Ashley M. Kopec

MAPK establishes a molecular context that defines effective training patterns for long-term memory formation.

Although the importance of spaced training trials in the formation of long-term memory (LTM) is widely appreciated, surprisingly little is known about the molecular mechanisms that support interactions between individual trials. The intertrial dynamics of ERK/MAPK activation have recently been correlated with effective training patterns for LTM. However, whether and how MAPK is required to mediate intertrial interactions remains unknown. Using a novel two-trial training pattern which induces LTM in Aplysia, we show that the first of two training trials recruits delayed protein synthesis-dependent nuclear MAPK activity that establishes a unique molecular context involving the recruitment of CREB kinase and ApC/EBP and is an essential intertrial signaling mechanism for LTM induction. These findings provide the first demonstration of a requirement for MAPK in the intertrial interactions during memory formation and suggest that the kinetics of MAPK activation following individual experiences determines effective training intervals for LTM formation.

Pattern and predictability in memory formation: From molecular mechanisms to clinical relevance

Most long-term memories are formed as a consequence of multiple experiences. The temporal spacing of these experiences is of considerable importance: experiences distributed over time (spaced training) are more easily encoded and remembered than either closely spaced experiences, or a single prolonged experience (massed training). In this article, we first review findings from studies in animal model systems that examine the cellular and molecular properties of the neurons and circuits in the brain that underlie training pattern sensitivity during long-term memory (LTM) formation. We next focus on recent findings which have begun to elucidate the mechanisms that support inter-trial interactions during the induction of LTM. Finally, we consider the implications of these findings for developing therapeutic strategies to address questions of direct clinical relevance.

Growth Factor Signaling and Memory Formation: Temporal and Spatial Integration of a Molecular Network.

Growth factor (GF) signaling is critically important for developmental plasticity. It also plays a crucial role in adult plasticity, such as that required for memory formation. Although different GFs interact with receptors containing distinct types of kinase domains, they typically signal through converging intracellular cascades (e.g., Ras-MEK-MAPK) to mediate overlapping functional endpoints. Several GFs have been implicated in memory formation, but due to a high level of convergent signaling, the unique contributions of individual GFs as well as the interactions between GF signaling cascades during the induction of memory is not well known. In this review, we highlight the unique roles of specific GFs in dendritic plasticity, and discuss the spatial and temporal profiles of different GFs during memory formation. Collectively, the data suggest that the roles of GF signaling in long-lasting behavioral and structural plasticity may be best viewed as interactive components in a complex molecular network.

A Novel Cysteine-Rich Neurotrophic Factor in "Aplysia" Facilitates Growth, MAPK Activation, and Long-Term Synaptic Facilitation.

Neurotrophins are critically involved in developmental processes such as neuronal cell survival, growth, and differentiation, as well as in adult synaptic plasticity contributing to learning and memory. Our previous studies examining neurotrophins and memory formation in Aplysia showed that a TrkB ligand is required for MAPK activation, long-term synaptic facilitation (LTF), and long-term memory (LTM) for sensitization. These studies indicate that neurotrophin-like molecules in Aplysia can act as key elements in a functionally conserved TrkB signaling pathway. Here we report that we have cloned and characterized a novel neurotrophic factor, Aplysia cysteine-rich neurotrophic factor (apCRNF), which shares classical structural and functional characteristics with mammalian neurotrophins. We show that apCRNF (1) is highly enriched in the CNS, (2) enhances neurite elongation and branching, (3) interacts with mammalian TrkB and p75(NTR), (4) is released from Aplysia CNS in an activity-dependent fashion, (5) facilitates MAPK activation in a tyrosine kinase dependent manner in response to sensitizing stimuli, and (6) facilitates the induction of LTF. These results show that apCRNF is a native neurotrophic factor in Aplysia that can engage the molecular and synaptic mechanisms underlying memory formation.

Opioid Self-Administration is Attenuated by Early-Life Experience and Gene Therapy for Anti-Inflammatory IL-10 in the Nucleus Accumbens of Male Rats

Early-life conditions can contribute to the propensity for developing neuropsychiatric disease, including substance abuse disorders. However, the long-lasting mechanisms that shape risk or resilience for drug addiction remain unclear. Previous work has shown that a neonatal handling procedure in rats (which promotes enriched maternal care) attenuates morphine conditioning, reduces morphine-induced glial activation, and increases microglial expression of the anti-inflammatory cytokine interleukin-10 (IL-10). We thus hypothesized that anti-inflammatory signaling may underlie the effects of early-life experience on later-life opioid drug-taking. Here we demonstrate that neonatal handling attenuates intravenous self-administration of the opioid remifentanil in a drug-concentration-dependent manner. Transcriptional profiling of the nucleus accumbens (NAc) from handled rats following repeated exposure to remifentanil reveals a suppression of pro-inflammatory cytokine and chemokine gene expression, consistent with an anti-inflammatory phenotype. To determine if anti-inflammatory signaling alters drug-taking behavior, we administered intracranial injections of plasmid DNA encoding IL-10 (pDNA-IL-10) into the NAc of non-handled rats. We discovered that pDNA-IL-10 treatment reduces remifentanil self-administration in a drug-concentration-dependent manner, similar to the effect of handling. In contrast, neither handling nor pDNA-IL-10 treatment alters self-administration of food or sucrose rewards. These collective observations suggest that neuroimmune signaling mechanisms in the NAc are shaped by early-life experience and may modify motivated behaviors for opioid drugs. Moreover, manipulation of the IL-10 signaling pathway represents a novel approach for influencing opioid reinforcement.

Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

BACKGROUND Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets. NEW METHOD We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer. RESULTS Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples. COMPARISON WITH EXISTING METHOD(S) Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation. CONCLUSIONS This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects.

Gut-immune-brain dysfunction in Autism: Importance of sex

Autism Spectrum Disorder (ASD) is characterized by social behavior deficits, stereotypies, cognitive rigidity, and in some cases severe intellectual impairment and developmental delay. Although ASD is most widely identified by its neurological deficits, gastrointestinal issues are common in ASD. An intimate and complex relationship exists between the gut, the immune system, and the brain, leading to the hypothesis that ASD may be a systems-level disease affecting the gut and immune systems, in addition to the brain. Despite significant advances in understanding the contribution of the gut and immune systems to the etiology of ASD, there is an intriguing commonality among patients that is not well understood: they are predominantly male. Virtually no attention has been given to the potential role of sex-specific regulation of gut, peripheral, and central immune function in ASD, despite the 4:1 male-to-female bias in this disorder. In this review, we discuss recent revelations regarding the impact of gut-immune-brain relationships on social behavior in rodent models and in ASD patients, placing them in the context of known or putative sex specific mechanisms.

Microglial elimination of dopamine D1 receptors defines sex-specific changes in nucleus accumbens development and social play behavior during adolescence

Adolescence is a developmental period in which the mesolimbic dopaminergic ‘reward’ circuitry of the brain, including the nucleus accumbens (NAc), undergoes significant developmental plasticity and neural circuit maturation. Dopamine D1 receptors (D1rs) in the NAc have recently been demonstrated to be critical modulators of social behavior, but how these receptors are regulated in adolescence to mediate social behavior is not well understood. In this report, we used multi-plexed immunohistochemistry with volumetric reconstructions, co-immunoprecipitation, ex vivo, and in vivo stereotaxic, microglial manipulation, and social behavior assessment to demonstrate that microglia and complement-mediated phagocytic activity shapes sex-specific NAc development. Moreover, we report for the first time that microglia-mediated phagocytosis is required for natural developmental changes in behavior, specifically, adolescent male social play behavior. These data have broad implications for understanding how experience interacts with the developing reward circuity, sex-specific responses to stimuli in adolescence, and how neuropsychiatric disorders may arise in a sexually dimorphic manner.

The Contribution of Spatial and Temporal Molecular Networks in the Induction of Long-term Memory and Its Underlying Synaptic Plasticity.

The ability to form long-lasting memories is critical to survival and thus is highly conserved across the animal kingdom. By virtue of its complexity, this same ability is vulnerable to disruption by a wide variety of neuronal traumas and pathologies. To identify effective therapies with which to treat memory disorders, it is critical to have a clear understanding of the cellular and molecular mechanisms which subserve normal learning and memory. A significant challenge to achieving this level of understanding is posed by the wide range of distinct temporal and spatial profiles of molecular signaling induced by learning-related stimuli. In this review we propose that a useful framework within which to address this challenge is to view the molecular foundation of long-lasting plasticity as composed of unique spatial and temporal molecular networks that mediate signaling both within neurons (such as via kinase signaling) as well as between neurons (such as via growth factor signaling). We propose that evaluating how cells integrate and interpret these concurrent and interacting molecular networks has the potential to significantly advance our understanding of the mechanisms underlying learning and memory formation.

Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

Adolescence is a developmental period in which the mesolimbic dopaminergic “reward” circuitry of the brain, including the nucleus accumbens (NAc), undergoes significant plasticity. Dopamine D1 receptors (D1rs) in the NAc are critical for social behavior, but how these receptors are regulated during adolescence is not well understood. In this report, we demonstrate that microglia and complement-mediated phagocytic activity shapes NAc development by eliminating D1rs in male, but not female rats, during adolescence. Moreover, immune-mediated elimination of D1rs is required for natural developmental changes in male social play behavior. These data demonstrate for the first time that microglia and complement-mediated immune signaling (i) participate in adolescent brain development in a sex-specific manner, and (ii) are causally implicated in developmental changes in behavior. These data have broad implications for understanding the adolescent critical period of development, the molecular mechanisms underlying social behavior, and sex differences in brain structure and function.Dopaminergic circuits in the nucleus accumbens regulate reward, including social play behavior in adolescent rodents. Here, the authors show that in male but not female rats, dopamine receptors are tagged by complement for microglial phagocytosis, thus mediating changes in social behavior.