The Neurobiology of Infant Attachment-Trauma and Disruption of Parent-Infant Interactions.

Current clinical literature and supporting animal literature have shown that repeated and profound early-life adversity, especially when experienced within the caregiver-infant dyad, disrupts the trajectory of brain development to induce later-life expression of maladaptive behavior and pathology. What is less well understood is the immediate impact of repeated adversity during early life with the caregiver, especially since attachment to the caregiver occurs regardless of the quality of care the infant received including experiences of trauma. The focus of the present manuscript is to review the current literature on infant trauma within attachment, with an emphasis on animal research to define mechanisms and translate developmental child research. Across species, the effects of repeated trauma with the attachment figure, are subtle in early life, but the presence of acute stress can uncover some pathology, as was highlighted by Bowlby and Ainsworth in the 1950s. Through rodent neurobehavioral literature we discuss the important role of repeated elevations in stress hormone corticosterone (CORT) in infancy, especially if paired with the mother (not when pups are alone) as targeting the amygdala and causal in infant pathology. We also show that following induced alterations, at baseline infants appear stable, although acute stress hormone elevation uncovers pathology in brain circuits important in emotion, social behavior, and fear. We suggest that a comprehensive understanding of the role of stress hormones during infant typical development and elevated CORT disruption of this typical development will provide insight into age-specific identification of trauma effects, as well as a better understanding of early markers of later-life pathology.

Neurobiology of Parental Regulation of the Infant and Its Disruption by Trauma Within Attachment.

The complex process of regulating physiological functions and homeostasis during external and internal disruptions develops slowly in altricial species, with parental care functioning as a co-regulator of infant physiological and emotional homeostasis. Here, we review our current understanding of the infant's use of parental behaviors for neurobehavioral regulation and its disruption with harsh parental care. Taking a cross-species view, we briefly review the human developmental literature that highlights the importance of the caregiver in scaffolding the child's physiological and emotional regulation, especially under threat and stress. We then use emerging corresponding animal literature within the phylogenetically preserved attachment system to help define neural systems supporting caregiver regulation and its supporting causal mechanism to provide translational bridges to inform causation and mechanisms impossible to define in children. Next, we briefly review animal research highlighting the impact of specific sensory stimuli imbedded in parental care as important for infant physiological and emotion regulation. We then highlight the importance of parental sensory stimuli gaining hedonic value to go beyond simple sensory stimuli to further impact neurobehavioral regulation, with poor quality of care compromising the infant's ability to use these cues for regulation. Clinically, parental regulation of the infant is correlated with later-life neurobehavioral outcome and quality of life. We suggest an understanding of this parental regulation of the infant's immediate neurobehavioral functioning within the context of attachment quality, that may provide insights into the complex processes during early life, initiating the pathway to pathology.

Bidirectional control of infant rat social behavior via dopaminergic innervation of the basolateral amygdala.

Social interaction deficits seen in psychiatric disorders emerge in early-life and are most closely linked to aberrant neural circuit function. Due to technical limitations, we have limited understanding of how typical versus pathological social behavior circuits develop. Using a suite of invasive procedures in awake, behaving infant rats, including optogenetics, microdialysis, and microinfusions, we dissected the circuits controlling the gradual increase in social behavior deficits following two complementary procedures-naturalistic harsh maternal care and repeated shock alone or with an anesthetized mother. Whether the mother was the source of the adversity (naturalistic Scarcity-Adversity) or merely present during the adversity (repeated shock with mom), both conditions elevated basolateral amygdala (BLA) dopamine, which was necessary and sufficient in initiating social behavior pathology. This did not occur when pups experienced adversity alone. These data highlight the unique impact of social adversity as causal in producing mesolimbic dopamine circuit dysfunction and aberrant social behavior.

Vulnerable and Resilient Phenotypes in a Mouse Model of Anorexia Nervosa.

BACKGROUND: Increased physical activity is a common feature of anorexia nervosa (AN). Although high activity levels are associated with greater risk of developing AN, particularly when combined with dieting, most individuals who diet and exercise maintain a healthy body weight. It is unclear why some individuals develop AN while most do not. A rodent model of resilience and vulnerability to AN would be valuable to research. Dopamine, which is believed to play a crucial role in AN, regulates both reward and activity and may modulate vulnerability. METHODS: Adolescent and young adult female C57BL/6N mice were tested in the activity-based anorexia (ABA) model, with an extended period of food restriction in adult mice. ABA was also tested in dopamine transporter knockdown mice and wild-type littermates. Mice that adapted to conditions and maintained a stable body weight were characterized as resilient. RESULTS: In adults, vulnerable and resilient phenotypes emerged in both the ABA and food-restricted mice without wheels. Vulnerable mice exhibited a pronounced increase in running throughout the light cycle, which dramatically peaked prior to requiring removal from the experiment. Resilient mice exhibited an adaptive decrease in total running, appropriate food anticipatory activity, and increased consumption, thereby achieving stable body weight. Hyperdopaminergia accelerated progression of the vulnerable phenotype. CONCLUSIONS: Our demonstration of distinct resilient and vulnerable phenotypes in mouse ABA significantly advances the utility of the model for identifying genes and neural substrates mediating AN risk and resilience. Modulation of dopamine may play a central role in the underlying circuit.

Contextual fear memory modulates PSD95 phosphorylation, AMPAr subunits, PKMζ and PI3K differentially between adult and juvenile rats.

It is well known that young organisms do not maintain memories as long as adults, but the mechanisms for this ontogenetic difference are undetermined. Previous work has revealed that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAr) subunits are trafficked into the synaptic membrane following memory retrieval in adults. Additionally, phosphorylated PSD-95-pS295 promotes AMPAr stabilization at the synapse. We investigated these plasticity related proteins as potential mediators in the differential contextual stress memory retrieval capabilities observed between adult and juvenile rats. Rats were assigned to either pedestal stress (1 h) or no stress control (home cage). Each animal was placed alone in an open field for 5 min at the base of a 6 × 6 sq inch pedestal (4ft high). Stress subjects were then placed on this pedestal for 1hr and control subjects were placed in their home cage following initial exploration. Each animal was returned to the open field for 5 min either 1d or 7d following initial exposure. Freezing postures were quantified during the memory retrieval test. The 1d test shows adult (P90) and juvenile (P26) stressed rats increase their freezing time compared to controls. However, the 7d memory retrieval test shows P90 stress rats but not P26 stress rats freeze while in the fear context. Twenty minutes after the memory retrieval test, hippocampi and amygdala were micro-dissected and prepared for western blot analysis. Our results show that 1d fear memory retrieval induced an upregulation of PSD-95 and pS295 in the adult amygdala but not in the juvenile. However, the juvenile animals upregulated PKMζ, PI3K and GluA2/3, GluA1-S845 in the dorsal hippocampus (DH), but the adults did not. Following the 7d memory retrieval test, adults upregulated GluA2 in the amygdala but not the juveniles. In the DH, adults increased PSD-95 and pS295 but not the juveniles. The adults appear to preferentially increase amygdala-driven processing at 1d and increase DH-driven context specific processing at 7d. These data identify molecular processes that may underlie the reduced fear-memory retrieval capability of juveniles. Together these data provide a potential molecular target that could be beneficial in treatment of anxiety disorders and PTSD.

Neurobiology of maternal regulation of infant fear: the role of mesolimbic dopamine and its disruption by maltreatment.

Child development research highlights caregiver regulation of infant physiology and behavior as a key feature of early life attachment, although mechanisms for maternal control of infant neural circuits remain elusive. Here we explored the neurobiology of maternal regulation of infant fear using neural network and molecular levels of analysis in a rodent model. Previous research has shown maternal suppression of amygdala-dependent fear learning during a sensitive period. Here we characterize changes in neural networks engaged during maternal regulation and the transition to infant self-regulation. Metabolic mapping of 2-deoxyglucose uptake during odor-shock conditioning in postnatal day (PN)14 rat pups showed that maternal presence blocked fear learning, disengaged mesolimbic circuitry, basolateral amygdala (BLA), and plasticity-related AMPA receptor subunit trafficking. At PN18, when maternal presence only socially buffers threat learning (similar to social modulation in adults), maternal presence failed to disengage the mesolimbic dopaminergic system, and failed to disengage both the BLA and plasticity-related AMPA receptor subunit trafficking. Further, maternal presence failed to block threat learning at PN14 pups following abuse, and mesolimbic dopamine engagement and AMPA were not significantly altered by maternal presence-analogous to compromised maternal regulation of children in abusive relationships. Our results highlight three key features of maternal regulation: (1) maternal presence blocks fear learning and amygdala plasticity through age-dependent suppression of amygdala AMPA receptor subunit trafficking, (2) maternal presence suppresses engagement of brain regions within the mesolimbic dopamine circuit, and (3) early-life abuse compromises network and molecular biomarkers of maternal regulation, suggesting reduced social scaffolding of the brain.

Developmental transitions in amygdala PKC isoforms and AMPA receptor expression associated with threat memory in infant rats.

Although infants learn and remember, they rapidly forget, a phenomenon known as infantile amnesia. While myriad mechanisms impact this rapid forgetting, the molecular events supporting memory maintenance have yet to be explored. To explore memory mechanisms across development, we used amygdala-dependent odor-shock conditioning and focused on mechanisms important in adult memory, the AMPA receptor subunits GluA1/2 and upstream protein kinases important for trafficking AMPAR, protein kinase M zeta (PKMζ) and iota/lambda (PKCι/λ). We use odor-shock conditioning in infant rats because it is late-developing (postnatal day, PN10) and can be modulated by corticosterone during a sensitive period in early life. Our results show that memory-related molecules did not change in pups too young to learn threat (PN8) but were activated in pups old enough to learn (PN12), with increased PKMζ-PKCι/λ and GluA2 similar to that observed in adult memory, but with an uncharacteristic decrease in GluA1. This molecular signature and behavioral avoidance of the conditioned odor was recapitulated in PN8 pups injected with CORT before conditioning to precociously induce learning. Blocking learning via CORT inhibition in older pups (PN12) blocked the expression of these molecules. PN16 pups showed a more adult-like molecular cascade of increased PKMζ-PKCι/λ and GluA1-2. Finally, at all ages, zeta inhibitory peptide (ZIP) infusions into the amygdala 24 hr after conditioning blocked memory. Together, these results identify unique features of memory processes across early development: AMPAR subunits GluA1/2 and PKC isoform expression are differentially used, which may contribute to mechanisms of early life forgetting.

Chronic voluntary oral methamphetamine induces deficits in spatial learning and hippocampal protein kinase Mzeta with enhanced astrogliosis and cyclooxygenase-2 levels.

Methamphetamine (MA) is an addictive drug with neurotoxic effects on the brain producing cognitive impairment and increasing the risk for neurodegenerative disease. Research has focused largely on examining the neurochemical and behavioral deficits induced by injecting relatively high doses of MA [30 mg/kg of body weight (bw)] identifying the upper limits of MA-induced neurotoxicity. Accordingly, we have developed an appetitive mouse model of voluntary oral MA administration (VOMA) based on the consumption of a palatable sweetened oatmeal mash containing a known amount of MA. This VOMA model is useful for determining the lower limits necessary to produce neurotoxicity in the short-term and long-term as it progresses over time. We show that mice consumed on average 1.743 mg/kg bw/hour during 3 hours, and an average of 5.23 mg/kg bw/day over 28 consecutive days on a VOMA schedule. Since this consumption rate is much lower than the neurotoxic doses typically injected, we assessed the effects of long-term chronic VOMA on both spatial memory performance and on the levels of neurotoxicity in the hippocampus. Following 28 days of VOMA, mice exhibited a significant deficit in short-term spatial working memory and spatial reference learning on the radial 8-arm maze (RAM) compared to controls. This was accompanied by a significant decrease in memory markers protein kinase Mzeta (PKMζ), calcium impermeable AMPA receptor subunit GluA2, and the post-synaptic density 95 (PSD-95) protein in the hippocampus. Compared to controls, the VOMA paradigm also induced decreases in hippocampal levels of dopamine transporter (DAT) and tyrosine hydroxylase (TH), as well as increases in dopamine 1 receptor (D1R), glial fibrillary acidic protein (GFAP) and cyclooxygenase-2 (COX-2), with a decrease in prostaglandins E2 (PGE2) and D2 (PGD2). These results demonstrate that chronic VOMA reaching 146 mg/kg bw/28d induces significant hippocampal neurotoxicity. Future studies will evaluate the progression of this neurotoxic state.

Estradiol rapidly increases GluA2-mushroom spines and decreases GluA2-filopodia spines in hippocampus CA1.

Hippocampal dendritic spine density rapidly increases following estradiol (E2 ) treatment, but the types of spines and trafficking of synaptic markers have received little investigation. We assessed rapid effects of E2 over time on the density of four spine types (stubby, filopodial, long thin, and mushroom) and trafficking of AMPA receptor subunit GluA2 and PSD95 on tertiary, apical dendrites in CA1. Castrated male rats received 20 µg kg-1 of E2 or vehicle and were sacrificed 30 or 120 min later. Images of Golgi-Cox impregnated and PSD95/GluA2 stained dendrites were captured under the confocal microscope and quantified with IMARIS-XT. Stubby and filopodial spine densities did not change following treatment. Long-thin spines significantly decreased at 30 min while mushroom spines significantly increased at 120 min. GluA2, PSD95, and GluA2/PSD95 colocalization levels in stubby or long thin spines did not change, but filopodial spines had significantly reduced GluA2 levels at 30 min. Mushroom spines showed significantly increased levels for GluA2, PSD95 and GluA2/PSD95 colocalization at 120 min. Because GluA2 is important for memory consolidation, current results present novel data suggesting that trafficking of GluA2 to mushroom spines provides one mechanism contributing to estradiol's ability to enhance learning and memory by the PI3 signaling pathway.

Social defeat stress induces depression-like behavior and alters spine morphology in the hippocampus of adolescent male C57BL/6 mice.

Social stress, including bullying during adolescence, is a risk factor for common psychopathologies such as depression. To investigate the neural mechanisms associated with juvenile social stress-induced mood-related endophenotypes, we examined the behavioral, morphological, and biochemical effects of the social defeat stress model of depression on hippocampal dendritic spines within the CA1 stratum radiatum. Adolescent (postnatal day 35) male C57BL/6 mice were subjected to defeat episodes for 10 consecutive days. Twenty-four h later, separate groups of mice were tested on the social interaction and tail suspension tests. Hippocampi were then dissected and Western blots were conducted to quantify protein levels for various markers important for synaptic plasticity including protein kinase M zeta (PKMζ), protein kinase C zeta (PKCζ), the dopamine-1 (D1) receptor, tyrosine hydroxylase (TH), and the dopamine transporter (DAT). Furthermore, we examined the presence of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor subunit GluA2 as well as colocalization with the post-synaptic density 95 (PSD95) protein, within different spine subtypes (filopodia, stubby, long-thin, mushroom) using an immunohistochemistry and Golgi-Cox staining technique. The results revealed that social defeat induced a depression-like behavioral profile, as inferred from decreased social interaction levels, increased immobility on the tail suspension test, and decreases in body weight. Whole hippocampal immunoblots revealed decreases in GluA2, with a concomitant increase in DAT and TH levels in the stressed group. Spine morphology analyses further showed that defeated mice displayed a significant decrease in stubby spines, and an increase in long-thin spines within the CA1 stratum radiatum. Further evaluation of GluA2/PSD95 containing-spines demonstrated a decrease of these markers within long-thin and mushroom spine types. Together, these results indicate that juvenile social stress induces GluA2- and dopamine-associated dysregulation in the hippocampus - a neurobiological mechanism potentially underlying the development of mood-related syndromes as a consequence of adolescent bullying.

Environmental Enrichment Increases Glucocorticoid Receptors and Decreases GluA2 and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism?

Environmental enrichment (EE) housing paradigms have long been shown beneficial for brain function involving neural growth and activity, learning and memory capacity, and for developing stress resiliency. The expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2, which is important for synaptic plasticity and memory, is increased with corticosterone (CORT), undermining synaptic plasticity and memory. Thus, we determined the effect of EE and stress on modulating GluA2 expression in Sprague-Dawley male rats. Several markers were evaluated which include: plasma CORT, the glucocorticoid receptor (GR), GluA2, and the atypical protein kinase M zeta (PKMζ). For 1 week standard-(ST) or EE-housed animals were treated with one of the following four conditions: (1) no stress; (2) acute stress (forced swim test, FST; on day 7); (3) chronic restraint stress (6 h/day for 7 days); and (4) chronic + acute stress (restraint stress 6 h/day for 7 days + FST on day 7). Hippocampi were collected on day 7. Our results show that EE animals had reduced time immobile on the FST across all conditions. After chronic + acute stress EE animals showed increased GR levels with no change in synaptic GluA2/PKMζ. ST-housed animals showed the reverse pattern with decreased GR levels and a significant increase in synaptic GluA2/PKMζ. These results suggest that EE produces an adaptive response to chronic stress allowing for increased GR levels, which lowers neuronal excitability reducing GluA2/PKMζ trafficking. We discuss this EE adaptive response to stress as a potential underlying mechanism that is protective for retaining synaptic plasticity and memory function.