Elizabeth J. Crofton

Inoculation stress hypothesis of environmental enrichment

One hallmark of psychiatric conditions is the vast continuum of individual differences in susceptibility vs. resilience resulting from the interaction of genetic and environmental factors. The environmental enrichment paradigm is an animal model that is useful for studying a range of psychiatric conditions, including protective phenotypes in addiction and depression models. The major question is how environmental enrichment, a non-drug and non-surgical manipulation, can produce such robust individual differences in such a wide range of behaviors. This paper draws from a variety of published sources to outline a coherent hypothesis of inoculation stress as a factor producing the protective enrichment phenotypes. The basic tenet suggests that chronic mild stress from living in a complex environment and interacting non-aggressively with conspecifics can inoculate enriched rats against subsequent stressors and/or drugs of abuse. This paper reviews the enrichment phenotypes, mulls the fundamental nature of environmental enrichment vs. isolation, discusses the most appropriate control for environmental enrichment, and challenges the idea that cortisol/corticosterone equals stress. The intent of the inoculation stress hypothesis of environmental enrichment is to provide a scaffold with which to build testable hypotheses for the elucidation of the molecular mechanisms underlying these protective phenotypes and thus provide new therapeutic targets to treat psychiatric/neurological conditions.

Overexpression of DeltaFosB in nucleus accumbens mimics the protective addiction phenotype, but not the protective depression phenotype of environmental enrichment

Environmental enrichment produces protective addiction and depression phenotypes in rats. ΔFosB is a transcription factor that regulates reward in the brain and is induced by psychological stress as well as drugs of abuse. However, the role played by ΔFosB in the protective phenotypes of environmental enrichment has not been well studied. Here, we demonstrate that ΔFosB is differentially regulated in rats reared in an isolated condition (IC) compared to those in an enriched condition (EC) in response to restraint stress or cocaine. Chronic stress or chronic cocaine treatment each elevates ΔFosB protein levels in the nucleus accumbens (NAc) of IC rats, but not of EC rats due to an already elevated basal accumulation of ΔFosB seen under EC conditions. Viral-mediated overexpression of ΔFosB in the NAc shell of pair-housed rats (i.e., independent of environmental enrichment/isolation) increases operant responding for sucrose when motivated by hunger, but decreases responding in satiated animals. Moreover, ΔFosB overexpression decreases cocaine self-administration, enhances extinction of cocaine seeking, and decreases cocaine-induced reinstatement of intravenous cocaine self-administration; all behavioral findings consistent with the enrichment phenotype. In contrast, however, ΔFosB overexpression did not alter responses of pair-housed rats in several tests of anxiety- and depression-related behavior. Thus, ΔFosB in the NAc the shell mimics the protective addiction phenotype, but not the protective depression phenotype of environmental enrichment.

Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

&NA; Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico‐limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction‐related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen‐synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno‐associated viral vector (AAV2) and assessed changes in anxiety‐ and depression‐like behavior and cocaine self‐administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety‐like behavior while increasing depression‐like behavior and cocaine self‐administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV‐shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward‐related behaviors. Loose‐patch recordings showed that TANs transduced by AAV‐shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole‐cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization‐induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression‐ and addiction‐related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub‐types. HighlightsSpecific knockdown of GSK3 beta in the NAc shell induces an anxiolytic‐like effect.Knockdown of GSK3 beta in the NAcSh induces depression‐like behavior.Viral‐mediated knockdown of GSK3 beta increases cocaine self‐administration.Knockdown also reduces spontaneous firing and alters intrinsic excitability of TANs.

Transcriptomics of Environmental Enrichment Reveals a Role for Retinoic Acid Signaling in Addiction

There exists much variability in susceptibility/resilience to addiction in humans. The environmental enrichment paradigm is a rat model of resilience to addiction-like behavior, and understanding the molecular mechanisms underlying this protective phenotype may lead to novel targets for pharmacotherapeutics to treat cocaine addiction. We investigated the differential regulation of transcript levels using RNA sequencing of the rat nucleus accumbens after environmental enrichment/isolation and cocaine/saline self-administration. Ingenuity Pathways Analysis and Gene Set Enrichment Analysis of 14,309 transcripts demonstrated that many biofunctions and pathways were differentially regulated. New functional pathways were also identified for cocaine modulation (e.g., Rho GTPase signaling) and environmental enrichment (e.g., signaling of EIF2, mTOR, ephrin). However, one novel pathway stood out above the others, the retinoic acid (RA) signaling pathway. The RA signaling pathway was identified as one likely mediator of the protective enrichment addiction phenotype, an interesting result given that nine RA signaling-related genes are expressed selectively and at high levels in the nucleus accumbens shell (NAcSh). Subsequent knockdown of Cyp26b1 (an RA degradation enzyme) in the NAcSh of rats confirmed this role by increasing cocaine self-administration as well as cocaine seeking. These results provide a comprehensive account of enrichment effects on the transcriptome and identify RA signaling as a contributing factor for cocaine addiction.

Convergent transcriptomics and proteomics of environmental enrichment and cocaine identifies novel therapeutic strategies for addiction.

Transcriptomic and proteomic approaches have separately proven effective at identifying novel mechanisms affecting addiction-related behavior; however, it is difficult to prioritize the many promising leads from each approach. A convergent secondary analysis of proteomic and transcriptomic results can glean additional information to help prioritize promising leads. The current study is a secondary analysis of the convergence of recently published separate transcriptomic and proteomic analyses of nucleus accumbens (NAc) tissue from rats subjected to environmental enrichment vs. isolation and cocaine self-administration vs. saline. Multiple bioinformatics approaches (e.g. Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA)) were used to interrogate these rich data sets. Although there was little correspondence between mRNA vs. protein at the individual target level, good correspondence was found at the level of gene/protein sets, particularly for the environmental enrichment manipulation. These data identify gene sets where there is a positive relationship between changes in mRNA and protein (e.g. glycolysis, ATP synthesis, translation elongation factor activity, etc.) and gene sets where there is an inverse relationship (e.g. ribosomes, Rho GTPase signaling, protein ubiquitination, etc.). Overall environmental enrichment produced better correspondence than cocaine self-administration. The individual targets contributing to mRNA and protein effects were largely not overlapping. As a whole, these results confirm that robust transcriptomic and proteomic data sets can provide similar results at the gene/protein set level even when there is little correspondence at the individual target level and little overlap in the targets contributing to the effects.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway

SUMMARY Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3β (GSK3β) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions—models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3β and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3β and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3β prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3β with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3β. A GSK3β-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3β regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

The Proteomics of Cocaine in the Nucleus Accumbens

Cocaine addiction is a major health issue with no FDA-approved medication, so novel therapeutic target identification is necessary. Recent technological advances provide novel target identification using large-scale discovery-based quantification of the proteome in response to cocaine. This approach improves upon historically utilized methods as proteins are quantified regardless of whether the protein is regulated by the manipulation. Additionally, it avoids experimenter biases and assumptions and reduces labor and cost requirements. In this chapter we discuss proteome-wide effects of cocaine in the nucleus accumbens in rats that self-administered cocaine or saline, a paradigm with excellent reproducibility and strong validity as a model of drug-taking and -seeking behavior. Also discussed are the proteomic effects of environmental enrichment, which provides a unique opportunity for novel target identification as enrichment produces a robust protective behavioral addiction phenotype and therefore a strong potential for identification of novel targets with protective effects.