Repeated recall and PKMζ maintain fear memories in juvenile rats.

We examined the neural substrates of fear memory formation and maintenance when repeated recall was used to prevent forgetting in young animals. In contrast to adult rats, juveniles failed to show contextual fear responses at 4 d post-fear conditioning. Reconsolidation sessions 3 and 6 d after conditioning restored contextual fear responses in juveniles 7 d after initial training. In juveniles that received reconsolidation sessions, protein kinase M zeta (PKMζ) increased in the amygdala, but not in the hippocampus. These data suggest that repeated reminders and increased PKMζ maintain fear responses in juvenile animals that otherwise would not exhibit this behavior.

Medial prefrontal cortex processes threatening stimuli in juvenile rats.

To survive, all mammalian species must recognize and respond appropriately to threatening stimuli. In adults, the prelimbic medial prefrontal cortex (mPFC) appears to be involved in fear expression, whereas the infralimbic mPFC mediates fear extinction. In juvenile rats (PN26), the mPFC receives information on potential predators but does not act on it. To test whether the prefrontal cortex is capable of fear regulation in the young organism, we exposed juvenile rats to a threatening or nonthreatening stimulus and assessed fear and brain Fos activation of the mPFC subdivisions, amygdala and periaqueductal gray (PAG). In response to the threat, juveniles froze more, spent more time far from the threat, and had elevated numbers of Fos-positive cells in the prelimbic mPFC, the medial amygdala, and ventral PAG. To test the hypothesis that the mPFC has a dual role in modulating the amygdala and PAG in juveniles, we pharmacologically disinhibited each of the two subdivisions of the mPFC and assessed freezing and downstream activation to the threat. Juvenile rats infused with picrotoxin into the prelimbic mPFC and exposed to a threatening stimulus froze less, spent less time far from the threat, and increased Fos expression. Infusion of picrotoxin into the infralimbic mPFC also reduced fear responding to the threatening stimulus but had no effect on Fos expression. In sum, it appears that the mPFC can process threatening stimuli in juveniles at this age, even though it is normally not involved in the fear responses.

Norepinephrine mediates contextual fear learning and hippocampal pCREB in juvenile rats exposed to predator odor.

Predator odors induce unconditioned fear in the young animal and provide the opportunity to study the mechanisms underlying unlearned and learned fear. In the current study, cat odor produced unlearned, innate fear in infant (postnatal age 14; PN14) and juvenile (PN26) rats, but contextual fear learning occurred only in juveniles. It was hypothesized that contextual fear learning in juveniles is mediated by norepinephrine. Consistent with this hypothesis, pre-training injection of the ß-adrenergic antagonist propranolol reduced the unlearned fear response while post-training injection inhibited contextual fear learning in juvenile rats exposed to cat odor. We suggest that NE mediates the formation of contextual fear memories by activation of the transcription factor CREB in the hippocampus in juveniles but not in infants. Levels of phosphorylated CREB (pCREB) were increased in the dorsal and ventral hippocampi of juvenile rats exposed to cat odor. These levels were not increased in infants or juveniles exposed to a control odor. Further, propranolol blocked these increases in pCREB. In conclusion, although innate fear occurs within the neonatal period, contextual fear learning is a relatively late-occurring event, is hippocampal dependent, and mediated by norepinephrine.

The role of the medial prefrontal cortex in innate fear regulation in infants, juveniles, and adolescents.

In adult animals, the medial prefrontal cortex (mPFC) plays a significant role in regulating emotions and projects to the amygdala and periaqueductal gray (PAG) to modulate emotional responses. However, little is known about the development of this neural circuit and its relevance to unlearned fear in pre-adulthood. To address these issues, we examined the mPFC of 14-d-old (infants), 26-d-old (juveniles), and 38- to 42-d-old (adolescents) rats to represent different developmental and social milestones. The expression patterns of the neuronal marker FOS were used to assess neurological activity. Muscimol, a GABA agonist, was used to inactivate the prelimbic and infralimbic mPFC subdivisions (400 ng in 200 nl). Animals were exposed to either a threatening or nonthreatening stimulus that was ecologically relevant and age specific. Freezing was measured as an indicator of innate fear behavior. The data indicated that the mPFC is neither active nor responsive to innate fear in infant rats. In juveniles, the prelimbic mPFC became responsive in processing aversive sensory stimulation but did not regulate freezing behavior. Finally, during adolescence, inactivation of the prelimbic mPFC significantly attenuated freezing and decreased FOS expression in the ventral PAG. Surprisingly, across all ages, there were no significant differences in FOS levels in the medial and basolateral/lateral amygdala when either mPFC subdivision was inactivated. Together, unlearned fear has a unique developmental course with different brain areas involved in unlearned fear in the immature animal than the adult. In particular, the mPFC neural circuitry is different in young animals and progressively develops more capacities as the animal matures.

Effects of the stimulus and chamber size on unlearned fear across development.

Predator odors have been found to induce unconditioned fear in adult animals and provide the opportunity to study the mechanisms underlying unlearned and learned fear. Predator threats change across an animal's lifetime, as do abilities that enable the animal to learn or engage in different defensive behaviors. Thus, the objective of this study was to determine the combination of factors that successfully induce unlearned fear to predator odor across development. Infant, juvenile, adolescent, and adult rats were exposed to one of the three odor stimuli (control odor, cat urine, or cat fur) in either a small or large chamber. Though all ages displayed fear-related behavior to cat odors, differences were reflected only in freezing behavior and not, as expected, risk-assessment. Infant and juvenile animals also increased freezing to cat urine compared to the control odor, possibly because these age groups possess limited defensive options to cope with threat and so may respond with freezing to all predator stimuli. Unexpectedly, chamber size had no effect on either freezing or risk-assessment in this study. Once the parameters of unconditioned fear are understood, they can be exploited to develop a learning paradigm to predator odors that could be used in early life.

Plasticity of defensive behavior and fear in early development.

Animals have the ability to respond to threatening situations with sets of defensive behaviors. This review demonstrates that defensive behaviors change during early life in mammals. First, unlearned responses are reorganized during early ontogeny and expressed in an age-specific way. Second, the expression of defensive responses is influenced by early experience prior to the first encounter with a threat. Third, once animals have been exposed to a threatening stimulus they subsequently modify their behavior. The neural bases of defensive behavior and the processes that alter them during development are discussed. Maturation of components and connections of the fear circuit seem to contribute to changes in unlearned fear responses. Early experience and learning modify these developmental processes and shape the expression of defensive behavior. Continuous reorganization of the neural substrate and defensive behavior during ontogeny seems to allow the animal to adjust to the conditions it encounters at a given age in a given environment. It is proposed that the developmental changes in defensive behavior can be conceptualized as phenotypic plasticity.

Developing a framework for development: a discussion.

A multitude of scientific disciplines study the development of behavior. Their use of different methodological and conceptual approaches makes integration of findings difficult. In a symposium at the 38th Annual Meeting of the International Society for Developmental Psychobiology in Washington DC, the question was discussed if a general theory of development could unify the field. The three participants explain their views and discuss the possibility of a theoretical framework for development.

Cortisol levels and hippocampus volumes in healthy preadolescent children.

BACKGROUND: Research in animal models has demonstrated that elevated levels of glucocorticoids can inflict damage within the hippocampus. In adult humans, elevated cortisol levels have been associated with reduced hippocampal volumes; however, normative data in children are not available. The objective of this study was to examine possible associations of serum cortisol levels with hippocampal volumes and morphology in healthy children. METHODS: Morning serum cortisol levels and hippocampus magnetic resonance imaging were measured in 17 healthy children (8 girls, 9 boys) between 7 and 12 years of age. RESULTS: Cortisol levels were not associated with total hippocampal volumes; however, with an analysis of surface morphology, significant associations were found for regionally specific portions of the hippocampus. Positive associations were detected for the anterior segment of the hippocampus and inverse associations along the lateral aspects of the hippocampus. CONCLUSIONS: Associations of cortisol levels with regionally specific variations in hippocampal morphology were detected during early development in healthy preadolescent children.

The role of the amygdala and olfaction in unconditioned fear in developing rats.

Early in ontogeny, young rats must be able to detect dangerous stimuli and to exhibit appropriate defensive behaviors. Different nuclei of the amygdala mediate unconditioned and conditioned fear responses to threat in adult rats. The aim of this study was to determine the role of the amygdala in unlearned fear behavior in young rats. When exposed to an unfamiliar adult male rat, preweaning rat pups freeze, with peak levels on postnatal day 14 and declining levels on day 18. Pups were made anosmic to block olfactory input to the amygdala, and amygdala activation was assessed by quantifying the neuronal marker c-fos. Anosmic pups did not freeze in the presence of the male rat and had decreased c-fos expression in the medial amygdala on day 14 and in the medial and lateral amygdala on day 18. However, the decrease in freezing between days 14 and 18 was not associated with a decrease in c-fos expression in the medial amygdala. The medial and lateral amygdala were then inactivated by local muscimol infusion on day 14. Muscimol infusion into the medial amygdala decreased freezing to the male rat but not to a loud noise, whereas infusion into the lateral amygdala blocked freezing to a loud noise but not to the male. These findings indicate that different nuclei of the amygdala process sensory information of different modalities, mediate unconditioned freezing, and may be involved in developmental changes in the fear response in young rats.

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats.

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.

Adaptations or pathologies? Long-term changes in brain and behavior after a single exposure to severe threat.

The experience of a single threatening situation may alter the behavior of an animal in a long-lasting way. Long-lasting changes in behavior have been induced in laboratory animals to model and investigate the development and neural substrate of human psychopathologies. Under natural conditions, however, changes in behavior after an aversive experience may be adaptive because behavioral modifications allow animals to adjust to a threat for extended periods of time. In the laboratory setting, properties of the aversive situation and the potential of the animal to respond to the threat may be altered and lead to extensive, prolonged changes, indicating a failure in behavioral regulation. Such long-term changes seem to be mediated by neuronal alterations in components of the fear pathway. To understand psychopathologies, determinants of exaggerated responsivity and the underlying molecular and neural processes have to be analyzed in a comparative way under conditions that produce normal and abnormal fear and anxiety.

Rat pups reduce ultrasonic vocalization after exposure to an adult male rat.

We examined how the experience of a threatening stimulus alters subsequent behavior in a situation where the immediate threat is absent. A small huddle of 12-day-old rats was exposed to a potentially infanticidal adult male rat for 5 min. During male exposure, pups were significantly more immobile than control pups. Thirty, 60, and 180 min after male exposure, the pups were isolated for 5 min from litter and dam in an unfamiliar environment. When isolated, pups that had been previously exposed to the male emitted significantly fewer ultrasonic vocalizations than controls, but did not differ in immobility. Low levels of vocalization were apparent 30 and 60 min after male exposure and were not evident at 180 min. The pups seemed to have adjusted their behavior to a potential male threat in a different context for a limited period of time.

Mother lowers glucocorticoid levels of preweaning rats after acute threat.

Exposure to a deadly threat, an adult male rat, induced the release of corticosterone in 14-day-old rat pups. The endocrine stress response was decreased when the pups were reunited with their mother immediately after exposure. These findings demonstrate that social variables can reduce the consequences of an aversive experience.