Targeting AMPA Receptor Modulation during Early Life Adversity: A Mediator for Threat Associated Memories

Early life adversity (ELA) is the exposure to a single or to multiple traumatic events before the age of 18 that go beyond the child’s coping. These adverse events are often exacerbated during adolescence particularly when cognitive performance is compromised. Adolescents who experienced ELA may show symptoms of post-traumatic stress disorder (PTSD), while not vividly recalling the early life trauma. These individuals show atypical connectivity between prefrontal-amygdala and hippocampus, all of which is associated with an increased risk of experiencing a traumatic event again later in life. While clinical research has increasingly stressed the importance in addressing the long-lasting consequences of ELA, treatment availability for ELA is low. Yet, animal models of the threat response during development have given clues into ways in which early adverse experiences transition later in life. Our previous research has provided behavioral evidence that there are differences in infant and juvenile threat processing, reflecting age-specific wiring in the brain. However, research still lacks how molecular markers important for synaptic plasticity and memory are modulated across the development after threat exposure. The overall GOAL of these studies was in identifying the role of AMPAr on transitional threat memory processing at three ages of infancy, and the juvenile stage, to understand how the brain at different stages of life responds and processes threat. Adult rats exposed to threat exhibit impaired long-term memory retrieval for tasks learned prior to threat exposure. AMPAr and PKMz expression, markers important for long-term memory processing, are also dysregulated in acute and chronically threat exposed adults, suggesting threat memories may override or conflict with previously established memories. While previous research has provided evidence that synaptic plasticity is dysregulated in the adult brain after threat exposure, research still lacks on how molecular markers important for synaptic plasticity and memory are modulated across development after threat exposure. Thus, the studies presented in this dissertation identified the role of threat associated AMPAr on developmental synaptic plasticity by 1. Utilization of age-specific threat conditioning paradigms 2. Monitoring biochemical changes 3. Examining the influence of stress hormones. These experiments focused on identifying AMPAr as a mediator for early life adversity associated memories. To achieve this goal, two specific aims were carried out: Specific Aim 1: Identify the role of AMPA receptor expression in the hippocampus and amygdala in threat memory retrieval during the juvenile or adult stage of life [Chapter 2] Previous research on ELA has focused primarily on the alterations of the developing amygdala-prefrontal circuitry, however, the hippocampus is also an important region in the threat circuit, where the contextual aspect of the threat memory is stored. While the amygdala has been shown involve in threat memories during the juvenile stage starting at PN17, the hippocampus is still a region yet to reach full maturation. Rats at this stage will demonstrate a threat response 24 hours later when tested in the initial training context, but; will not retain this threat memory as early as 4 days after the initial exposure. However, juvenile rats exposed to the initial context at 3 days and then 6 days’ post-threat retain a threat response, suggesting consistent reconsolidation of memories in juveniles is needed for the sustainment of threat memories. The need for consistent reconsolidation of memories is a developmental feature not present in the adult rodent brain. Studies focusing on the natural phenomena, infantile amnesia, the inability to maintain memories during infancy and early childhood, have considered possible reasons for forgetting and recall failure seen during this stage of development. Previous research shows that while adults and juveniles both have developed projections between the basolateral amygdala (BLA) and hippocampus, juveniles do not show activation of BLA to hippocampus projections during threat conditioning and extinction. This difference projection strength suggests the hippocampus and its projections to regions like the prefrontal cortex and amygdala are immature, making these immature projections a potential driving force behind increased forgetting in juveniles. To identify the role of the AMPA receptor associated with threat memory retrieval in juvenile rats, we focus on both the amygdala and hippocampus in understanding how neural mechanisms important for threat memory processing differentiate between juveniles and adults. Groups of male juvenile and adult rats were exposed to the pedestal stress paradigm and tested for contextual threat memory retrieval either 1d or 7d later. Rats were sacrificed 30 minutes after test and the dorsal hippocampus and amygdala were evaluated for GluA1-3, PKMz and PSD95. We found that: (1) As expected, both juvenile and adults have intact threat memory retrieval 24 hours post-training. However, at 7 days post-training only adults exhibit a sustained threat memory (2) GluA1-2 AMPAr subunits increase at 1d post-training in the hippocampus of juvenile rats and phosphorylated Serine 845 GluA1 AMPAr subunit increases 7d post-training in the hippocampus of juvenile rats (3) PSD-95 and phosphorylated Serine295 PSD-95 increase only 1d post-training in the amygdala and 7d post-training in the hippocampus of only adult rats Overall, these results indicate long-term dysregulation in the juvenile brain associated with adversity induced GluA1-AMPAr subunit expression. Additionally, the increase in PSD-95 and its phosphorylated state observed only in adults, highlights the lack of mature dendritic spines and increased neurogenesis in juveniles seen in previous literature on infantile amnesia and ELA. Specific Aim 2: Identify the role of AMPA receptor expression in the amygdala in threat memory retrieval during infancy [Chapter 3] Adolescents who have experienced maltreatment early in life respond faster to fearful facial expressions and show an overgeneralized threat response. This rapid response to these fearful facial expressions was also associated with activation of the prefrontal cortex, suggesting dysregulated development of the amygdala-prefrontal circuitry. Furthermore, longitudinal fMRI studies on adolescent individuals who experience ELA show atypical connectivity between the prefrontal cortex to the amygdala and prefrontal cortex to hippocampus. This pattern suggests that trauma has long-term consequences resulting in atypical development of brain regions important for short and long-term memory storage as well as emotional memory storage. In rodents, altered connectivity between these brain regions is observed in models of maltreatment during infancy, resulting in depressive and anxiety like symptoms, and deficits in social behavior later on in adolescence. Furthermore, infant rats maltreated by the mother show increased levels of the stress hormone, corticosterone (CORT), suggesting CORT may play a role in the altered threat circuitry shown later in adolescent rats. Increased CORT levels in the amygdala during infancy prematurely activates the amygdala threat circuitry, a function that usually does not emerge until PN10. With previous literature highlighting the effects of trauma on brain regions important for not only threat memory processing but also non-threat memory processing, suggesting that altered and atypical development in these brain regions may also serve as a driving force for the various impairments seen in trauma exposed adolescent youths

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

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 (PKMz). For one 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); (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/PKMz. ST-housed animals showed the reverse pattern with decreased GR levels and a significant increase in synaptic GluA2/PKM. These results suggest that EE produces an adaptive response to chronic stress allowing for increased GR levels, which lowers neuronal excitability reducing GluA2/PKMz trafficking. We discuss this EE adaptive response to stress as a potential underlying mechanism that is protective for retaining synaptic plasticity and memory function

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

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

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-4isoxazolepropionic acidreceptor (AMPAr)subunits aretraffickedinto the synaptic membranefollowing 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 (1h) or no stress control (home cage). Each animal was placed alone in an open field for 5minat 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 homecage following initial exploration. Each animal was returned to the open field for 5min 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

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 longterm 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 postsynaptic 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

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

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

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