A sphingolipid mechanism for behavioral extinction.

Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a reduction of acid sphingomyelinase activity during extinction of conditioned behavior in rats. This reduction was associated with expression of re-learning-related behavior, but not with emotional behaviors. Read the Editorial Highlight for this article on page 485.

Chronic corticosterone treatment enhances extinction-induced depression in aged rats.

Withdrawal and avoidance behavior are common symptoms of depression and can appear as a consequence of absence of reward, i.e. extinction-induced depression (EID). This is particularly relevant for the aged organism subjected to pronounced loss of former rewards. Avoidance of the former site of reward and increased withdrawal into a distant compartment accompany extinction of food-rewarded behavior in rodent models. During extinction, behavioral markers for re-learning dissociate from indicators of extinction-induced depression. Here we examined the effect of a chronic treatment with corticosterone (CORT), a well-known inducer of depression-related behavior, on EID in adult and aged rats. Adult (3-4months) and aged (18months) male rats were treated with CORT via drinking water for 3weeks prior to extinction of a cued food-reward task. CORT treatment increased the distance from the site of reward and decreased goal tracking behavior during extinction, especially in the aged rats. Plasma hormone levels measured before and after restraint stress showed a decline in basal ACTH- and CORT-levels after chronic CORT treatment in aged animals. The treatment significantly impaired the HPA-axis activation after acute stress in both, adult and aged animals, alike. Altogether, these findings show an enhancement of EID after chronic CORT treatment in the aged organism, which may be mediated by an impaired HPA-axis sensitivity. These findings may have special relevance for the investigation of human geriatric depression.

Indices of extinction-induced "depression" after operant learning using a runway vs. a cued free-reward delivery schedule.

Loss of reward is one of the etiological factors leading to affective disorders, such as major depression. We have proposed several variants of an animal model of depression based on extinction of reinforced behavior of rats. A number of behaviors emitted during extinction trials were found to be attenuated by antidepressant treatment and, thus, qualified as indices of extinction-induced "despair". These include increases in immobility in the Morris water maze and withdrawal from the former source of reward as well as biting behavior in operant chambers. Here, we assess the effects of reward omission on behaviors after learning of (a) a cued free-reward delivery in an operant chamber and (b) food-reinforced runway behavior. Sixty adult male Wistar rats were either trained to receive food reinforcement every 90 s (s) after a 5s lasting cue light (FI 90), or to traverse an alley to gain food reward. Daily drug treatment with either the selective serotonin reuptake inhibitor citalopram or the tricyclic antidepressant imipramine (each 10mg/kg) or vehicle was begun either 25 days (operant chamber) or 3 days (runway) prior to extinction. The antidepressants suppressed rearing behavior in both paradigms specifically during the extinction trials, which indicates this measure as a useful marker of depression-related behavior, possibly indicating vertical withdrawal. In the operant chamber, only marginal effects on operant learning responses during extinction were found. In the runway, the operant learned responses run time and distance to the goal, as well as total distance moved, grooming and quiescence were also influenced by the antidepressants, providing a potential set of markers for extinction-induced "depression" in the runway. Both paradigms differ substantially with respect to the anticipation of reward, behaviors that are learned and that accompany extinction. Accordingly, antidepressant treatment influenced different sets of behaviors in these two learning tasks.

Toward an animal model of extinction-induced despair: focus on aging and physiological indices.

Behaviors that are under the control of positive or negative reinforcers undergo extinction when the anticipated reward/reinforcer is withheld. Despair, an important symptom of environmentally determined depression in humans, can be generated by extinction, or the failure of expected reward to accrue. Although well known to clinicians dealing with depressive patients, an animal model has not been available for extinction-induced depression. We have made a beginning towards validating such a model, based on the extinction of negatively reinforced behavior in the rat, i.e., upon removal of the possibility to escape onto a safety platform in the water maze. As a marker for despair, we employed behavioral immobility, i.e., the cessation of swimming in the attempt to find safety from the water, presumably, a type of learned helplessness. This measure was sensitive to antidepressants and correlated with neurotransmitter contents, neurotrophins and hypothalamus-pituitary adrenal axis markers in selected sites of the brain. Given that some cases of depression in the elderly may be biologically distinct from others and from early-onset depression, and since particularly the aged are prone to experience extinction-induced despair, we compared aged (ca. 24 months old) animals with adults in most of our studies. We found a number of distinct differences in behavioral and biological measures, indicative of differences in propensity to, as well as response to, extinction-induced despair between aged and adults. Our results add to the body of evidence for differences in the neurobiological substrates of depressive disorders between aged and adults, with the implication for the requirement of different treatment strategies in these two populations.

Extinction-induced "despair" in aged and adult rats: links to neurotrophins in frontal cortex and hippocampus.

In the search for animal models of human geriatric depression, we found that operant extinction of escape from water results in the expression of immobility in different age groups, indicative of behavioral "despair", which was also associated with the resistance-to-extinction (RTE) expressed by these animals. With respect to the neurotrophin hypothesis of depression, nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) protein levels in frontal cortex (FC) and hippocampus (HP) were examined and related to behavioral immobility and RTE in the water maze in aged and adult Wistar rats. Age-related increases in levels of NGF were found in HP and of NT-3 in FC. Indices of immobility showed relationships in the aged with NGF and, in adults, with BDNF, pointing to a dissociation of neurotrophic involvement in extinction trial-induced "despair" in aged and adult rats. The present results support the hypothesis, that extinction-induced immobility in the water maze reflects a state akin to behavioral despair and point to age-related differences of neurotrophic involvement in depressive-like symptoms. The concept of extinction-induced behavioral "despair" in the aged subsumes several aspects of human geriatric depression, such as co-morbidity of learning impairment and anxiety, and, thus could represent a useful paradigm to examine the neuronal mechanisms underlying depression, especially in aged rodents.

Differential susceptibility to extinction-induced despair and age-dependent alterations in the hypothalamic-pituitary-adrenal axis and neurochemical parameters.

Clinical studies point to structural differences in the neurobiological mechanisms underlying early versus late onset of depression. However, studies examining the neuropathology of depressive-like behavior induced in the aged rodent are sparse. Extinction of learned behavior induces be- havioral 'despair', and is held to provide a conceptual and empirical model of human depression resulting from the withdrawal of reinforcement. We tested whether the neuroendocrinological and chemical concomitants of susceptibility to extinction-induced despair in aged animals differed from adult ones. Following the withholding of reinforcement (extinction of escape from a water maze), a number of aged and adult rats are prone to develop depressive-like behavior, i.e. immobility. Analysis of hypothalamus-pituitary-adrenal (HPA) axis markers revealed an increase in the mineralocorticoid/glucocorticoid receptor (MR/GR) mRNA ratio in the CA1 region of the hippocampus in aged and adult despair animals; however, in dependence on age, divergent changes contributed to the enhanced ratio. While aged despair rats had less GR mRNA, adult despair rats had more MR mRNA. Furthermore, age- and despair-related interactions with hippocampal and cortical steroid receptor co-activators and neurotransmitter contents in diverse brain areas were found. For instance, adult despair rats had an increased, and aged despair rats a decreased, DOPAC/dopamine turnover compared to the respective non-despair group. These results show that neurobiological underpinnings of depression in the aged differ from those of adults, and underline the importance of investigating age-related alterations in HPA axis dynamics in conjunction with neurotransmitter systems to advance our knowledge about neuronal mechanisms of late-life and/or late-onset depression.

Behavioral actions of intranasal application of dopamine: effects on forced swimming, elevated plus-maze and open field parameters.

BACKGROUND: Recently, we found evidence that intra-nasally administered dopamine (DA), can enter the brain, leading to an immediate increase in extracellular DA levels in striatal subregions. This offers a potential alternative approach to target the brain with exogenous DA, which otherwise cannot cross the blood-brain barrier. Here, we examined whether intra-nasally applied DA also exerts behavioral activity on mesocortical and nigrostriatal dopaminergic functions. METHOD: Male Wistar rats (3-4 months) were tested for potential behavioral effects of intra-nasally applied DA (0.03, 0.3 or 3.0 mg/kg) in the forced swimming test (FST) for antidepressant-like activity, elevated plus-maze for anxiety-related behavior, and on motor activity in a novel and familiar environment. RESULTS: Intra-nasally administered dopamine in a dose of 0.3 mg/kg exerted antidepressant-like activity in the FST, but had neither anxiolytic-like nor anxiogenic-like effects in the elevated plus-maze. Furthermore, intra-nasal dopamine stimulated locomotor activity in a familiar, but not novel, open field. CONCLUSIONS: These results support the view that intra-nasally applied DA can act on the central nervous system by entering the brain via the nose-brain pathway, making this kind of application procedure a promising alternative for targeting the brain, and thus treating disorders involving mesocortical and/or nigrostriatal dopaminergic disturbances.

Prolonged effects of intra-nasally administered testosterone on proceptive behavior in female capuchin monkeys (Cebus apella).

Sexual dysfunction in the female has been associated with a decrease in androgen levels, which can be reversed by testosterone treatment, however, bearing the risk of adverse side effects. Previously we found that intranasal application of testosterone led to an increase in proceptive behavior in female capuchin monkeys, which was still enhanced beyond treatment pointing to prolonged effects. Here, we sought to replicate our previous findings and to further examine possible prolonged effects of such treatment. During 5 days of baseline females were observed for their sexual and non-sexual behavior without drug. They then received daily intranasal testosterone (0.24 mg per nostril) for 7 days, followed by 15 days of intranasal testosterone application every third day or were left undisturbed (n=5 per group). Diverse sexual and non-sexual behaviors were scored. Blood samples were collected and analyzed for testosterone, estradiol, and progesterone using immunoenzymatic chemiluminescent immunoassay. In accord with our previous results an increase in sexual proceptive behavior was revealed during testosterone treatment, which persisted about 2 weeks beyond treatment in the group receiving testosterone every third day as well as in the group that no longer received any treatment. These behavioral results were accompanied by an increase in plasma testosterone levels. This study demonstrates the efficacy of testosterone in enhancing sexual behavior in female capuchin monkeys by means of intranasal application, and provided evidence that daily substance application is not necessary to maintain beneficial effects on sexual behavior. These findings render the intranasal application procedure to be a useful alternative compared to other forms of administration.

Effects of intra-nasally administered testosterone on sexual proceptive behavior in female capuchin monkeys (Cebus apella).

Sexual dysfunction in the female has been associated with a decrease in androgen levels, which can be reversed by testosterone treatment, however, bearing the risk of adverse side effects. Nasally administered testosterone could be an effective method for androgenic treatment avoiding the first-pass intestinal and hepatic metabolism and side effects. Here we examined the effects of chronic intranasal administration of testosterone on sexual behavior in female capuchin monkeys (Cebus apella) that lived with male cohorts. During 8 days of baseline 10 females were observed for their sexual and non-sexual behavior without drug. They then received daily intranasal testosterone (0.24 mg per nostril, n=5 per group) or placebo gel for 5 days, followed by 5 days of wash out, followed by 5 days of daily intranasal application, whereby the animals that had received testosterone before, now received placebo and vice versa. Diverse sexual and non-sexual behaviors were scored. Blood samples were collected and analyzed for testosterone, estradiol, dihydrotestosterone and progesterone using EIAs. The results revealed an increase in sexual behavior (eyebrow raising, chest rubbing, courtship behavior, masturbation) in the females during testosterone treatment, which seemed to be prolonged even when testosterone treatment was discontinued. These behavioral results were accompanied by an increase in plasma testosterone levels. This study demonstrates the efficacy of testosterone in enhancing sexual behavior in female capuchin monkeys by means of intranasal application, which may be a useful alternative compared to other forms of administration.

Aged and adult rats compared in acquisition and extinction of escape from the water maze: focus on individual differences.

Individual differences in water maze and open-field performance of aged and adult rats were compared in a cross-sectional study. Three- and 24-month-old rats were classified into superior, moderate, and inferior groups on the basis of escape latencies during hidden platform acquisition and were compared regarding water maze acquisition and extinction, and open-field behavior. Unexpectedly, subgroup differences were invariant across age: The inferior and superior maze learners differed in (a) thigmotactic swimming during water maze acquisition and extinction and (b) open-field rearings. Thus, although aging has a detrimental effect on water maze acquisition and extinction, the degree of impairment might be partly determined by individual novelty-induced rearing activity and thigmotactic swimming at adult ages.

The graded anxiety test: a novel test of murine unconditioned anxiety based on the principles of the elevated plus-maze and light-dark test.

Standard tests of murine unconditioned anxiety such as the elevated plus-maze and light-dark test are based on a dichotomy of avoidance behaviour (walled vs. open arms and dark vs. light compartments). We combined the principles of both tests by modifying the elevated plus-maze as follows: one walled arm was made transparent and had a white floor (WTW), whereas the other walled arm was opaque-gray having a black floor (WOB). Furthermore, one open arm had a white floor (OW), while the other had a black one (OB). These modifications allow the distinction between more than two sub-compartments that elicit different degrees of avoidance behaviour, thus having a higher discriminative potency. Additionally, the paradigm was thought to permit the within-task detection of pharmacological side effects on the perception of the anxiogenic stimuli provided. The degree of avoidance of the sub-compartments exhibited by saline-treated mice for the distal parts of the four arms was distributed as follows: WOB

What's conditioned in conditioned place preference?

Conditioned place preference (CPP) is a learned behavior shown in many vertebrates, including humans. CPP occurs when a subject comes to prefer one place more than others because the preferred location has been paired previously with rewarding events. The CPP paradigm is widely used to explore the reinforcing effects of natural and pharmacological stimuli, including drugs of addiction. There is a general assumption that an acquired place preference is based on classical conditioning derived 'incentive motivation'. However, this may be an oversimplification of the multiple learning processes involved. We argue that although CPP may appear as an incentive-driven behavior related to secondary reinforcers, it may also be a result of operant conditioning of behavior prevailing at the conditioning site, as well as a result of conditioned treatment effects. Here, we outline alternative explanations for an observed CPP, which may fundamentally affect the interpretation of results with this paradigm in its use as a screening tool for rewarding properties of treatments.

Animal models of extinction-induced depression: loss of reward and its consequences.

The absence or loss of rewards or reinforcers holds a major role in the development of depression in humans. In spite of the prevalence of extinction-induced depression (EID) in humans, few attempts have been made to establish animal models thereof. Here we present the concept of extinction-related depression and summarize the results of two sets of studies in our attempt to create animal models of EID, one set based on extinction after positive reinforcement in the Skinner-box, the other on extinction after negative reinforcement - escape from water. We found various behaviors emitted during the extinction trials that responded to treatment with antidepressant drugs: Accordingly, the important behavioral marker for EID during extinction of escape from the water was immobility. During extinction after positive reinforcement the important indices for extinction-induced depression are the withdrawal from the former site of reward, biting behavior and rearing up on the hind legs. Avoidance behavior and biting may model aspects of human depressive behavior, which may include withdrawal or avoidance as well as aggressive-like behaviors.

Aging-induced proteostatic changes in the rat hippocampus identify ARP3, NEB2 and BRAG2 as a molecular circuitry for cognitive impairment.

Disturbed proteostasis as a particular phenotype of the aging organism has been advanced in C. elegans experiments and is also conceived to underlie neurodegenerative diseases in humans. Here, we investigated whether particular changes in non-disease related proteostasis can be identified in the aged mammalian brain, and whether a particular signature of aberrant proteostasis is related to behavioral performance of learning and memory. Young (adult, n = 30) and aged (2 years, n = 50) Wistar rats were tested in the Morris Water Maze (MWM) to distinguish superior and inferior performers. For both young and old rats, the best and worst performers in the MWM were selected and the insoluble proteome, termed aggregome, was purified from the hippocampus as evidence for aberrant proteostasis. Quantitative proteomics (iTRAQ) was performed. The aged inferior performers were considered as a model for spontaneous, age-associated cognitive impairment. Whereas variability of the insoluble proteome increased with age, absolute changes in the levels of insoluble proteins were small compared to the findings in the whole C. elegans insoluble proteome. However, we identified proteins with aberrant proteostasis in aging. For the cognitively impaired rats, we identified a changed molecular circuitry of proteins selectively involved in F-actin remodeling, synapse building and long-term depression: actin related protein 3 (ARP3), neurabin II (NEB2) and IQ motif and SEC7 domain-containing protein 1 (BRAG2). We demonstrate that aberrant proteostasis is a specific phenotype of brain aging in mammals. We identify a distinct molecular circuitry where changes in proteostasis are characteristic for poor learning and memory performance in the wild type, aged rat. Our findings 1. establish the search for aberrant proteostasis as a successful strategy to identify neuronal dysfunction in deficient cognitive behavior, 2. reveal a previously unknown functional network of proteins (ARP3, NEB2, BRAG2) involved in age-associated cognitive dysfunction.

Intranasal application of dopamine reduces activity and improves attention in Naples High Excitability rats that feature the mesocortical variant of ADHD.

Based on findings of a profound action of intranasally applied dopamine (DA) on dopamine release in the striatum, we examined the possibility that intranasal application of DA would influence indices of attention and activity in juvenile male rats of the Naples High Excitability line. This rat model features the main aspects of Attention Deficit/Hyperactivity Disorder (ADHD). Juvenile NHE rats received an intranasal application of either DA (0.075 mg/kg, 0.15 mg/kg and 0.3 mg/kg) or vehicle into both nostrils daily for 15 days. On day 14, 1 h after treatment, they were tested in the Làt maze, and one day later, in the eight arm radial maze. Activity in the Làt maze: The highest dose of DA (0.3 mg/kg) decreased horizontal (HA) and vertical (VA) activity during the first 10 min of the test. No effect was found for rearing duration (RD), which indexes non-selective attention (NSA). Activity in the radial maze: No treatment effects were found for HA and VA components, and for RD. Attention indices: The intermediate dose of DA (0.15 mg/kg) significantly improved the number of arms visited before the first repetitive arm entry in the radial maze, an index of selective spatial attention (SSA). In conclusion, intranasal application of DA reduced hyperactivity at the highest dose used, whereas the intermediate dose improved attention in an animal model of ADHD. These results suggest the potential of employing intranasal DA for therapeutic purposes.

"Despair" induced by extinction trials in the water maze: relationship with measures of anxiety in aged and adult rats.

We have previously reported that extinction of escape behavior in the water maze due to the removal of the platform coincided with the development of behavioral "despair" in aged and adult rats, as assessed by immobility. The present study examines further predictions derived from the hypothesis that the withholding of reinforcement induces behaviors akin to depression. We tested for correlations between extinction performance and immobility, as well as between immobility and measures of anxiety in aged and adult rats. Age comparisons were also performed on these variables. Forty aged and 29 adult male Wistar rats (24 and 3 months old, respectively) were examined in the open field, black/white box and elevated-plus maze followed by 6 days of training in the water maze hidden platform task and 8 days of extinction without the platform. Indices of immobility increased over trials of extinction, with the aged showing higher levels, earlier onsets and larger slope increases of immobility than the adults. A lower resistance-to-extinction was predictive of more "despair" in both age groups. Between-group differences in the open field, black/white box and elevated-plus maze indicated that the aged showed more anxiety-like behavior than the adults and/or explored these environments less. Within the aged group, indicators of fearfulness in the three tests were predictive of higher levels of "despair". The extinction-despair model is held to provide the promise of a conceptual and empirical model of human depression that is the consequence of withdrawal of reinforcement.

Extinction-induced immobility in the water maze and its neurochemical concomitants in aged and adult rats: a possible model for depression?

Extinction of escape behavior in the water maze due to the removal of the platform, was hypothesized to induce a negative state, including the development of immobility, which is held to reflect a state of "despair" when measured in the forced swimming test. 27 aged and 8 adult animals (26 and 3 months old Wistar rats, respectively) were tested in the water maze during nine days with a platform hidden, followed by 7 days of extinction trials with the platform absent. As expected, both age groups developed immobility over the extinction trials, with the aged showing more than the adults. To examine whether the age difference in immobility was related to performance differences during acquisition, the aged were subdivided into superior, intermediate and inferior learners (n = 9 per group) on the basis of overall times to platform during acquisition, and compared with each other and the adults. Results showed that the aged inferior learners displayed the highest levels of immobility among the aged. Immobility scores were then correlated with post-mortem neurotransmitter contents in the hippocampus and ventral striatum. In the ventral striatum, levels of immobility were correlated with levels of acetylcholine, dopamine and the metabolite dihydroxyphenylacetic acid in the aged, and with norepinephrine in the adults. The data support the hypothesis that multiple extinction trials in the water maze result in immobility that may indicate "behavioral despair," and that striatal neurotransmitter systems correlate with the degree of its expression. The concept of extinction-induced despair is held to provide the promise of a conceptual and empirical model of human depression that is the consequence of loss of reinforcers.

Histidine-decarboxylase knockout mice show deficient nonreinforced episodic object memory, improved negatively reinforced water-maze performance, and increased neo- and ventro-striatal dopamine turnover.

The brain's histaminergic system has been implicated in hippocampal synaptic plasticity, learning, and memory, as well as brain reward and reinforcement. Our past pharmacological and lesion studies indicated that the brain's histamine system exerts inhibitory effects on the brain's reinforcement respective reward system reciprocal to mesolimbic dopamine systems, thereby modulating learning and memory performance. Given the close functional relationship between brain reinforcement and memory processes, the total disruption of brain histamine synthesis via genetic disruption of its synthesizing enzyme, histidine decarboxylase (HDC), in the mouse might have differential effects on learning dependent on the task-inherent reinforcement contingencies. Here, we investigated the effects of an HDC gene disruption in the mouse in a nonreinforced object exploration task and a negatively reinforced water-maze task as well as on neo- and ventro-striatal dopamine systems known to be involved in brain reward and reinforcement. Histidine decarboxylase knockout (HDC-KO) mice had higher dihydrophenylacetic acid concentrations and a higher dihydrophenylacetic acid/dopamine ratio in the neostriatum. In the ventral striatum, dihydrophenylacetic acid/dopamine and 3-methoxytyramine/dopamine ratios were higher in HDC-KO mice. Furthermore, the HDC-KO mice showed improved water-maze performance during both hidden and cued platform tasks, but deficient object discrimination based on temporal relationships. Our data imply that disruption of brain histamine synthesis can have both memory promoting and suppressive effects via distinct and independent mechanisms and further indicate that these opposed effects are related to the task-inherent reinforcement contingencies.