The impact of chronic fluoxetine treatment in adolescence or adulthood on context fear memory and perineuronal nets.

Selective serotonin reuptake inhibitors, such as fluoxetine (Prozac), are commonly prescribed pharmacotherapies for anxiety. Fluoxetine may be a useful adjunct because it can reduce the expression of learned fear in adult rodents. This effect is associated with altered expression of perineuronal nets (PNNs) in the amygdala and hippocampus, two brain regions that regulate fear. However, it is unknown whether fluoxetine has similar effects in adolescents. Here, we investigated the effect of fluoxetine exposure during adolescence or adulthood on context fear memory and PNNs in the basolateral amygdala (BLA), the CA1 subregion of the hippocampus, and the medial prefrontal cortex in rats. Fluoxetine impaired context fear memory in adults but not in adolescents. Further, fluoxetine increased the number of parvalbumin (PV)-expressing neurons surrounded by a PNN in the BLA and CA1, but not in the medial prefrontal cortex, at both ages. Contrary to previous reports, fluoxetine did not shift the percentage of PNNs toward non-PV cells in either the BLA or CA1 in the adults, or adolescents. These findings demonstrate that fluoxetine differentially affects fear memory in adolescent and adult rats but does not appear to have age-specific effects on PNNs.

It's all about who you know: Memory retention of a rat's cagemates during infancy negatively predicts adulthood hippocampal FGF2.

Recent research has demonstrated that individual differences in infant fear memory positively predict adulthood anxiety-like behavior and conditioned fear expression. However, the physiological mechanisms underlying this relationship and the effect of environmental (e.g., social) influences on the stability of this relationship have not been explored. In the present study, we examined whether individual differences in infant fear memory predict levels of endogenous fibroblast growth factor-2 (FGF2; a biomarker of fear/anxiety) in adulthood, and whether the mean memory retention of a rat's cagemates predicts conditioned fear expression and FGF2 in adulthood. We conditioned infant rats to associate a white noise with shock, and tested their memory of the association 1 week later. They were then weaned and randomly assigned to cage/cagemates. In adulthood, rats received weak context conditioning (i.e., a single shock) and were tested for fear of the context the following day. Rats were then euthanized and their brains extracted to measure levels of hippocampal FGF2 protein. Across 2 experiments, an individual rat's fear memory during infancy positively predicted their own fear expression in adulthood, but the mean memory retention of their cagemates did not predict fear expression. In contrast, the mean memory retention of a rat's cagemates during infancy negatively predicted hippocampal FGF2 protein in adulthood, but an individual rat's memory retention did not predict their own levels of FGF2. These data support the idea that variations in the fearfulness of a rat's cagemates predict individual differences on physiological measures in adulthood.

Maternal care, infant fear memory retention, and the moderating role of variations in separation-induced ultrasonic vocalizations.

Individual differences in parental care predict variations in offspring anxiety across species. Here, we examined whether between- and within-litter variations in maternal licking (a measure of rodent maternal care) predict infant rats' retention of an aversive association (a predictor of later anxiety-like behavior) and whether the relationship between maternal licking and infant fear memory is moderated by variations in infants' solicitation of maternal care. Unique marks were drawn on each pup, coded for fading, and touched up daily across the first week of life. Mark fading was used as an index of maternal licking where greater fading suggested more maternal licking the previous day. Separation-induced ultrasonic vocalizations (USVs) were recorded to measure individual differences in solicitation of maternal care. Infants were fear conditioned at postnatal day (P) 17 and tested for fear of the conditioned stimulus (CS) 1 week later. Across litters, mark fading negatively predicted CS-elicited fear at test for male, but not female, offspring. This relationship was moderated by number of USVs emitted at P1, such that mark fading only predicted CS-elicited fear for males that emitted a low number of USVs. These results suggest that offspring solicitation may moderate the relationship between maternal care and fear/anxiety.

Early-life stress leads to sex-dependent changes in pubertal timing in rats that are reversed by a probiotic formulation.

Puberty marks the beginning of a period of dramatic physical, hormonal, and social change. This instability has made adolescence infamous as a time of "storm and stress" and it is well-established that stress during adolescence can be particularly damaging. However, prior stress may also shape the adolescent experience. In the present series of experiments, we observed sex-specific effects of early-life maternal separation stress on the timing of puberty onset in the rat. Specifically, stressed females exhibited earlier pubertal onset compared to standard-reared females, whereas stressed males matured later than their standard-reared counterparts. Further, we demonstrated that a probiotic treatment restores the normative timing of puberty onset in rodents of both sexes. These results are in keeping with previous findings that probiotics reverse stress-induced changes in learned fear behaviors and stress hormone levels, highlighting the remarkable and wide-ranging restorative effects of probiotics in the context of early-life stress.

Behavioral tagging in infant rats.

Recent studies have shown that exposure to a novel environment may stabilize the persistence of weak memories, a phenomenon often attributed to a process referred to as "behavioral tagging." While this phenomenon has been repeatedly demonstrated in adult animals, no studies to date have examined whether it occurs in infant animals, which is surprising given that infants exhibit an impaired ability to form long-term memories (LTMs). In the present study, infant (i.e., postnatal day (P) 17) rats were placed in a context and repeatedly shocked. Infant rats given brief open field exposure 1 h, but not 2 h, prior to conditioning exhibited enhanced retention when tested 1 d later (Experiments 1 and 2), but comparable retention when tested shortly after training (Experiment 2). Thus, exploration of an open field facilitates subsequent context fear memories by enhancing the persistence of the memory rather than strengthening the context-shock association at encoding. While exploration of an open field did not lead to better memory when animals were tested 3 d later (Experiment 3), a brief pretest shock led to a more pronounced reinstatement effect in rats exposed to the open field 1 h before conditioning (Experiment 4). Finally, unlike what has been reported in adults, Experiments 5 and 6 suggest that familiarization of the open field before subsequent exposure does not abolish the behavioral tagging effect in infants. Overall, while these findings suggest that similar behavioral tagging mechanisms to those reported in adults might be involved in the formation of LTMs in infant rats, they also suggest that there may be developmental differences in the retention of familiarization experiences.

Elucidating the mechanisms of fear extinction in developing animals: a special case of NMDA receptor-independent extinction in adolescent rats.

NMDA receptors (NMDARs) are considered critical for the consolidation of extinction but recent work challenges this assumption. Namely, NMDARs are not required for extinction retention in infant rats as well as when extinction training occurs for a second time (i.e., reextinction) in adult rats. In this study, a possible third instance of NMDAR-independent extinction was tested. Although adolescents typically exhibit impaired extinction retention, rats that are conditioned as juveniles and then given extinction training as adolescents (JuvCond-AdolesExt) have good extinction retention. Unexpectedly, this good extinction retention is not associated with an up-regulation of a synaptic plasticity marker in the medial prefrontal cortex, a region implicated in extinction consolidation. In the current study, rats received either the noncompetitive NMDAR antagonist MK801 (0.1 mg/kg, s.c.) or saline before extinction training. In several experiments, rats conditioned and extinguished as juveniles, adolescents, or adults exhibited impaired extinction retention after MK801 compared to saline, but this effect was not observed in JuvCond-AdolesExt rats. Further experiments ruled out several alternative explanations for why NMDAR antagonism did not affect extinction retention in adolescents extinguishing fear learned as a juvenile. These results illustrate yet another circumstance in which NMDARs are not required for successful extinction retention and highlight the complexity of fear inhibition across development.

The impact of chronic fluoxetine on conditioned fear expression and hippocampal FGF2 in rats: Short- and long-term effects.

Phenotypic differences in conditioned fear expression may be a marker of vulnerability to the development of anxiety disorders. Hippocampal FGF2 correlates negatively with conditioned fear expression, and antidepressants, the first-line pharmacological treatment for anxiety, increase hippocampal FGF2. In the present study, we assessed whether treatment with the selective serotonin reuptake inhibitor fluoxetine reduces conditioned fear expression in rats that naturally express lower (Low fear) or higher (High fear) levels of fear following a single-trial conditioning session. Experiment 1 demonstrated that phenotypic differences in fear expression were highly stable over a two-week interval. Experiment 2 demonstrated that two weeks of fluoxetine treatment (administered via drinking water) reduced conditioned fear expression in both Low and High fear rats. Experiment 3 demonstrated that two weeks of fluoxetine reduced conditioned fear expression even when treatment commenced two weeks after fear conditioning, but not when conditioned fear was assessed following a two-week drug-free washout period. Additionally, fluoxetine increased hippocampal FGF2 levels relative to vehicle-treated rats, but only when measured immediately after fluoxetine treatment, and not two weeks after treatment termination. Together, these results provide further indirect evidence that endogenous hippocampal FGF2 may mediate individual differences in conditioned fear expression. They also suggest that fluoxetine may be effective in reducing conditioned fear expression in both vulnerable and resilient populations when administered immediately, or sometime after, aversive experiences, but these effects do not persist once treatment is terminated.

Ghosts of mother's past: Previous maternal stress leads to altered maternal behavior following a subsequent pregnancy in rats.

A rodent model was used to explore whether mothers that experienced a postnatal stressor in the past (i.e., daily separations from her previous litter) exhibited altered maternal behavior during a typical, subsequent rearing experience. Stress-naïve female rats were bred and then separated from their pups (maternal separation) or remained with their pups (standard-rearing). After those pups were weaned, mothers were bred again with all pups from the subsequent litter being standard-reared. In the first week of life, various maternal behaviors directed towards these subsequent offspring were observed, including levels of nursing and pup retrieval. After weaning, mothers were tested for anxiety-like behavior, as well as memory on the object-recognition and object-placement tasks. The results show that previously stressed mothers retrieve their offspring significantly faster compared to mothers with no stress history, which may reflect a more "overprotective" mothering style. No other differences on maternal care were observed. Also, while previously stressed mothers were no more anxious than control mothers, they had impaired spatial memory on the object-placement task. This was not due to a general memory impairment as mothers performed equally on the object-recognition task, suggesting that previous maternal stress has specific effects on hippocampal-dependent tasks. That is, stress exerts lasting effects on types of behavior that are proposed to be beneficial to mothers and their offspring (i.e., efficient foraging and navigation abilities). Taken together, these results provide evidence that stress has specific and persistent effects on caregivers.

Differences in the persistence of spatial memory deficits induced by a chronic stressor in adolescents compared to juveniles.

Adolescence is thought of as a stress-sensitive developmental period. While many studies have compared adolescent responses to stress relative to that of adults, a growing body of work has examined stress responses in juveniles. Here we investigated if a chronic stressor has a differential effect on spatial memory in rats depending on whether it occurs during adolescence or the juvenile period. Male rats were exposed to the stress hormone corticosterone (Cort) in their drinking water, a vehicle control (2.5% ethanol), or water, for 7 days before being tested on a novel Object/Place task 6 days or 6 weeks later. Exposure to Cort or ethanol at either age impaired spatial memory at the 6-day test. The ethanol induced impairment was attenuated 6 weeks later. However, rats given Cort during adolescence, but not the juvenile period, were still impaired. Together, these results suggest that adolescence is indeed a stress-sensitive period.

Individual differences in fear extinction and anxiety-like behavior.

There is growing appreciation for the substantial individual differences in the acquisition and inhibition of aversive associations, and the insights this might give into identifying individuals particularly vulnerable to stress and psychopathology. We examined whether animals that differed in rate of extinction (i.e., Fast versus Slow) were different in their response to an acute stress in adulthood or following a chronic stress that occurred either early or later in life. We found that Slow Extinguishers had significantly poorer extinction retention than Fast Extinguishers, but an acute stressor did not differentially affect anxiety-like behavior in the two groups. Further, while exposure to chronic stress in adulthood did not impact on the extinction phenotypes or anxiety-like behavior, exposure to chronic stress early in life affected both extinction retention and anxiety-like behavior. These findings have implications for the development of a more nuanced approach to identifying those most at risk of anxiety disorders.

Pharmacological evidence that a failure to recruit NMDA receptors contributes to impaired fear extinction retention in adolescent rats.

Adolescents, both humans and rodents, exhibit a marked impairment in extinction of fear relative to younger and older groups which could be caused by a failure to efficiently recruit NMDA receptors (NMDARs) in adolescence. It is well-established that systemic administration of NMDAR antagonists (e.g., MK801) before extinction training impairs the retention of extinction in adult and juvenile rodents, but it is unknown whether this is also the case for adolescents. Therefore, in the present study we investigated the effect of pharmacologically manipulating the NMDAR on extinction retention in adolescent rats. When extinction retention is typically impaired (i.e., after one session of extinction training) adolescent male rats given d-cycloserine (a partial NMDAR agonist) showed enhanced extinction retention relative to saline-treated animals while animals given MK801 (a non-competitive antagonist) did not exhibit any further impairment of extinction retention relative to the controls. In a further two experiments we demonstrated that when two sessions of extinction training separated by either 4 or 24h intervals were given to adolescent rats, saline-treated animals exhibited good extinction retention and the animals given MK801 before the second session exhibited impaired extinction retention. These findings suggest that extinction in adolescence does not initially involve NMDARs and this is a likely mechanism that contributes to the impaired fear inhibition observed at this age. However, NMDARs appear to be recruited with extended extinction training or after administration of a partial agonist, both of which lead to effective extinction retention.

Individual differences in the expression of conditioned fear are associated with endogenous fibroblast growth factor 2.

These experiments examined the relationship between the neurotrophic factor fibroblast growth factor 2 (FGF2) and individual differences in the expression of conditioned fear. Experiments 1 and 2 demonstrated that rats naturally expressing low levels of contextual or cued fear have higher levels of hippocampal FGF2 relative to rats that express high levels of conditioned fear and nonconditioned rats. Experiment 3 demonstrated that hippocampal FGF2 is not increased in rats that exhibit pharmacological-induced amnesia of conditioned fear. Together, these experiments provide evidence that FGF2 may be an endogenous regulator of fear responses to conditioned stimuli.

Individual differences in conditioned fear expression are associated with enduring differences in endogenous Fibroblast Growth Factor-2 and hippocampal-mediated memory performance.

Rodent studies of individual differences in fear expression following Pavlovian fear conditioning are thought to provide useful means by which to examine the factors associated with vulnerability and resilience to anxiety and trauma- and stressor-related disorders in humans. We have recently demonstrated that rats that naturally exhibit low levels of conditioned fear have greater hippocampal expression of the neurotrophic factor Fibroblast Growth Factor-2 (FGF2), relative to rats that naturally exhibit high levels of conditioned fear. In the present study we determined whether individual variance in conditioned fear expression is associated with distinct behavioral profiles across a range of tasks designed to assess expression of trait anxiety and non-emotional memory performance, and whether the differences in hippocampal FGF2 are relatively stable across time. Results indicated that, relative to rats naturally exhibiting low levels of fear, rats naturally exhibiting high levels of fear in the presence of a previously conditioned cue and context also showed heightened levels of trait anxiety, reduced ability to discriminate between a previously conditioned context and a safe context, and impaired performance on the hippocampal-mediated place recognition task, but not on the non-hippocampal-mediated object recognition task. Moreover, differences in hippocampal FGF2 expression were evident between high and low fear rats even three months following the tests for conditioned fear expression. Together, these results suggest that individual differences in conditioned fear expression may be mediated partly by enduring differences in hippocampal functioning.

Habituation and extinction of fear recruit overlapping forebrain structures.

Establishing the neurocircuitry involved in inhibiting fear is important for understanding and treating anxiety disorders. To date, extinction procedures have been predominately used to examine the inhibition of learned fear, where fear is reduced to a conditioned stimulus (CS) by presenting it in the absence of the unconditioned stimulus (US). However, learned fear can also be reduced by habituation procedures where the US is presented in the absence of the CS. Here we used expression of the activity marker c-Fos in rats to compare the recruitment of several forebrain structures following fear habituation and extinction. Following fear conditioning where a tone CS was paired with a loud noise US, fear was then reduced the following day by either presentation of the CS or US alone (i.e. CS extinction or US habituation, respectively). This extinction and habituation training recruited several common structures, including infralimbic cortex, basolateral amygdala, midline thalamus and medial hypothalamus (orexin neurons). Moreover, this overlap was shared when examining the neural correlates of the expression of habituation and extinction, with common recruitment of infralimbic cortex and midline thalamus. However, there were also important differences. Specifically, acquisition of habituation was associated with greater recruitment of prelimbic cortex whereas expression of habituation was associated with greater recruitment of paraventricular thalamus. There was also less recruitment of central amygdala for habituation compared to extinction in the retention phase. These findings indicate that largely overlapping neurocircuitries underlie habituation and fear extinction and imply common mechanisms for reducing fear across different inhibitory treatments.

Treating Generational Stress: Effect of Paternal Stress on Development of Memory and Extinction in Offspring Is Reversed by Probiotic Treatment.

Early-life adversity is a potent risk factor for mental-health disorders in exposed individuals, and effects of adversity are exhibited across generations. Such adversities are also associated with poor gastrointestinal outcomes. In addition, emerging evidence suggests that microbiota-gut-brain interactions may mediate the effects of early-life stress on psychological dysfunction. In the present study, we administered an early-life stressor (i.e., maternal separation) to infant male rats, and we investigated the effects of this stressor on conditioned aversive reactions in the rats' subsequent infant male offspring. We demonstrated, for the first time, longer-lasting aversive associations and greater relapse after extinction in the offspring (F1 generation) of rats exposed to maternal separation (F0 generation), compared with the offspring of rats not exposed to maternal separation. These generational effects were reversed by probiotic supplementation, which was effective as both an active treatment when administered to infant F1 rats and as a prophylactic when administered to F0 fathers before conception (i.e., in fathers' infancy). These findings have high clinical relevance in the identification of early-emerging putative risk phenotypes across generations and of potential therapies to ameliorate such generational effects.

Forming competing fear learning and extinction memories in adolescence makes fear difficult to inhibit.

Fear inhibition is markedly impaired in adolescent rodents and humans. The present experiments investigated whether this impairment is critically determined by the animal's age at the time of fear learning or their age at fear extinction. Male rats (n = 170) were tested for extinction retention after conditioning and extinction at different ages. We examined neural correlates of impaired extinction retention by detection of phosphorylated mitogen-activated protein kinase immunoreactivity (pMAPK-IR) in several brain regions. Unexpectedly, adolescent rats exhibited good extinction retention if fear was acquired before adolescence. Further, fear acquired in adolescence could be successfully extinguished in adulthood but not within adolescence. Adolescent rats did not show extinction-induced increases in pMAPK-IR in the medial prefrontal cortex or the basolateral amygdala, or a pattern of reduced caudal central amygdala pMAPK-IR, as was observed in juveniles. This dampened prefrontal and basolateral amygdala MAPK activation following extinction in adolescence occurred even when there was no impairment in extinction retention. In contrast, only adolescent animals that exhibited impaired extinction retention showed elevated pMAPK-IR in the posterior paraventricular thalamus. These data suggest that neither the animal's age at the time of fear acquisition or extinction determines whether impaired extinction retention is exhibited. Rather, it appears that forming competing fear conditioning and extinction memories in adolescence renders this a vulnerable developmental period in which fear is difficult to inhibit. Furthermore, even under conditions that promote good extinction, the neural correlates of extinction in adolescence are different than those recruited in animals of other ages.

D-cycloserine enhances generalization of fear extinction in children.

BACKGROUND: For exposure therapy to be successful, it is essential that fear extinction learning extends beyond the treatment setting. D-cycloserine (DCS) may facilitate treatment gains by increasing generalization of extinction learning, however, its effects have not been tested in children. We examined whether DCS enhanced generalization of fear extinction learning across different stimuli and contexts among children with specific phobias. METHODS: The study was a double-blind placebo-controlled randomized controlled trial among dog or spider phobic children aged 6-14. Participants ingested either 50 mg of DCS (n = 18) or placebo (n = 17) before receiving a single prolonged exposure session to their feared stimulus. Return of fear was examined 1 week later to a different stimulus (a different dog or spider), presented in both the original treatment context and an alternate context. Avoidance and fear were measured with Behavior Approach Tests (BATs), where the child was asked to increase proximity to the stimulus while reporting their fear level. RESULTS: There were no differences in BAT performance between groups during the exposure session or when a new stimulus was later presented in the treatment context. However, when the new stimulus was presented in a different context, relative to placebo, the DCS group showed less avoidance (P = .03) and less increase in fear (P = .04) with moderate effect sizes. CONCLUSIONS: DCS enabled children to better retain their fear extinction learning. This new learning generalized to different stimuli and contexts.

Teens that fear screams: A comparison of fear conditioning, extinction, and reinstatement in adolescents and adults.

This study investigated differences between adolescents and adults on fear conditioning, extinction, and reinstatement (i.e., the recovery of conditioned fear following re-exposure to the unconditioned stimulus [US] post-extinction). Participants underwent differential conditioning (i.e., the Screaming Lady) where one neutral face (CS+) was followed by the same face expressing fear and a loud scream (US) while another neutral face (CS-) remained neutral. Extinction involved non-reinforced presentations of both CSs, after which participants were reinstated (2xUSs) or not. On two self-report measures, both ages showed conditioning, good extinction learning and retention, and reinstatement-induced relapse. However, only adolescents showed conditioning, extinction, and reinstatement on the eye tracking measure; relapse on this measure could not be assessed in adults given they did not show initial conditioning. Lastly, higher levels of depression predicted stronger conditioning and weaker extinction in adolescents only. These findings are discussed in terms of their implications for adolescent anxiety disorders.

Infantile amnesia: forgotten but not gone.

Unlike adult memories that can be remembered for many years, memories that are formed early in life are more fragile and susceptible to being forgotten (a phenomenon known as "infantile" or "childhood" amnesia). Nonetheless, decades of research in both humans and nonhuman animals demonstrate the importance of early life experiences on later physical, mental, and emotional functioning. This raises the question of how early memories can be so influential if they cannot be recalled. This review presents one potential solution to this paradox by considering what happens to an early memory after it has been forgotten. Specifically, we describe evidence showing that these forgotten early-acquired memories have not permanently decayed from storage. Instead, there appears to be a memory "trace" that persists in the face of forgetting which continues to affect a variety of behavioral responses later in life. Excitingly, the discovery of this physical trace will allow us to explore previously untestable issues in new ways, from whether forgetting is due to a failure in retrieval or storage to how memories can be recovered after extended periods of time. A greater understanding of the characteristics of this memory trace will provide novel insights into how some memories are left behind in childhood while others are carried with us, at least in some form, for a lifetime.