Corticosterone delivery to the amygdala increases corticotropin-releasing factor mRNA in the central amygdaloid nucleus and anxiety-like behavior

The present study examined the effects of stereotaxic delivery of corticosterone to the amygdala on anxiety-like behavior and corticotropin-releasing factor (CRF) mRNA level in the central nucleus of the amygdala (CeA). Micropellets (30 microg) of crystalline corticosterone or cholesterol (control) were implanted bilaterally at the dorsal margin of the CeA in Wistar rats. Seven days post-implantation, anxiety-like behavior was accessed using an elevated plus-maze. CRF mRNA level in the CeA was determined by in situ hybridization 4 h after being tested on the elevated plus-maze. Corticosterone implants increased indices of anxiety on the elevated plus-maze and produced a concomitant increase in both basal level of CRF mRNA per neuron and the number of neurons with CRF hybridization signal in the CeA. The plus-maze increased CRF mRNA levels in the CeA of cholesterol implanted rats to the elevated basal levels observed in corticosterone treated animals. Exposure to the plus-maze did not increase CRF mRNA level in the CeA of corticosterone implanted rats beyond elevated basal levels. Taken together, these findings support the involvement of the amygdala in anxiety-like behaviors in response to chronically elevated corticosterone and suggests that elevated glucocorticoids may increase anxiety by inducing CRF expression in the CeA.     

The smell of danger: a behavioral and neural analysis of predator odor-induced fear

The odors of predators used in animal models provide, in addition to electric footshock, an important means to investigate the neurobiology of fear. Studies indicate that cat odor and trimethylthiazoline (TMT), a synthetic compound isolated from fox feces, are often presented to rodents to induce fear-related responses including freezing, avoidance, stress hormone and, in some tests, risk assessment behavior. Furthermore, we report that different amounts of cat odor impregnated on small-, medium-, or large-sized cloths impact the display of fear-related behavior when presented to rats. That is, rats exposed to a large cat odor containing cloth exhibit an increase in fear behavior, particularly freezing, which remains at high levels in habituation tests administered over a period of 7 days. The large cloth also induces a long-lasting increase in avoidance behavior during repeated habituation and extinction tests. A review of the brain regions involved in predator odor-induced fear behavior indicates a modulatory role of the medial amygdala, bed nucleus of the stria terminalis, and dorsal premammillary nucleus. In addition, the basolateral amygdala is involved in fear behavior induced by cat odor but not TMT, and the central amygdala does not appear to play a major behavioral role in predator odor-induced fear. Future research involving the use of predator odor is likely to rapidly expand knowledge on the neurobiology of fear, which has implications for understanding fear-related psychopathology.     

The role of the amygdala in human fear: automatic detection of threat

Behavioral data suggest that fear stimuli automatically activate fear and capture attention. This effect is likely to be mediated by a subcortical brain network centered on the amygdala. Consistent with this view, brain imaging studies show that masked facial stimuli activate the amygdala as do masked pictures of threatening animals such as snakes and spiders. When the stimulus conditions allow conscious processing, the amygdala response to feared stimuli is enhanced and a cortical network that includes the anterior cingulate cortex and the anterior insula is activated. However, the initial amygdala response to a fear-relevant but non-feared stimulus (e.g. pictures of spiders for a snake phobic) disappears with conscious processing and the cortical network is not recruited. Instead there is activation of the dorsolateral and orbitofrontal cortices that appears to inhibit the amygdala response. The data suggest that activation of the amygdala is mediated by a subcortical pathway, which passes through the superior colliculi and the pulvinar nucleus of the thalamus before accessing the amygdala, and which operates on low spatial frequency information.     

Functional emergence of the hippocampus in context fear learning in infant rats

The hippocampus is a part of the limbic system and is important for the formation of associative memories, such as acquiring information about the context (e.g., the place where an experience occurred) during emotional learning (e.g., fear conditioning). Here, we assess whether the hippocampus is responsible for pups' newly emerging context learning. In all experiments, postnatal day (PN) 21 and PN24 rat pups received 10 pairings of odor‐0.5 mA shock or control unpaired odor‐shock, odor only, or shock only. Some pups were used for context, cue or odor avoidance tests, while the remaining pups were used for c‐Fos immunohistochemistry to assess hippocampal activity during acquisition. Our results show that cue and odor avoidance learning were similar at both ages, while contextual fear learning and learning‐associated hippocampal (CA1, CA3, and dentate gyrus) activity (c‐Fos) only occurred in PN24 paired pups. To assess a causal relationship between the hippocampus and context conditioning, we infused muscimol into the hippocampus, which blocked acquisition of context fear learning in the PN24 pups. Muscimol or vehicle infusions did not affect cue learning or aversion to the odor at PN21 or PN24. The results suggest that the newly emerging contextual learning exhibited by PN24 pups is supported by the hippocampus. © 2009 Wiley‐Liss, Inc.     

Neural responses to dynamic expressions of fear in schizophrenia

Abnormalities in social functioning are a significant feature of schizophrenia. One critical aspect of these abnormalities is the difficulty these individuals have with the recognition of facial emotions, particularly negative expressions such as fear. The present work focuses on fear perception and its relationship to the paranoid symptoms of schizophrenia, specifically, how underlying limbic system structures (i.e. the amygdala) react when probed with dynamic fearful facial expressions. Seven paranoid and eight non-paranoid subjects (all males) with a diagnosis of schizophrenia took part in functional magnetic resonance imaging study (1.5T) examining neural responses to emerging fearful expressions contrasted with dissipating fearful expressions. Subjects viewed emerging and dissipating expressions while completing a gender discrimination task. Their brain activation was compared to that of 10 healthy male subjects. Increased hippocampal activation was seen in the non-paranoid group, while abnormalities in the bilateral amygdalae were observed only in the paranoid individuals. These patterns may represent trait-related hippocampal dysfunction, coupled with state (specifically paranoia) related amygdala abnormalities. The findings are discussed in light of models of paranoia in schizophrenia.     

Corticosterone effects on corticotropin-releasing hormone mRNA in the central nucleus of the amygdala and the parvocellular region of the paraventricular nucleus of the hypothalamus

Using in situ hybridization histochemistry, we report differential expression of corticotropin-releasing hormone (CRH) mRNA in the central nucleus of the amygdala (CEA) and the parvocellular region of the paraventricular nucleus of the hypothalamus (PVN) following systemic treatment with corticosterone (CORT) in adrenally-intact rats. Both injection of low (1 mg/kg/day) and high (5 mg/day) CORT reduced CRH mRNA expression in the PVN in a dose-dependent manner, although it returned to normal at the low dose by 14 days. By contrast, the high dose of CORT increased CRH mRNA transiently in the CEA at 4 days, although the low dose of CORT decreased it at 14 days. In a second experiment, we implanted a slowly-releasing CORT pellet for 2 weeks (200 mg, 60 day release) subcutaneously. This treatment produced an elevation of CRH mRNA in the CEA both at 1 and 2 weeks, whereas CRH mRNA in the PVN was decreased to a large extent as seen in the high CORT group of the first experiment. These results suggest that glucocorticoids can facilitate CRH mRNA expression in the CEA, a site implicated in anxiety and fear, while restraining the hypothalamic-pituitary-adrenal axis as indicated by the reduction in CRH mRNA in the PVN.     

The effects of predator odors in mammalian prey species: a review of field and laboratory studies

Prey species show specific adaptations that allow recognition, avoidance and defense against predators. For many mammalian species this includes sensitivity towards predator-derived odors. The typical sources of such odors include predator skin and fur, urine, feces and anal gland secretions. Avoidance of predator odors has been observed in many mammalian prey species including rats, mice, voles, deer, rabbits, gophers, hedgehogs, possums and sheep. Field and laboratory studies show that predator odors have distinctive behavioral effects which include (1) inhibition of activity, (2) suppression of non-defensive behaviors such as foraging, feeding and grooming, and (3) shifts to habitats or secure locations where such odors are not present. The repellent effect of predator odors in the field may sometimes be of practical use in the protection of crops and natural resources, although not all attempts at this have been successful. The failure of some studies to obtain repellent effects with predator odors may relate to (1) mismatches between the predator odors and prey species employed, (2) strain and individual differences in sensitivity to predator odors, and (3) the use of predator odors that have low efficacy. In this regard, a small number of recent studies have suggested that skin and fur-derived predator odors may have a more profound lasting effect on prey species than those derived from urine or feces. Predator odors can have powerful effects on the endocrine system including a suppression of testosterone and increased levels of stress hormones such as corticosterone and ACTH. Inhibitory effects of predator odors on reproductive behavior have been demonstrated, and these are particularly prevalent in female rodent species. Pregnant female rodents exposed to predator odors may give birth to smaller litters while exposure to predator odors during early life can hinder normal development. Recent research is starting to uncover the neural circuitry activated by predator odors, leading to hypotheses about how such activation leads to observable effects on reproduction, foraging and feeding.     

Hippocampal involvement in the expression of kindling-induced fear in rats

Kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides a superb opportunity to study the nature and neural mechanisms of fear sensitization. Interestingly, these changes in behavior are accompanied by increased binding to inhibitory receptors and decreased binding to excitatory receptors in the CA1 and dentate gyrus regions of the hippocampus. This led us to hypothesize that kindling-induced fear may result from an increased inhibitory tone within hippocampal circuits. To test this hypothesis, we investigated FOS protein immunoreactivity in hippocampal and amygdalar regions of kindled rats that were exposed to an unfamiliar open field. We found that FOS immunoreactivity was significantly decreased in the CA1 region, dentate gyrus, and perirhinal cortex of kindled rats compared to sham-stimulated rats. These results support our hypothesis that kindling-induced fear may be produced by inhibition within hippocampal circuits. They also suggest that neural changes within the hippocampus may be important for the sensitization of fear.     

Abnormal development pattern of the amygdala and hippocampus from childhood to adulthood with autism

Using magnetic resonance imaging to determine neuropathology in autism spectrum disorders, we report findings on the volume of the amygdala and hippocampus in autistic children. The volumes of amygdala, hippocampus and total brain were obtained by volbrain and their volumes were measured in young people (6.5–27.0 years of age) that comes from ABIDE dataset. Although there was no significant difference in total brain capacity between groups, autistic children (6.5–12.0 years of age) had larger right and left absolute and relative amygdala volumes than the control group. There was no difference in amygdala volume between adolescence (13–19 years old) and adults (20–27 years old). Interestingly, the volume of the amygdala in typical developing children increased significantly from 6.5 to 27 years of age. Thus, amygdala in children with autism was initially small, but no age-related increases were observed in normal developing children. The right absolute hippocampal volume of autistic patients was also larger than that of normal adults, but not after controlling the total brain volume. These cross-sectional findings suggest that abnormal patterns of hippocampal and amygdala development continue into adolescence in autistic patients.     

An electrophysiological characterization of the projection from the central nucleus of the amygdala to the periaqueductal gray of the rat: the role of opioid receptors

The midbrain periaqueductal gray (PAG) and the central nucleus of the amygdala (CNA) are both known to be involved in fear and anxiety, analgesia, vocalization, cardiovascular and respiratory changes, and freezing. Anatomical studies have shown that a connection between these two regions exists but little is known about the physiology or the neurochemical constituents of this pathway. The goals of this study were to characterize the projection from the CNA to the PAG using electrophysiological techniques and to determine whether μ- and/or δ-opioid receptors, which play a large role in a majority of the functions of the PAG, are involved in this pathway. Of the 38 PAG cells tested with single shock stimulation of the CNA, 44% responded; of those, 46% were excited and 54% were inhibited. The latency to onset of response for the inhibitory cells (12.71 ± 6.61 ms) was shorter than that of the excitatory cells (22.33 ± 4.04 ms). Forty-six percent of the 129 PAG cells tested with train electrical stimulation of the CNA responded; 44% were excited and 56% were inhibited. Chemical stimulation of the CNA (10 mMd,l-homocysteic acid) produced similar results; 48% (62/128) of PAG cells responded; 45% of cells were excited and 55% were inhibited. The baseline firing rate of the inhibitory cells was significantly higher compared to the excitatory cells. Chemical stimulation of the CNA produced an increase in blood pressure in 12 animals, a decrease in two animals, and had no effect on the blood pressure of 68 animals. The blood pressure changes ranged between 8.5 and 26.3 mmHg with a mean of 16.2 ± 2.2 mmHg. The effect of naloxone (given either on site in the PAG or systemically) on the response to CNA stimulation was tested in 21 cells. Twenty-five percent of the excitatory cells (2/8) and 77% (10/13) of the inhibitory cells were blocked by naloxone with the majority of the blocked cells located in the ventrolateral PAG. It is concluded that: (1) Approximately 50% of cells in the lateral and ventrolateral columns of the PAG respond to CNA stimulation; (2) the inhibitory response is mediated by a faster conducting or a more direct pathway than the pathway that mediates the excitatory response; (3) neurons that are inhibited by CNA stimulation have a significantly higher baseline firing rate than neurons that are excited, suggesting that they may be tonically active interneurons; and (4) at least one link in the CNA-PAG pathway utilizes μ- or δ-opioid receptors.     

Circadian modulation of fos responses to odor of the red fox, a rodent predator, in the rat olfactory system

We have previously shown that neuronal responses to a biologically neutral odor, cedar wood oil, in the olfactory system are greater in the subjective night compared to subjective day. In the present study, we confirm these results and extend them to a biologically relevant odor, the urine of the red fox, a rodent predator. Fos induced by exposure of rats to fox urine or a neutral odor, mineral oil, was markedly enhanced during the subjective night compared to subjective day in the main olfactory bulb, primary olfactory cortex, and other structures related to olfaction. These results show that neuronal responses to an ethologically relevant odor follow a circadian rhythm similar to biologically neutral odors. Fos responses induced by fox urine were observed to be of greater magnitude than a neutral odor in brain areas involved in fear responses, suggesting that fox urine activates fear circuitry.     

Neurotensin immunoreactive structures in the human infant striatum, septum, amygdala and cerebral cortex

Neurotensin immunoreactive (NT-IR) neuronal perikarya are present in small numbers in the bed nucleus of the stria terminalis, lateral olfactory stria, substantia innominata, caudate nucleus and putamen of the human infant forebrain. Larger numbers of perikarya are present in the amygdala and related structures. NT-IR axons are present in the medial septal area, bed nucleus of the stria terminalis, caudate nucleus, putamen and amygdala. The cerebral cortex contains a rich network of NT axons with an accentuation in layer II. This network appears to be derived from bundles of axons which traverse the deep white matter from the thalamus.     

The effect of experimental epilepsy induced by injection of tetanus toxin into the amygdala of the rat on eating behaviour and response to novelty

A minute dose of tetanus toxin injected into the amygdala of rats produced an apparently reversible epileptiform syndrome similar to that previously described after injection of the toxin into the hippocampus. During the active epilepsy the toxin-injected rats occasionally exhibited ‘paroxysmal eating’ and also sometimes ran round in circles attempting to bite their own tails. When presented with a novel but palatable food (chocolate buttons or harvest crunch) the toxin-injected rats showed less neophobia than their controls—they ate sooner and ate more. This was found both during the active epilepsy and several weeks later when they had recovered. A similar effect of amygdala injections was found in a second experiment, in which the effect was compared with that of toxin injection in the hippocampus. These rats were tested also on the playground maze on their approach response to a neutral novel object (in a familiar environment in the context of seven familiar objects). The amygdala rats did not show any increase in their novelty response; thus their reduction in neophobia was specific to an appetitive behaviour. In contrast, the hippocampally-injected rats did not exhibit a novelty response in the playground maze, but showed normal neophobia to a new food.     

Repeated restraint stress and corticosterone injections during late pregnancy alter GAP-43 expression in the hippocampus and prefrontal cortex of rat pups

In the offspring of prenatal stress animals, overactivity and impaired negative feedback regulation of the hypothalamic–pituitary–adrenal axis are consistent finding. However, little was known about how prenatal stress can permanently alter developmental trajectories of pup's brain. Growth-associated protein-43 (GAP-43) is a presynaptic membrane phosphoprotein whose expression increases during developmental events such as axonal outgrowth or remodeling and synaptogenesis. Phosphorylation of GAP-43 by protein kinase C was correlated with enhanced axonal growth and transmitter release. In adult animals, increase of GAP-43 correlated with monoaminergic deficit in neuropsychiatric disorders. The present study examines the effects of repeated maternal restraint stress on the level of GAP-43 in the brain of rat pups. The results showed that prenatal stress significantly increased GAP-43 level in the PFC of rat pup during PND 7–14 as compared to control but not significant difference when observed at PND 21. Increased GAP-43 expression was also observed in the pup's hippocampus during the same postnatal periods. However, when observed at PND 60, pups born from stressed mother showed a significant lower (p < 0.001) GAP-43 expression as compare with control group. These changes indicate the direct effect of corticosteroid hormone, since repeated maternal injection with corticosterone (CORT, 40 mg/kg) during GD 14–21 also gave the same results. PND 7–14 is the peak period of synaptogenesis in these brain areas and abnormal axon sprouting and reorganization may lead to a defect in synaptic pruning at later stage of life. The results suggested that maternal stress is harmful to the developing brain and upregulation of GAP-43 indicated a protective mechanism against the toxicity of maternal stress hormone. Prenatal stress alter the normal developmental trajectories in the pup's brain may underlies the mechanism link between early life stress and neuropsychopathology in later life.     

Neural plasticity and stress induced changes in defense in the rat

We investigated the effects of predator stress on behavior and amygdala afferent and efferent neural transmission in rats. Pathways studied were: ventral angular bundle input to the basolateral amygdala; central and basolateral amygdala output to the periaqueductal gray (PAG). Predator stress was ‘anxiogenic’ in elevated plus maze, light/dark box and acoustic startle tests one week after stress. Lasting changes were also observed in neural transmission. Predator stress appeared to potentiate right and depotentiate left hemisphere afferent amygdala transmission. In contrast, predator stress potentiated amygdala efferent transmission to right and left PAG, depending on the amygdala nucleus stimulated. Paired pulse and intensity series analysis suggests that transmission changes may be postsynaptic or presynaptic, depending on the pathway. Path analysis relating brain and behavioral changes suggests that potentiation and depotentiation in both hemispheres participate jointly in effecting some, but not all, of the behavioral changes produced by predator stress. Potentiation in left hemisphere amygdala afferents and efferents predicts anxiolytic-like effects, while potentiation in the right hemisphere amygdala afferents predicts anxiogenic-like effects. Path analysis also supports the view that changes in different neural systems mediate changes in different behaviors. These findings have their parallel in studies in the cat, but there are species differences.     

Opposing roles of the amygdala and dorsolateral periaqueductal gray in fear-potentiated startle

The whole-body acoustic startle response is a short-latency reflex mediated by a relatively simple neural circuit in the lower brainstem and spinal cord. The amplitude of this reflex is markedly enhanced by moderate fear levels, and less effectively increased by higher fear levels. Extensive evidence indicates that the amygdala plays a key role in the potentiation of startle by moderate fear. More recent evidence suggests that the periaqueductal gray is involved in the loss of potentiated startle at higher levels of fear. The influence of both structures may be mediated by anatomical connections with the acoustic startle circuit, perhaps at the level of the nucleus reticularis pontis caudalis. The mediated by anatomical connections with the acoustic startle circuit, perhaps at the level of the nucleus reticularis pontis caudalis. The present chapter reviews these data.     

Changes in extracellular levels of amygdala amino acids in genetically fast and slow kindling rat strains

A neurochemical basis for many of the epilepsies has long been suspected to result from an imbalance between excitatory and inhibitory neurotransmitter mechanisms. Data supporting changes in extrasynaptic amino acid levels during epileptogenesis, however, remain controversial. In the present study, we used in vivo microdialysis to measure the levels of extracellular GABA (gamma-aminobutyric acid) and glutamate during seizure development in rats with a genetic predisposition for (Fast), or against (Slow), amygdala kindling. Dialysates were collected from both amygdalae before, during, and up to 12 min after a threshold-triggered amygdala afterdischarge (AD). One hour later, samples were again collected from both amygdalae in response to a hippocampal threshold AD. Daily amygdala kindling commenced the next day but without dialysis. After the rats were fully kindled, the same protocol was again employed. Amino acid levels were not consistently increased above baseline with triggered seizures in either strain. Instead, before kindling, a focal seizure in the Slow rats was associated with a large decrease in GABA in the non-stimulated amygdala, while amino acid levels in the Fast rats remained near baseline in both amygdalae. Similar results were seen after kindling. By contrast, before and after kindling, hippocampal stimulation caused large decreases in all amino acid levels in both amygdalae in both strains. These data suggest that, in response to direct stimulation, extracellular amino acid concentrations remain stable in tissues associated with either greater natural (Fast) or induced (kindled Fast/Slow) excitability, but are lowered with indirect stimulation (hippocampus) and/or low excitability.     

Brain sites involved in fear memory reconsolidation and extinction of rodents

Fear memory is a motivational system essential for organisms survival having a central role in organization of defensive behaviors to threat. In the last years there has been a growing interest on conditioned fear memory reconsolidation and extinction, two specific phases of memorization process, both induced by memory retrieval. Understanding the mechanisms underlying these two mnemonic processes may allow to work out therapeutic interventions for treatment of human fear and anxiety disorders, such as specific phobias and post-traumatic stress disorder. Based on the use of one-trial conditioning paradigms, which allow to follow the evolution of a mnemonic trace in its various phases, the present paper has attempted to reorganize the current literature relative to the rodents highlighting both the role of several brain structures in conditioned fear memory reconsolidation and extinction and the selective cellular processes involved. A crucial role seems to be play by medial prefrontal cortex, in particular by prelimbic and infralimbic cortices, and by distinct connections between them and the amygdala, hippocampus and entorhinal cortex.     

Deep brain stimulation of the amygdala alleviates post-traumatic stress disorder symptoms in a rat model

Post-traumatic stress disorder (PTSD) is an anxiety disorder triggered by a life-threatening event causing intense fear. Recently, functional neuroimaging studies have suggested that amygdala hyperactivity is responsible for the symptoms of PTSD. Deep brain stimulation (DBS) can functionally reduce the activity of a cerebral target by delivering an electrical signal through an electrode. We tested whether DBS of the amygdala could be used to treat PTSD symptoms. Rats traumatized by inescapable shocks, in the presence of an unfamiliar object, develop the tendency to bury the object when re-exposed to it several days later. This behavior mimics the symptoms of PTSD. 10 Sprague–Dawley rats underwent the placement of an electrode in the right basolateral nucleus of the amygdala (BLn). The rats were then subjected to a session of inescapable shocks while being exposed to a conspicuous object (a ball). Five rats received DBS treatment while the other 5 rats did not. After 7 days of treatment, the rats were re-exposed to the ball and the time spent burying it under the bedding was recorded. Rats treated with BLn DBS spent on average 13 times less time burying the ball than the sham control rats. The treated rats also spent 18 times more time exploring the ball than the sham control rats. In conclusion, the behavior of treated rats in this PTSD model was nearly normalized. We argue that these results have direct implications for patients suffering from treatment-resistant PTSD by offering a new therapeutic strategy.