conditions with daily renewal of test solutions. Following exposure, we conducted a set of behavioural assays in individual fish, aimed at assessing the effects of FLX on their locomotor activity and behavioural responses. We found that FLX impaired swimming behaviour at high concentrations (25 µg/L and 50 µg/L) but not at low concentrations close to environmental levels (1 µg/L and 5 µg/L). When swimming activity was assessed 5 min after transfer of the focal fish to the testing tank, 50 µg/L FLX was the only concentration showing significant effects. However, when the same trials were performed 24 h later, 25 µg/L FLX turned out to be an effect concentration in addition to 50 µg/L. Interestingly, these concentrations would elicit fish plasma concentrations comprised within the range of human therapeutic doses. When subjected to a light/dark preference test, fish showed tendency to remain less time in the dark area at high FLX concentrations, thus suggesting an anti-anxiety response. Shoaling behaviour was not affected by FLX exposure. Our study contributes to the growing body of literature evaluating the effects of FLX on animal behaviour. Regarding the experimental design used in behavioural testing, our findings suggest that focal fish should be subjected to long habituation periods, namely of at least a few hours, in order to better assess the effects of drug exposure.

Review: Neuropathology and behavioural features of transgenic murine models of Alzheimer's disease.

Our understanding of the underlying biology of Alzheimer's disease (AD) has been steadily progressing; however, this is yet to translate into a successful treatment in humans. The use of transgenic mouse models has helped to develop our understanding of AD, not only in terms of disease pathology, but also with the associated cognitive impairments typical of AD. Plaques and neurofibrillary tangles are often among the last pathological changes in AD mouse models, after neuronal loss and gliosis. There is a general consensus that successful treatments need to be applied before the onset of these pathologies and associated cognitive symptoms. This review discusses the different types of AD mouse models in terms of the temporal progression of the disease, how well they replicate the pathological changes seen in human AD and their cognitive defects. We provide a critical assessment of the behavioural tests used with AD mice to assess cognitive changes and decline, and discuss how successfully they correlate with cognitive impairments in humans with AD. This information is an important tool for AD researchers when deciding on appropriate mouse models, and when selecting measures to assess behavioural and cognitive change.

New 5-HT1A, 5HT2A and 5HT2C receptor ligands containing a picolinic nucleus: Synthesis, in vitro and in vivo pharmacological evaluation.

Picolinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors was evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine), known to play critical roles in affinity for serotoninergic receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide (3o) with Ki=0.046nM, was the most affine and selective derivative for the 5-HT1A receptor compared to other serotoninergic dopaminergic and adrenergic receptors. N-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)picolinamide (3b), instead, showed a subnanomolar affinity towards 5-HT2A with Ki=0.0224nM, whereas N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)ethyl)picolinamide (3s) presented an attractive 5-HT2C affinity with Ki=0.8nM. Moreover, the compounds having better affinity and selectivity binding profiles towards 5-HT2A were selected and tested on rat ileum, to determine their effect on 5HT induced contractions. Those more selective towards 5-HT1A receptors were studied in vivo on several behavioral tests.

Bergamot Essential Oil Attenuates Anxiety-Like Behaviour in Rats.

Preclinical studies have recently highlighted that bergamot essential oil (BEO) is endowed with remarkable neurobiolological effects. BEO can affect synaptic transmission, modulate electroencephalographic activity and it showed neuroprotective and analgesic properties. The phytocomplex, along with other essential oils, is also widely used in aromatherapy to minimize symptoms of stress-induced anxiety and mild mood disorders. However, only limited preclinical evidences are actually available. This study examined the anxiolytic/sedative-like effects of BEO using an open field task (OFT), an elevated plus-maze task (EPM), and a forced swimming task (FST) in rats. This study further compared behavioural effects of BEO to those of the benzodiazepine diazepam. Analysis of data suggests that BEO induces anxiolytic-like/relaxant effects in animal behavioural tasks not superimposable to those of the DZP. The present observations provide further insight to the pharmacological profile of BEO and support its rational use in aromatherapy.

Ozone degrades floral scent and reduces pollinator attraction to flowers.

In this work we analyzed the degradation of floral scent volatiles from Brassica nigra by reaction with ozone along a distance gradient and the consequences for pollinator attraction. For this purpose we used a reaction system comprising three reaction tubes in which we conducted measurements of floral volatiles using a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS) and GC-MS. We also tested the effects of floral scent degradation on the responses of the generalist pollinator Bombus terrestris. The chemical analyses revealed that supplementing air with ozone led to an increasing reduction in the concentrations of floral volatiles in air with distance from the volatile source. The results revealed different reactivities with ozone for different floral scent constituents, which emphasized that ozone exposure not only degrades floral scents, but also changes the ratios of compounds in a scent blend. Behavioural tests revealed that floral scent was reduced in its attractiveness to pollinators after it had been exposed to 120 ppb O3 over a 4.5 m distance. The combined results of chemical analyses and behavioural responses of pollinators strongly suggest that high ozone concentrations have significant negative impacts on pollination by reducing the distance over which floral olfactory signals can be detected by pollinators.

Three methods of behavioural testing to measure anxiety - A review.

Behavioural test is very useful to assess the anxiety activity, screen new anxiolytic drugs, explore the pathogenesis of anxiety disorders. Methods of behavioural testing that reflects different aspects of anxiety emotionality simultaneously have always been a critical issue for academics. In this paper, we reviewed previous methods to use behavioural test to evaluate the anxiety activity. A single test was used to measure only one aspect of anxiety emotionality. A battery of behavioural tests could get a comprehensive information of anxiety profile. In one single trial, open field test, elevated plus maze and light/dark box are integrated to assess different types of emotional behaviours. This new paradigm is useful for evaluating multiple dimensions of behaviours simultaneously, minimizing general concerns about previous test experience and inter-test intervals between tests. It is proposed as a promising alternative to using test battery.

Lambda-cyhalothrin alters locomotion, mood and memory abilities in Swiss mice.

Lambda-cyhalothrin (LCT) is a type II pyrethroid widely used in agriculture for plant protection against pests. However, pyrethroids represents a risk for rural female farmworkers, and few studies addressed LCT-behavioural alterations in mice. The present study evaluates the effect of LCT on behaviour of eight weeks aged female mice. Mice were divided into three groups including treated mice that received through gavage (i) 0.5 mg/kg bw and (ii) 2 mg/kg of LCT dissolved in corn oil, and (iii) the vehicle controls. Behavioural tests assess the locomotor activity using open field test, the anxiety by the dark-light box test, the learning memory with novel object recognition test, the memory retention by the elevated plus maze test, and the spatial working memory using the Y-maze test. Subacute treatment with low doses of LCT decreases total distance travelled, induces anxiogenic effect by reducing the time spent in the enlightened compartment, alters memory retention by increasing the latency time, and also affects learning memory by reducing the recognition index parameter. However, LCT does not significantly alter spatial working memory. In conclusion, LCT-treated female mice show an alteration in locomotor activity, mood state and memory abilities probably related to oxidative stress and altered neurotransmission.

Sex-dependent impact of perinatal 5G electromagnetic field exposure in the adolescent rat behavior.

The fifth generation (5G) network is currently being worldwide spread out, raising questions about the potential impact of this new technology, particularly on immature organisms. The current study aimed to investigate the effects of daily 5G electromagnetic field (EMF) perinatal exposure on the neurodevelopment of rats. The exposure level was set to the limit of whole-body public exposure defined by the International Commission on Non-Ionizing Radiation Protection. The mother rat specific absorption rate (SAR) was 0.07 W/kg for 22 h/day at 3500 MHz continuous wave from gestational day (GD) 8 to post-natal day (PND) 21. Clinical observations were performed on weight, length, sex ratio, number of pups per litter, and number of stillborn in sham and EMF-exposed groups (n = 7). The age of pinna ear detachment, incisor eruption, and eye opening were recorded. Behavior was assessed on righting, gripping, and negative geotaxis reflexes at PND 3 or 7 and on stereotyped and horizontal movements in the open field at PND 43. Our results indicated that both male and female pups showed delayed incisor eruption in the EMF-exposed group compared to the sham group (+ 1 day). Regarding activity in the open field, adolescent females showed less stereotyped movements (- 70%), while adolescent males showed more stereotyped movements (+ 50%) compared to the sham-exposed adolescent rats. Thus, the present study suggested that perinatal exposure to 5G at SAR level below reglementary threshold led to perturbations in the descendants seen in juveniles and adolescents.

Assessing Auditory Processing in Children with Listening Difficulties: A Pilot Study.

BACKGROUND: Auditory processing disorders (APD) may be one of the problems experienced by children with listening difficulties (LiD). The combination of auditory behavioural and electrophysiological tests could help to provide a better understanding of the abilities/disabilities of children with LiD. The current study aimed to quantify the auditory processing abilities and function in children with LiD. METHODS: Twenty children, ten with LiD (age = 8.46; SD = 1.39) and ten typically developing (TD) (age = 9.45; SD = 1.57) participated in this study. All children were evaluated with auditory processing tests as well as with attention and phonemic synthesis tasks. Electrophysiological measures were also conducted with click and speech auditory brainstem responses (ABR). RESULTS: Children with LiD performed significantly worse than TD children for most behavioural tasks, indicating shortcomings in functional auditory processing. Moreover, the click-ABR wave I amplitude was smaller, and the speech-ABR waves D and E latencies were longer for the LiD children compared to the results of TD children. No significant difference was found when evaluating neural correlates between groups. CONCLUSIONS: Combining behavioural testing with click-ABR and speech-ABR can highlight functional and neurophysiological deficiencies in children with learning and listening issues, especially at the brainstem level.

Environmental Enrichment as Part of the Improvement of the Welfare of Japanese Quails.

The aim of this study was to evaluate the indicators of the behavioural and physiological welfare of Japanese quails (Coturnix japonica) as possible responses to the enrichment of the birds' habitat. The study sample consisted of 280 Japanese quails (224 ♀ and 56 ♂, respectively). Birds of 5 weeks of age were randomly divided into seven equally sized groups and then divided into replication subgroups (four per group, 10 birds in each replication). Birds were maintained in 0.5 m2 cages with unrestricted access to water and food. The experimental factor was the presence or absence of enrichment of the birds' cages: the nest box, scratcher, plastic corrugated pipe (tunnel), limestone cubes, sandbathing box and feeder box with a drilled cover. Quails were subjected to behavioural tests (tonic immobility and open field tests) and, after 6 weeks, blood samples were taken from them to determine their biochemical indices as well as their cortisol and corticosterone levels. An additional element was the assessment of fertility indices. The presence of enrichment was shown to reduce behavioural disturbances in Japanese quails. This study also found that the colour and shape of an object were very important regarding the birds' interest in it. Additionally, individuals kept in enriched cages, who were allowed to exhibit their natural behavioural patterns, had lower stress levels.

Effects of corticosterone on BDNF expression and mood behaviours in mice.

Stress hormones such as cortisol play a critical role in depressive disorders. Therefore, corticosterone has been used to develop a depression model in animals. Our previous studies found that the precursor of brain-derived neurotrophic factor (proBDNF) and its receptors are upregulated in depression in human and animal models. In the present study, we aimed to examine whether proBDNF and mature BDNF (mBDNF) are altered in the corticosterone-induced depression model in mice. Male and female mice were given corticosterone dissolved in 0.3% hydroxypropyl- ß-cyclodextrin (ß-CD) or vehicle (ß-CD) in drinking water for 33 days. We have found that corticosterone induced depressive-like behaviours as reflected by increased immobility time in the tail suspension test and decreased grooming time in the splash test. Corticosterone also induced anxiety-like behaviours as represented by decreased entries into the central zone of the open field test and the open arms of the elevated plus maze test. We found that corticosterone administration resulted in differential changes of proBDNF and mature BDNF in different brain regions and peripheral tissues. ProBDNF was increased in the hippocampus and cerebellum, but no change was found in the prefrontal cortex and hypothalamus. Both proBDNF and mBDNF were significantly increased in the pituitary gland. In contrast, proBDNF was significantly decreased in the adrenal gland.  There were no significant changes in proBDNF or mBDNF in other peripheral tissues, including the liver and sex organs. We conclude that the stress hormone corticosterone causes depressive behaviours but differentially regulates the processing of proBDNF in mice. ProBDNF may participate in the development of depression behaviours in corticosterone treated animals.

Post-acute delivery of α5-GABAA antagonist, S 44819, improves functional recovery in juvenile rats following stroke.

Hypo-excitability was reported in the peri-infarct tissue following stroke, an effect counteracted by a blockage of α5-GABAA receptors in adult rodents. Our present study aims to evaluate the effect of a selective α5-GABAA receptor antagonist, S 44819, in stroke in juvenile animals. We have set up and characterized an original model of transient ischemic stroke in 28 day-old Sprague-Dawley rats (45-min occlusion of the middle cerebral artery by intraluminal suture). In this model, S 44819 (1, 3 and 10 mg/kg, b.i.d) was orally administered from day 3 to day 16 after stroke onset. Sensorimotor recovery was assessed on day 1, day 9 and day 16 after stroke onset. Results show that rats treated with S 44819 at the doses of 3 and 10 mg/kg displayed a significant improvement of the neurological deficits (neuroscore) on day 9 and day 16, when compared with animals treated with vehicle. Grip-test data analysis reveals that rats treated with S 44819 at the dose of 3 mg/kg displayed a better recovery on day 9 and day 16. These results are in agreement with those previously observed in adult rats, demonstrating that targeting α5-GABAA receptors improves neurological recovery after stroke in juvenile rats.

Deficiency of CHAMP1, a gene related to intellectual disability, causes impaired neuronal development and a mild behavioural phenotype.

CHAMP1 is a gene associated with intellectual disability, which was originally identified as being involved in the maintenance of kinetochore-microtubule attachment. To explore the neuronal defects caused by CHAMP1 deficiency, we established mice that lack CHAMP1. Mice that are homozygous knockout for CHAMP1 were slightly smaller than wild-type mice and died soon after birth on pure C57BL/6J background. Although gross anatomical defects were not found in CHAMP1 -/- mouse brains, mitotic cells were increased in the cerebral cortex. Neuronal differentiation was delayed in CHAMP1 -/- neural stem cells in vitro, which was also suggested in vivo by CHAMP1 knockdown. In a behavioural test battery, adult CHAMP1 heterozygous knockout mice showed mild memory defects, altered social interaction, and depression-like behaviours. In transcriptomic analysis, genes related to neurotransmitter transport and neurodevelopmental disorder were downregulated in embryonic CHAMP1 -/- brains. These results suggest that CHAMP1 plays a role in neuronal development, and CHAMP1-deficient mice resemble some aspects of individuals with CHAMP1 mutations.

Reliability of common mouse behavioural tests of anxiety: A systematic review and meta-analysis on the effects of anxiolytics.

The validity of widely used rodent behavioural tests of anxiety has been questioned, as they often fail to produce consistent results across independent replicate studies. In this study, we assessed the sensitivity of common behavioural tests of anxiety in mice to detect anxiolytic effects of drugs prescribed to treat anxiety in humans. We conducted a pre-registered systematic review of 814 studies reporting effects of 25 anxiolytic compounds using common behavioural tests for anxiety. Meta-analyses of effect sizes of treatments showed that only two out of 17 commonly used test measures reliably detected effects of anxiolytic compounds. We report considerable between-study variation in size and even direction of effects of most anxiolytics on most outcome variables. Our findings indicate a general lack of sensitivity of those behavioural tests and cast serious doubt on both construct and predictive validity of most of these tests. In view of scientifically valid and ethically responsible research, we call for a revision of behavioural tests of anxiety in mice and the development of more predictive tests.

Exploratory Behaviours of Primitive Horses Based on Konik: A Preliminary Study.

This study aimed at assessing the behaviour of Konik geldings and mares, kept in a stable and in a free-range system, during behavioural tests regarded as a determinant of the exploration urge. A total of 19 Konik horses kept in individual stables and in a free-range system were included in the study. The experiment was conducted in five phases separated by five-day breaks. A one-stage passive human test was performed during the first phase, a three-stage active human test-2nd phase, a three-stage unknown object test-3rd phase, a two-stage unknown surface test-4th phase, and a one-stage test of social isolation-5th phase. Ten attributes were analysed, including the horse sex and the keeping system. The results were also correlated with one another. Konik horses were found to show the urge to explore, although their behavioural responses are individual and stimulus dependent. In many cases, the horse sex and the keeping system influence the exploratory behaviour, although it is manifested by a greater intensity in geldings than in mares, and in free-range horses than in those kept in a stable. The study is regarded as preliminary due to the small number of horses in the study groups.