An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Interoceptive insular cortex participates in sensory processing of gastrointestinal malaise and associated behaviors.

The insular cortex plays a central role in the perception and regulation of bodily needs and emotions. Its modular arrangement, corresponding with different sensory modalities, denotes a complex organization, and reveals it to be a hub that is able to coordinate autonomic and behavioral responses to many types of stimuli. Yet, little is known about the dynamics of its electrical activity at the neuronal level. We recorded single neurons in behaving rats from the posterior insula cortex (pIC), a subdivision considered as a primary interoceptive cortex, during gastrointestinal (GI) malaise, a state akin to the emotion of disgust in humans. We found that a large proportion of pIC neurons were modulated during the rodent compensatory behaviors of lying on belly (LOB) and Pica. Furthermore, we demonstrated that LOB was correlated with low-frequency oscillations in the field potentials and spikes at the theta (8 Hz) band, and that low-frequency electrical microstimulation of pIC elicited LOB and Pica. These findings demonstrate that pIC neurons play a critical role in GI malaise perception, and that the pIC influences the expression of behaviors that alleviate GI malaise. Our model provides an accessible approach at the single cell level to study innate emotional behaviors, currently elusive in humans.

Effort Displayed During Appetitive Phase of Feeding Behavior Requires Infralimbic Cortex Activity and Histamine H1 Receptor Signaling.

The chances to succeed in goal-directed behaviors, such as food or water-seeking, improve when the subject is in an increased arousal state. The appetitive phase of these motivated behaviors is characterized by high levels of behavioral and vegetative excitation. The key decision of engaging in those particular behaviors depends primarily on prefrontal cortical areas, such as the ventromedial prefrontal cortex. We propose that the infralimbic cortex (ILC) located in the medial prefrontal cortex induces an increase in arousal during the appetitive phase of motivated behavior, and that this increase in arousal is, in turn, mediated by the activation of the brain histaminergic system, resulting in higher motivation for getting food rewards. To test this hypothesis, we conduct a progressive ratio operant conditioning to test the degree of motivation for food, while simultaneously manipulating the histaminergic system through pharmacologic interventions. We found that the behavioral responses to obtain food in hungry rats were disrupted when the ILC was inhibited through muscimol infusion, blocking brain H1 histamine receptors by intracerebroventricular infusion of pyrilamine or by satiety. In contrast, the consummatory behavior was not affected by ILC inhibition. The extracellular histamine levels in the ILC were increased in direct correlation with the degree of motivation measured in the progressive ratio test. ILC inhibition also prevented this increase in histamine levels. The rise in extracellular histamine levels during the progressive ratio test was similar (ca. 200%) during the active or the resting period of the day. However, different basal levels are observed for these two periods. Our findings suggest that increased histamine levels during this behavior are not simply explained by the awaked state, but instead, there is a motivation-related release of histamine, suggestive of a specific form of brain activation. Serotonin (another critical component of the ascending arousal system) was also tested. Interestingly, changes in levels of this neuromodulator were not detected during the progressive ratio test. In conclusion, our results suggest that ILC activation and subsequent increase in brain histamine release are both necessary for the normal performance of a motivated behavior such as feeding.

Interoceptive Insular Cortex Mediates Both Innate Fear and Contextual Threat Conditioning to Predator Odor.

The insular cortex (IC), among other brain regions, becomes active when humans experience fear or anxiety. However, few experimental studies in rats have implicated the IC in threat responses. We have recently reported that inactivation of the primary interoceptive cortex (pIC) during pre-training, or the intra-pIC blockade of protein synthesis immediately after training, impaired the consolidation of auditory fear conditioning. The present study was designed to investigate the role of the pIC in innate and learned defensive responses to predator odor. Freezing behavior was elicited by single or repetitive exposures to a collar that had been worn by a domestic cat. Sessions were video-recorded and later scored by video observation. We found that muscimol inactivation of the pIC reduced the expression of freezing reaction in response to a single or repeated exposure to cat odor. We also found that pIC inactivation with muscimol impaired conditioning of fear to the context in which rats were exposed to cat odor. Furthermore, neosaxitoxin inactivation of the pIC resulted in a prolonged and robust reduction in freezing response in subsequent re-exposures to cat odor. In addition, freezing behavior significantly correlated with the neural activity of the IC. The present results suggest that the IC is involved in the expression of both innate and learned fear responses to predator odor.

The activity of discrete sets of neurons in the posterior insula correlates with the behavioral expression and extinction of conditioned fear.

The interoceptive insular cortex is known to be involved in the perception of bodily states and emotions. Increasing evidence points to an additional role for the insula in the storage of fear memories. However, the activity of the insula during fear expression has not been studied. We addressed this issue by recording single units from the posterior insular cortex (pIC) of awake behaving rats expressing conditioned fear during its extinction. We found a set of pIC units showing either significant increase or decrease in activity during high fear expression to the auditory cue ("freezing units"). Firing rate of freezing units showed high correlation with freezing and outlasted the duration of the auditory cue. In turn, a different set of units showed either significant increase or decrease in activity during low fear state ("extinction units"). These findings show that expression of conditioned freezing is accompanied with changes in pIC neural activity and suggest that the pIC is important to regulate the behavioral expression of fear memory. NEW & NOTEWORTHY Here, we show novel single-unit data from the interoceptive insula underlying the behavioral expression of fear. We show that different populations of neurons in the insula codify expression and extinction of conditioned fear. Our data add further support for the insula as an important player in the regulation of emotions.

Plasticity in the Interoceptive System.

The most outstanding manifestations of the plastic capacities of brain circuits and their neuronal and synaptic components in the adult CNS are learning and memory. A reduced number of basic plastic mechanisms underlie learning capacities at many levels and regions of the brain. The interoceptive system is no exception, and some of the most studied behavioral changes that involve learning and memory engage the interoceptive pathways at many levels of their anatomical and functional organization.In this chapter, we will review four examples of learning, mostly in rats, where the interoceptive system has a role. In the case of conditioned taste aversion, the interoceptive system is of outstanding importance. In drug addiction, the role of the insular cortex - the highest level of the interoceptive system- is unusual and complex, as many forebrain regions are engaged by the process of addiction. In the third example, neophobia, the gustatory region of the insular cortex plays a major role. Finally, the role of different areas of the insular cortex in different processes of aversive memory, particularly fear conditioning, will be reviewed.

The Histaminergic Tuberomamillary Nucleus Is Involved in Appetite for Sex, Water and Amphetamine.

The histaminergic system is one component of the ascending arousal system which is involved in wakefulness, neuroendocrine control, cognition, psychiatric disorders and motivation. During the appetitive phase of motivated behaviors the arousal state rises to an optimal level, thus giving proper intensity to the behavior. Previous studies have demonstrated that the histaminergic neurons show an earlier activation during the appetitive phase of feeding, compared to other ascending arousal system nuclei, paralleled with a high increase in arousal state. Lesions restricted to the histaminergic neurons in rats reduced their motivation to get food even after 24 h of food deprivation, compared with intact or sham lesioned rats. Taken together, these findings indicate that the histaminergic system is important for appetitive behavior related to feeding. However, its role in other goal-directed behaviors remains unexplored. In the present work, male rats rendered motivated to obtain water, sex, or amphetamine showed an increase in Fos-ir of histaminergic neurons in appetitive behaviors directed to get those reinforcers. However, during appetitive tests to obtain sex, or drug in amphetamine-conditioned rats, Fos expression increased in most other ascending arousal system nuclei, including the orexin neurons in the lateral hypothalamus, dorsal raphe, locus coeruleus and laterodorsal tegmental neurons, but not in the ventral tegmental area, which showed no Fos-ir increase in any of the 3 conditions. Importantly, all these appetitive behaviors were drastically reduced after histaminergic cell-specific lesion, suggesting a critical contribution of histamine on the intensity component of several appetitive behaviors.

A role for the interoceptive insular cortex in the consolidation of learned fear.

A growing body of evidence suggests that learned fear may be related to the function of the interoceptive insular cortex. Using an auditory fear conditioning paradigm in rats, we show that the inactivation of the posterior insular cortex (pIC), the target of the interoceptive thalamus, prior to training produced a marked reduction in fear expression tested 24h later. Accordingly, post-training anisomycin infused immediately, but not 6h after, also reduced fear expression tested the following day, supporting a role for the pIC in consolidation of fear memory. The long-term (ca. a week) and reversible inactivation of the pIC with the sodium channel blocker neosaxitoxin, immediately after fear memory reactivation induced a progressive decrease in the behavioral expression of conditioned fear. In turn, we observed that fear memory reactivation is accompanied by an enhanced expression of Fos and Zif268, early genes involved in neural activity and plasticity. Taken together these data indicate that the pIC is involved in the regulation of fear memories.

Infralimbic cortex activation and motivated arousal induce histamine release.

Appetitive behaviours occur in a state of behavioural and physiological activation that allows the optimal performance of these goal-directed behaviours. Here, we tested the hypothesis that histamine neurons under the command of the infralimbic cortex are important to provide behavioural activation. Extracellular histamine and serotonin were measured by microdialysis of the medial prefrontal cortex in behaving rats in parallel with a picrotoxin microinjection into the infralimbic cortex. The injection aroused the rats behaviourally, increased histamine release and decreased serotonin levels. Inhibition of the infralimbic cortex with muscimol produced the opposite effects on neurotransmitter release. The behavioural activation induced by motivating hungry rats with caged food was paralleled by an immediate histamine release, whereas awakening induced by tapping their microdialysis bowl increased serotonin, but not histamine levels. In conclusion, picrotoxin injection into the infralimbic cortex produces behavioural activation together with histamine release; in a similar manner, induction of an appetitive state produced histamine release, likely related to increased behavioural activation characteristic of an appetitive behaviour.

Effect of insular cortex inactivation on autonomic and behavioral responses to acute hypoxia in conscious rats.

The present work was aimed to evaluate the contribution of interoception to the autonomic and behavioral responses to hypoxia. To address this issue, we studied whether the inactivation of the primary interoceptive posterior insular cortex (pIC) may disrupt the autonomic and behavioral effects of hypoxia in conscious rats. Rats were implanted with telemetric transmitters and microinjection cannulae placed bilaterally in the pIC. After one week, rats were injected with bupivacaine (26.5µM 1µL/side) and saline (1µL/side) into the pIC, and exposed to hypoxia (∼6% O2) for 150s, and autonomic and behavioral responses were recorded. Hypoxia produces hypertension, tachycardia followed by bradycardia, and hypothermia. When O2 dropped to ∼8%, rats showed escape behavior. Baseline cardiovascular variables and the pattern of hypoxia-induced autonomic and behavioral responses were not disrupted by pIC inactivation. However, pIC inactivation produced a modest but significant temperature decrease, higher bradycardic and hypertensive responses to hypoxia, and a minimal delay in escape onset. In addition, we measured the hypoxia-induced Fos activation in the nucleus tractus solitarius (NTS), the periaqueductal gray matter (PAG) and the pIC, which are key components of the interoceptive pathway. Hypoxia increased the number of Fos-positive neurons in the NTS and PAG, but not in the pIC. Present results suggest that pIC is not involved in the hypoxia-induced behavioral response, which seems to be processed in the NTS and PAG, but has a role in the efferent control of autonomic changes coping with hypoxia.

Histamine and motivation.

Brain histamine may affect a variety of different behavioral and physiological functions; however, its role in promoting wakefulness has overshadowed its other important functions. Here, we review evidence indicating that brain histamine plays a central role in motivation and emphasize its differential involvement in the appetitive and consummatory phases of motivated behaviors. We discuss the inputs that control histaminergic neurons of the tuberomamillary nucleus (TMN) of the hypothalamus, which determine the distinct role of these neurons in appetitive behavior, sleep/wake cycles, and food anticipatory responses. Moreover, we review evidence supporting the dysfunction of histaminergic neurons and the cortical input of histamine in regulating specific forms of decreased motivation (apathy). In addition, we discuss the relationship between the histamine system and drug addiction in the context of motivation.

A role for the insular cortex in long-term memory for context-evoked drug craving in rats.

Drug craving critically depends on the function of the interoceptive insular cortex, and may be triggered by contextual cues. However, the role of the insula in the long-term memory linking context with drug craving remains unknown. Such a memory trace probably resides in some neocortical region, much like other declarative memories. Studies in humans and rats suggest that the insula may include such a region. Rats chronically implanted with bilateral injection cannulae into the high-order rostral agranular insular cortex (RAIC) or the primary interoceptive posterior insula (pIC) were conditioned to prefer the initially aversive compartment of a 2-compartment place preference apparatus by repeatedly pairing it to amphetamine. We found a reversible but long-lasting loss (ca. 24 days) of amphetamine-conditioned place preference (CPP) and a decreased expression in the insula of zif268, a crucial protein in memory reconsolidation, when anisomycin (ANI) was microinjected into the RAIC immediately after the reactivation of the conditioned amphetamine/context memory. ANI infusion into the RAIC without reactivation did not change CPP, whereas ANI infusion into pIC plus caused a 15 days loss of CPP. We also found a 24 days loss of CPP when we reversibly inactivated pIC during extinction trials. We interpret these findings as evidence that the insular cortex, including the RAIC, is involved in a context/drug effect association. These results add a drug-related memory function to the insular cortex to the previously found role of the pIC in the perception of craving or malaise.

The histamine H3 receptor and eating behavior.

Interest in the histaminergic system as a potential target for the treatment of feeding disorders is driven by the unsatisfactory history of the pharmacotherapy of obesity. Eating behavior is regulated by a complex interplay of central neurotransmitter systems, peripheral endocrine stimuli, the circadian rhythm, and environmental cues, all factors that change the behavioral state and alter homeostatic aspects of appetite and energy expenditure. Key factors driving eating behavior are appetite and satiety that are regulated through different mechanisms. Brain histamine has long been considered a satiety signal in the nervous system. Recent observations, however, indicate that histamine does not meet the criteria for being a satiety signal, because augmented histamine release accompanies the appetitive phase of feeding behavior rather than food consumption and satiety. The appetitive phase requires a high and yet optimal arousal state, and the histaminergic system is crucial for sustaining a high degree of arousal during motivated behavior. Histamine H(1) receptors in the brain are crucial for the regulation of the diurnal rhythm of food intake and the regulation of obesity; however, from a therapeutic standpoint, no brain-penetrating H(1) receptor agonists have been identified that would have antiobesity effects. Despite conflicting preclinical data, insights are emerging into the potential role of histamine H(3) receptors as a target of antiobesity therapeutics. The aim of this review is to outline the relevance of the histaminergic system in controlling feeding behavior and evaluate the potential therapeutic use of histaminergic ligands for the treatment of eating disorders.

Orexin-B-saporin lesions in the lateral hypothalamus enhance photic masking of rapid eye movement sleep in the albino rat.

The 24-h distribution of rapid eye movement (REM) sleep is known to be deeply reshaped among albino rats with neurotoxic lesions in the lateral hypothalamus (LH) or among rodent models of human narcolepsy-cataplexy, with selective damage of orexinergic neurones. We explored the hypothesis that this phenomenon is explained by an enhancement of REM sleep photic masking, as a consequence of damage in the LH. Orexin-B-saporin neurotoxic lesions were induced in the LH of male Sprague-Dawley rats. LH-lesioned and control rats were sleep-recorded successively under 12:12 light/dark (LD) and skeleton photoperiod. Compared to controls, lesioned rats exhibited 50% less and 82% more REM sleep during rest and active phases, respectively, under the 12:12 LD schedule. After transference to a skeleton photoperiod, lesioned rats exhibited an 88% increase in REM sleep during the rest phase, recovering the characteristic rest phase preference of REM sleep observed among control rats. The increase in rest phase REM sleep during the skeleton photoperiod was correlated positively with the magnitude of the LH lesion. Our results suggest that changes in the temporal organization of sleep-wake states observed among rats with neurotoxic lesions in the lateral hypothalamus and rodent models of narcolepsy-cataplexy may be explained by the enhancement of photic masking.

The histaminergic tuberomammillary nucleus is critical for motivated arousal.

Obtaining food, shelter or water, or finding a mating partner are examples of motivated behaviors, which are essential to preserve the species. The full expression of such behaviors requires a high but optimal arousal state. We tested the idea that tuberomammillary nucleus (TMN) histamine neurons are crucial to generate such motivated arousal, using a model of the appetitive phase of feeding behavior. Hungry rats enticed with food within a wire mesh box showed intense goal-directed motor activity aimed at opening the box, an increase in core temperature, a fast histamine release in the hypothalamus and an early increase in Fos immunoreactivity in TMN and cortical neurons. Enticing with stronger-tasting food induced stronger motor, temperature and Fos immunoreactivity brain responses than ordinary food pellets. TMN lesion greatly decreased all of those responses. We conclude that histamine neurons increase arousal and vegetative activity, allowing the normal unfolding of voluntary, goal-directed behavior such as obtaining food.

The parietal association cortex of the rat

Spatial cognition is a complex higher function in mammals and is involved in a variety of tasks that can be explored in the laboratory. In this review we will discuss the role of the posterior parietal/anteromedial cortex of rodents, also known as the parietal association cortex, and the hippocampal formation in spatial navigation. We will also discuss other higher associational functions of the posterior parietal/anteromedial cortex as they relate to Dr. Pinto-Hamuy's contribution to understanding behavioral functions.

The parietal association cortex of the rat.

Spatial cognition is a complex higher function in mammals and is involved in a variety of tasks that can be explored in the laboratory. In this review we will discuss the role of the posterior parietal/anteromedial cortex of rodents, also known as the parietal association cortex, and the hippocampal formation in spatial navigation. We will also discuss other higher associational functions of the posterior parietal/anteromedial cortex as they relate to Dr. Pinto-Hamuy's contribution to understanding behavioral functions.

Antiribosomal-P autoantibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis.

The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P-mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

Inactivation of the interoceptive insula disrupts drug craving and malaise induced by lithium.

Addiction profoundly alters motivational circuits so that drugs become powerful reinforcers of behavior. The interoceptive system continuously updates homeostatic and emotional information that are important elements in motivational decisions. We tested the idea that interoceptive information is essential in drug craving and in the behavioral signs of malaise. We inactivated the primary interoceptive cortex in amphetamine-experienced rats, which prevented the urge to seek amphetamine in a place preference task. Interoceptive insula inactivation also blunted the signs of malaise induced by acute lithium administration. Drug-seeking and malaise both induced Fos expression, a marker of neuronal activation, in the insula. We conclude that the insular cortex is a key structure in the perception of bodily needs that provides direction to motivated behaviors.