Reduced Fear Responses Research Articles

Treatment of Nonsocial Environmental Fear in a Service Dog UsingFluoxetineand Behavior Modification.

Training and maintaining a service dog requires extensive time and financial resources. The emotional bond between service dogs and their handlers poses unique challenges when dogs develop behavioral issues. We present a case of an otherwise healthy adult service dog exhibiting acute environmental fears, hindering her job performance. The dog's fear responses escalated in various settings, including school and home environments. Diagnostic evaluation ruled out medical pathology, leading to a diagnosis of nonsocial environmental fear and generalized anxiety. A multimodal treatment approach involving environmental management, antidepressant medication (fluoxetine), and behavior modification was implemented. Desensitization and counterconditioning protocols targeted specific triggers, such as shiny floors and elevators. Eight weeks after treatment initiation, the dog showed improvement, enabling a modified behavior treatment plan to be implemented as she gradually returned to work with reduced fear responses. Follow-up revealed sustained progress with additional dosage adjustment of fluoxetine.

Contextual fear conditioning in zebrafish: Influence of different shock frequencies, context, and pharmacological modulation on behavior

Contextual fear conditioning is a protocol used to assess associative learning across species, including fish. Here, our goal was to expand the analysis of behavioral parameters that may reflect aversive behaviors in a contextual fear conditioning protocol using adult zebrafish (Danio rerio) and to verify how such parameters can be modulated. First, we analyzed the influence of an aversive stimulus (3 mild electric shocks for 5 s each at frequencies of 10, 100 or 1000 Hz) on fish behavior, and their ability to elicit fear responses in the absence of shock during a test session. To confirm whether the aversive responses are context-dependent, behaviors were also measured in a different experimental environment in a test session. Furthermore, we investigated the effects of dizocilpine (MK-801, 2 mg/kg, i.p.) on fear-related responses. Zebrafish showed significant changes in baseline activity immediately after shock exposure in the training session, in which 100 Hz induced robust contextual fear responses during the test session. Importantly, when introduced to a different environment, animals exposed to the aversive stimulus did not show any differences in locomotion and immobility-related parameters. MK-801 administered after the training session reduced fear responses during the test, indicating that glutamate NMDA-receptors play a key role in the consolidation of contextual fear-related memory in zebrafish. In conclusion, by further exploring fear-related behaviors in a contextual fear conditioning task, we show the effects of different shock frequencies and confirm the importance of context on aversive responses for associative learning in zebrafish. Additionally, our data support the use of zebrafish in contextual fear conditioning tasks, as well as for advancing pharmacological studies related to associative learning in translational neurobehavioral research.

Training dairy heifers with positive reinforcement: Effects on anticipatory behavior

Dairy cattle are often restrained for veterinary procedures, but restraint can cause fear responses that can make the procedure challenging for both the animal and the human handler. Positive reinforcement training (PRT) is used in other species to reduce fear responses and there is now evidence that this can also facilitate handling in cattle. The objectives of this study were to test the effect of PRT on anticipatory and play behavior in dairy heifers. We predicted that heifers trained with PRT would show more anticipatory and play behaviors than control heifers in the period before gaining access to a chute. We used 20 heifers (5 ± 0.6 mo old) that had been habituated to the chute area and had previous experience with handling. Heifers were randomly assigned to 2 treatments: control (n = 10) and PRT (n = 10). PRT heifers were subjected to a training protocol that included standard farm handling techniques, as well as target training with food reinforcement. Control heifers were moved to the chute using standard farm handling techniques only. As predicted, PRT heifers performed more behavioral transitions (7.6 ± 0.77 versus 4.4 ± 0.57 transitions for control heifers; F1,9 = 21.99, P < 0.01), and specifically performed more locomotory play such as jumping (2.1 ± 0.30 vs. 0.4 ± 0.19 jumps; F1,9 = 57.18, P < 0.01) and running (2.0 ± 0.40 s vs. 0.5 ± 0.16 s; F1,9 = 20.73, P < 0.01). These results indicate that PRT results in heifers having a more positive emotional state in anticipation of handling, and supports the use of training to improve the welfare of dairy cattle.

Explaining the Return of Fear with Revised Rescorla-Wagner Models.

Exposure therapy - exposure to a feared stimulus without harmful consequences - can reduce fear responses in many mental disorders. However, such relief is often partial and temporary: fear can return after the therapy has ended. Conditioning research has identified three mechanisms for the return of fear, viz. change in physical context (renewal), the passage of time (spontaneous recovery), and an encounter with the fear-producing unconditioned stimulus (reinstatement). To understand why fear returns and thereby develop more effective therapies, we develop mathematical learning models based on that of Rescorla and Wagner. According to this model, context cues present during extinction become conditioned inhibitors (i.e. safety signals) which prevent total erasure of the threat association. Adding various mechanisms to the model allows it to explain different facets of the return of fear. Among these mechanisms is decay of inhibitory associations, which provides a novel explanation for spontaneous recovery. To make the benefits of exposure robust and permanent, one must minimize the degree to which the extinction context becomes inhibitory in order to maximize unlearning. We simulate several experimental paradigms that reduce the return of fear and explain them according to this principle.

Aversive Contexts Reduce Activity in the Ventral Subiculum- BNST Pathway

Many anxiety disorders can be characterized by abnormalities in detecting and learning about threats, and the inability to reduce fear responses in non-threatening environments. PTSD may be the most representative of context processing pathology, as intrusive memories are experienced in “safe” contexts. The ventral subiculum (vSUB), the main output of the ventral hippocampus, encodes environmental cues and is critical for context processing. The bed nucleus of the stria terminalis (BNST) contributes to anxiety-like behaviors as well as context fear conditioning. Given the important roles of the BNST and the vSUB in these anxiety and fear-related behaviors, and the anatomical connections between the two brain regions, the major aims of this study were to characterize the anatomy and function of the vSUB–BNST pathway. First, using the retrograde tracer cholera toxin, we mapped the topographical arrangement of the vSUB–BNST pathway. Dual retrograde tracing experiments revealed neurons projecting to the BNST and those projecting to the basolateral amygdala are distinct populations. Second, we assessed whether activity in this pathway, as indexed by FOS immunohistochemistry, was modulated by context fear conditioning. Our data reveal less activation of the vSUB–BNST pathway in both males and females in aversive contexts and the greatest activation when animals explored a neutral familiar context. In addition, the vSUB of females contained fewer GABAergic neurons compared to males. These findings suggest that the vSUB–BNST pathway is involved in eliciting appropriate responses to contexts.

P713. Modeling the Role of Context Inhibition in the Return of Fear

Exposure therapy can reduce fear responses in many mental disorders, but such relief is often temporary. Conditioning research has identified three major mechanisms for the return of fear: change in physical context (renewal), the passage of time (spontaneous recovery), and an encounter with the fear-producing unconditioned stimulus (reinstatement). To better understand the return of fear, we develop computational models and use them to simulate benchmark conditioning experiments.

Dorsal Bed Nucleus of the Stria Terminalis-Subcortical Output Circuits Encode Positive Bias in Pavlovian Fear and Reward.

Opposite emotions like fear and reward states often utilize the same brain regions. The bed nucleus of the stria terminalis (BNST) comprises one hub for processing fear and reward processes. However, it remains unknown how dorsal BNST (dBNST) circuits process these antagonistic behaviors. Here, we exploited a combined Pavlovian fear and reward conditioning task that exposed mice to conditioned tone stimuli (CS)s, either paired with sucrose delivery or footshock unconditioned stimuli (US). Pharmacological inactivation identified the dorsal BNST as a crucial element for both fear and reward behavior. Deep brain calcium imaging revealed opposite roles of two distinct dBNST neuronal output pathways to the periaqueductal gray (PAG) or paraventricular hypothalamus (PVH). dBNST neural activity profiles differentially process valence and Pavlovian behavior components: dBNST-PAG neurons encode fear CS, whereas dBNST-PVH neurons encode reward responding. Optogenetic activation of BNST-PVH neurons increased reward seeking, whereas dBNST-PAG neurons attenuated freezing. Thus, dBNST-PVH or dBNST-PAG circuitry encodes oppositely valenced fear and reward states, while simultaneously triggering an overall positive affective response bias (increased reward seeking while reducing fear responses). We speculate that this mechanism amplifies reward responding and suppresses fear responses linked to BNST dysfunction in stress and addictive behaviors.

Oxytocin variation and brain region-specific gene expression in a domesticated avian species.

The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia (WRM). Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to WRMs. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We sequenced the OT from 10 wild munias and 11 Bengalese finches and identified intra-strain variability in both the untranslated and protein-coding regions of the sequence, with all the latter giving rise to synonymous mutations. Several of these changes fall in specific transcription factor-binding sites, and show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed a significantly higher OT mRNA expression in the cerebrum of the Bengalese finches relative to munias, but a significantly lower expression in their diencephalon. Our study thus points to a brain region-specific pattern of neurochemical expression in domesticated and wild avian strains, which could be linked to domestication and the behavioral changes associated with it.

Prefrontal inhibition drives formation and dynamic expression of probabilistic Pavlovian fear conditioning.

The association between cause and effect is usually probabilistic. Memories triggered by ambiguous cues may be altered or biased into a more negative perception in psychiatric diseases. Understanding the formation and modulation of this probabilistic association is important for revealing the nature of aversive memory and alterations in brain diseases. We found that 50% conditioned and unconditioned stimuli (CS-US) association during Pavlovian fear conditioning results in reduced fear responses and neural spiking in the dorsomedial prefrontal cortex (dmPFC) due to enhanced inhibition from dmPFC parvalbumin (PV) neurons. Formation of probabilistic memory is associated with increased synaptic inputs to PV-neurons and requires activation of ventral hippocampus, which detects CS-US mismatch during conditioning. Stress prior to conditioning impairs the formation of probabilistic memory by abolishing PV-neuronal plasticity, while stress prior to memory retrieval reverts enhanced PV-neuron activity. In conclusion, PV-neurons tailor learned responses to fit brain state at the moment of retrieval.

Augmentation of fear extinction by theta-burst transcranial magnetic stimulation of the prefrontal cortex in humans.

BackgroundFear extinction alone does not erase the original fear memory. Interventions that enhance extinction can be beneficial for the treatment of fear-related disorders. Repetitive transcranial magnetic stimulation has been shown to improve memory performance. The present study examined the effects of intermittent theta-burst stimulation (iTBS) on fear extinction and the return of fear memory in humans.MethodsNinety-one young healthy volunteers underwent 3 experiments using a randomized controlled experimental design. Participants first acquired fear conditioning, after which they received 30 Hz iTBS before and after extinction training. The iTBS was applied to 1 of 2 targets: the left dorsolateral prefrontal cortex (dlPFC) and the vertex (control). Fear responses were measured 24 hours later and 1 month later.ResultsDuring the spontaneous recovery and reinstatement tests, iTBS of the left dlPFC before and after extinction significantly reduced fear response, whereas iTBS of the vertex had no effect on fear memory performance. This combined approach had a relatively long-lasting effect (i.e., at least 1 month).LimitationsWe did not explore the effect of iTBS of the dlPFC on the expression of fear without extinction training. The neural mechanisms of iTBS with fear extinction to inhibit the fear response are unclear. Our results are preliminary and should be interpreted with caution.ConclusionThe present results showed that 30 Hz iTBS of the left dlPFC enhanced retention of fear extinction. Our study introduces a new intervention for fear memory and suggests that the left dlPFC may be a treatment target for fear-related disorders.

Prefrontal Inhibition Drives Formation and Dynamic Expression of Probabilistic Pavlovian Fear Conditionin

Under natural conditions, the association between cause and effect is usually not absolute but rather probabilistic, indicating the ambiguous nature of the cues. Responses or memory retrieval triggered by ambiguous cues are altered or biased in a more negative manner in certain psychiatric diseases, such as anxiety and depression. A better understanding of the formation and modulation of this probabilistic association is important for revealing the nature of aversive memory and its alterations in brain diseases. By using probabilistic Pavlovian fear conditioning as a model, we find that conditioning with 50% conditioned and unconditioned stimuli (CS-US) association results in reduced fear responses and neural spiking in dorsomedial prefrontal cortex (dmPFC) due to enhanced inhibition from dmPFC parvalbumin (PV) neurons. This PV-neuronal plasticity is associated with increased synaptic inputs to PV-neurons and requires activation of ventral hippocampus (vHPC). Ventral HPC is required for probabilistic memory formation by detecting CS-US mismatching during conditioning but not for its expression. Stress prior to conditioning impairs the formation of probabilistic memory by abolishing PV-neuronal plasticity, while stress prior to memory retrieval reverts established PV-neuron modulation. Therefore, PV-neurons act as a hub for integration and tailor learned responses to fit brain states at the moment of retrieval.

Handling and milking frequency affect milk yield and behavior in dairy ewes

The effects of human handling (friendly versus aversive) and milking frequency (once versus twice daily) on productive and behavioral parameters of two Greek dairy sheep breeds (Karagouniko and Chios) were examined. The experiment started a week after lamb weaning (50 ± 5 days after lamb birth) and lasted for ten weeks. Milk yield, milk flow rate and number of kick responses during milking were weekly recorded. Each milking was alternatively performed by a friendly or an aversive milker. At the end of 4th and 9th week, an isolation - flight distance test was also applied. As shown, Karagouniko ewes had lower daily milk yield but higher milk flow rate than Chios ewes. Higher milk yield was also observed in ewes milked twice compared to those milked once per day. Aversive handling increased the number of kick responses during milking and decreased milk yield. At the same time, ewes exhibited a preference for a specific position in the milking parlor. During the isolation - flight distance test, Karagouniko ewes bleated less, but had longer flight distances compared to Chios ones. Multiparous had a shorter flight distance than the primiparous ewes. Finally, ewes milked once per day displayed more jumps and less bleats compared to those milked twice. It is suggested that specific training programs targeted at the handling improvement with the intention to reduce fear responses could improve productivity in dairy ewes.

Exploring the neural basis of fear produced by mental imagery: imaginal exposure in individuals fearful of spiders.

Imaginal exposure, i.e. reducing fear using exposure to mental imagery, is a widely used psychological treatment technique for dysfunctional fears. Yet, little is known about its underlying neural mechanisms. The present study examines the neural basis of imaginal exposure using a novel experimental procedure consisting of repeated exposure to flashpoint mental imagery of phobic (spiders) and neutral (gloves) stimuli. Whether the 10 min long imaginal exposure procedure could reduce fear responses was examined one week later. Thirty participants fearful of spiders underwent the experimental procedure. Neural activity was assessed using functional magnetic resonance imaging (session 1). Subjective fear and skin conductance responses were measured throughout the study (sessions 1 and 2). Imaginal exposure evoked intense fear and heightened skin conductance responses, and indicated robust activation in several brain regions, including amygdala, midcingulate cortex and insula. Findings demonstrate that neural activity in fear-processing brain areas can be elicited solely by generating a mental image of a phobic stimulus, that is, in the absence of the percept. Relevant for treatment development, results reveal that a single 10 min session of brief exposures to flashpoint mental imagery can lead to lasting reductions in phobic fear at both the subjective and physiological levels. This article is part of the theme issue 'Offline perception: voluntary and spontaneous perceptual experiences without matching external stimulation'.

Comparing neural correlates of conditioned inhibition between children with and without anxiety disorders – A preliminary study

Cognitive-behavioral therapy (CBT), a first-line treatment for pediatric anxiety disorders, is based on principles of threat learning and extinction. However, CBT does not work sufficiently for up to 40% of clinically anxious youth. The neural and behavioral correlates of conditioned inhibition might provide promising targets for attempts to improve CBT response. During conditioned inhibition, threat and safety cues appear together, forming a safety compound. Here, we test whether this safety compound elicits a reduced fear response compared to pairing the threat cue with a novel cue (novel compound). The current pilot study compares behavioral, physiological, and neural correlates of conditioned inhibition between children with (n = 17, Mage = 13.09, SDage = 3.05) and without (n = 18, Mage = 14.49, SDage = 2.38) anxiety disorders. Behavioral and physiological measures did not differ between children with and without anxiety disorders during fear acquisition. During testing, children with anxiety disorders showed overall higher skin conductance response and expected to hear the aversive sound following the novel compound more often than children without anxiety disorders. Children with anxiety disorders showed more activity in the right ventromedial prefrontal cortex (vmPFC) to the safety versus novel compound. Children without anxiety disorders showed the opposite pattern – more right vmPFC activity to the novel versus safety compound (F(1,31) = 5.40, p = 0.03). No group differences manifested within the amygdala, dorsal anterior cingulate cortex, or hippocampus. These pilot findings suggest a feasible approach for examining conditioned inhibition in pediatric anxiety disorders. If replicated in larger samples, findings may implicate perturbed conditioned inhibition in pediatric anxiety disorders and provide targets for CBT.

Oxytocin Promotes Accurate Fear Discrimination and Adaptive Defensive Behaviors.

The nonapeptide, oxytocin (OT), known for its role in social bonding and attachment formation, has demonstrated anxiolytic properties in animal models and human studies. However, its role in the regulation of fear responses appears more complex, brain site-specific, sex-specific, and dependent on a prior stress history. Studies have shown that OT neurons in the hypothalamus are activated during cued and contextual fear conditioning and during fear recall, highlighting the recruitment of endogenous oxytocin system in fear learning. OT is released into the extended amygdala, which contains the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), both critical for the regulation of fear and anxiety-like behaviors. Behavioral studies report that OT in the CeA reduces contextual fear responses; whereas in the BNST, OT receptor (OTR) neurotransmission facilitates cued fear and reduces fear responses to un-signaled, diffuse threats. These ostensibly contrasting behavioral effects support growing evidence that OT works to promote fear discrimination by reducing contextual fear or fear of diffuse threats, yet strengthening fear responses to imminent and predictable threats. Recent studies from the basolateral nucleus of the amygdala (BLA) support this notion and show that activation of OTR in the BLA facilitates fear discrimination by increasing fear responses to discrete cues. Also, OTR transmission in the CeA has been shown to mediate a switch from passive freezing to active escape behaviors in confrontation with an imminent, yet escapable threat but reduce reactivity to distant threats. Therefore, OT appears to increase the salience of relevant threat-signaling cues yet reduce fear responses to un-signaled, distant, or diffuse threats. Lastly, OTR signaling has been shown to underlie emotional discrimination between conspecifics during time of distress, social transmission of fear, and social buffering of fear. As OT has been shown to enhance salience of both positive and negative social experiences, it can also serve as a warning system against potential threats in social networks. Here, we extend the social salience hypothesis by proposing that OT enhances the salience of relevant environmental cues also in non-social contexts, and as such promotes active defensive behaviors.

Face masks matter: Eurasian tree sparrows show reduced fear responses to people wearing face masks during the COVID-19 pandemic

Continuous exposure to human activity has led to considerable behavioural changes in some wildlife populations. Animals are more likely to survive in a changing environment by adjusting their behaviour to repeatedly occurring but harmless stimulations. During the COVID-19 pandemic, starting in late 2019, face masks were recommended to the public to prevent the spread of pathogens. In this context, we compared the flight initiation distance (FID) of the Eurasian tree sparrow (Passer montanus), a commonly seen bird across China, in Yibin and Dazhou, Sichuan, in response to people with or without face masks. After continuous exposure to people wearing face masks for nearly six months, sparrows evidently became adapted to people wearing face masks, and correspondingly showed shorter FIDs in response to people wearing masks. To our knowledge, this is the first study showing that birds show reduced fear responses to people wearing face masks during the COVID-19 pandemic. Our results suggest a novel aspect of short-term adaptation of wildlife to human behaviour, and that the learning ability of sparrows allows them to adjust their behaviours to adapt to such subtle changes in the environment.

Cognitive and Affective Processes in Body Integrity Dysphoria (BID): A Pilot Study

Individuals suffering from Body Integrity Dysphoria (BID) have the longstanding desire for amputation (BID-A) or palsy (BID-P). Most findings on mental aspects of BID are based on self-reports from sufferers. The aim of this pilot study is to examine cognitive and affective processes beyond what is accessible by self-reports. Therefore, n=5 BID-A, n=3 BID-P-sufferers, n=22 healthy controls and n=8 patients with body dysmorphic disorder (a further group with a strong desire for body modification) were tested. Selective attention bias (eye-tracking), tendency towards false memory (DRM-paradigm) and lack of affective involvement in the unwanted body part (induction and destruction of a rubber-hand/foot-illusion) were examined. Descriptive comparison of the groups showed that BID-A-sufferers fixated amputation stumps faster and longer than any other group and showed a reduced fear response when the body illusion was destroyed. There was no indication of a higher tendency towards false memory in either BID-group. Due to the small sample size, these results cannot be generalized. However, findings showed that BID-symptoms and underlying processes can be accessed in more ways than through self-reports. Moreover, results indicate that BID-A-sufferers selective attention and affective involvement differ from people not desiring an amputation. Understanding these processes may help developing an etiological model, identifying subtypes, and deriving treatment approaches.

Shifting from fear to safety through deconditioning-update

Aversive memories are at the heart of psychiatric disorders such as phobias and post-traumatic stress disorder (PTSD). Here, we present a new behavioral approach in rats that robustly attenuates aversive memories. This method consists of 'deconditioning' animals previously trained to associate a tone with a strong footshock by replacing it with a much weaker one during memory retrieval. Our results indicate that deconditioning-update is more effective than traditional extinction in reducing fear responses; moreover, such effects are long lasting and resistant to renewal and spontaneous recovery. Remarkably, this strategy overcame important boundary conditions for memory updating, such as remote or very strong traumatic memories. The same beneficial effect was found in other types of fear-related memories. Deconditioning was mediated by L-type voltage-gated calcium channels and is consistent with computational accounts of mismatch-induced memory updating. Our results suggest that shifting from fear to safety through deconditioning-update is a promising approach to attenuate traumatic memories.

Dogs, but Not Wolves, Lose Their Sensitivity Toward Novelty With Age.

Selection of behavioral traits holds a prominent role in the domestication of animals, and domesticated species are generally assumed to express reduced fear and reactivity toward novel stimuli compared to their ancestral species. However, very few studies have explicitly tested this proposed link between domestication and reduced fear responses. Of the limited number of studies experimentally addressing the alterations of fear during domestication, the majority has been done on canids. These studies on foxes, wolves, and dogs suggest that decreased expression of fear in domesticated animals is linked to a domestication-driven delay in the first onset of fearful behavior during early ontogeny. Thus, wolves are expected to express exaggerated fearfulness earlier during ontogeny compared to dogs. However, while adult dogs are less fearful toward novelty than adult wolves and wolf-dog hybrids, consensus is lacking on when differences in fear expression arise in wolves and dogs. Here we present the first extended examination of fear development in hand-raised dogs and European gray wolves, using repeated novel object tests from 6 to 26 weeks of age. Contrary to expectations, we found no evidence in support of an increase in fearfulness in wolves with age or a delayed onset of fear response in dogs compared to wolves. Instead, we found that dogs strongly reduced their fear response in the period between 6 and 26 weeks of age, resulting in a significant species difference in fear expression toward novelty from the age of 18 weeks. Critically, as wolves did not differ in their fear response toward novelty over time, the detected species difference was caused solely by a progressive reduced fear response in dogs. Our results thereby suggest that species differences in fear of novelty between wolves and dogs are not caused by a domestication-driven shift in the first onset of fear response. Instead, we suggest that a loss of sensitivity toward novelty with age in dogs causes the difference in fear expression toward novelty in wolves and dogs.

Effects of Regulating Visitor Viewing Proximity and the Intensity of Visitor Behaviour on Little Penguin (Eudyptula minor) Behaviour and Welfare

Simple SummarySome studies on zoo penguins have shown that the presence of visitors may be fear-provoking. To manage zoo visitor–animal interactions, it is important to determine what it is about visitors that affects zoo penguins. Consequently, we investigated the effects of regulating both zoo visitor viewing proximity to the penguin enclosure and the intensity of visitor behaviour on the behaviour and stress physiology of little penguins. A physical barrier was used to increase visitor viewing distance, by 2 m away from the enclosure, and signage and uniformed personnel was used to attempt to regulate the intensity of visitor behaviour. Signage and uniformed personnel had no effect on visitor number, noise level or visitor behaviour. However, the physical barrier reduced the frequency of potentially threatening visitor behaviours such as banging on enclosure features, looming over the pool, physical contact with the pool’s water and sudden movement. When visitors were further away from the enclosure, fewer penguins were huddling, vigilant, and retreating, and more penguins were close to the visitor viewing area, surface swimming and preening in the water. There were no effects of visitor viewing proximity or signage and uniformed personnel on the stress physiology of the little penguins. While there was no effect of signage and uniformed personnel in moderating visitor behaviour, the viewing proximity of visitors to the enclosure affected both visitor and penguin behaviour, where potentially threatening visitor behaviours were decreased and little penguins showed less avoidance behaviour when visitors were further away.We examined the effects of regulating the viewing proximity of visitors and the intensity of visitor behaviour on behaviours indicative of fear and stress physiology of 15 zoo-housed little penguins (Eudyptula minor). A 2 × 2 factorial fully randomised design was used to examine the effects of regulating: (1) the viewing proximity of visitors to enclosure, ‘normal viewing distance’ and ’increased viewing distance’ (using a physical barrier set up 2 m away from the enclosure), and (2) the intensity of visitor behaviour, ‘unregulated visitor behaviour’ and ‘regulated visitor behaviour’ (using signage and uniformed personnel). In addition, a treatment consisting of closing the enclosure to visitors was included. Penguin behaviour, visitor numbers and visitor behaviour were recorded by CCTV video recordings and direct observations, respectively. Penguin faecal glucocorticoid metabolites were also analysed as a measure of stress physiology. We found that increased viewing distance reduced (p < 0.05) all visitor behaviours except for loud vocalisations and tactile contact with penguins. However, there were no direct effects of signage and uniformed personnel on visitor behaviour (p > 0.05). As the regulation of viewing proximity increased from a closed exhibit to an open exhibit with increased viewing distance, and then to an open exhibit with normal viewing distance, this increased the proportion of penguins huddling (p = 0.0011), vigilant (p = 0.0060) and retreating (p = 0.00013), and decreased the proportion of penguins within 1 m of the visitor viewing area (p = 0.00066), surface swimming (p = 0.00091) and preening in the water (p = 0.042). There were also limited effects of regulating visitor behaviour on penguin behaviour. No treatment effects were found on faecal glucocorticoid metabolites (p > 0.05). These results indicate that regulating visitor viewing proximity affects penguin behaviours indicative of fear and visitor behaviour. This suggests that close visitor contact can be fear-provoking for little penguins but increasing the distance between visitors and penguins can reduce fear responses of penguins by regulating both viewing proximity and visitor behaviour. However, it is unclear whether these changes in penguin behaviour are due to the increased separation between visitors and penguins and/or specific visitor behaviours associated with close viewing proximity to the enclosure, such as leaning over the enclosure or tactile contact with the pool, which are impeded when visitors are further away.