Deveroux Ferguson

Interests

Research

Neuro-degenerative, developmental and psychiatric disease; Developmental, cell and molecular biology; Gene environment interactions and epigenetics

Courses

Scholarly Contributions

Journals/Publications

• Kim, H., Wei, J., Call, T., Ma, X., Quintus, N., Summers, A., Carotenuto, S., Johnson, R., Nguyen, A., Cui, Y., Park, J., Qiu, S., & Ferguson, D. (2024). SIRT1 Coordinates Transcriptional Regulation of Neural Activity and Modulates Depression-Like Behaviors in the Nucleus Accumbens. Biological Psychiatry, 96(6). doi:10.1016/j.biopsych.2024.03.017
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  Background: Major depression and anxiety disorders are significant causes of disability and socioeconomic burden. Despite the prevalence and considerable impact of these affective disorders, their pathophysiology remains elusive. Thus, there is an urgent need to develop novel therapeutics for these conditions. We evaluated the role of SIRT1 in regulating dysfunctional processes of reward by using chronic social defeat stress to induce depression- and anxiety-like behaviors. Chronic social defeat stress induces physiological and behavioral changes that recapitulate depression-like symptomatology and alters gene expression programs in the nucleus accumbens, but cell type–specific changes in this critical structure remain largely unknown. Methods: We examined transcriptional profiles of D1-expressing medium spiny neurons (MSNs) lacking deacetylase activity of SIRT1 by RNA sequencing in a cell type–specific manner using the RiboTag line of mice. We analyzed differentially expressed genes using gene ontology tools including SynGO and EnrichR and further demonstrated functional changes in D1-MSN–specific SIRT1 knockout (KO) mice using electrophysiological and behavioral measurements. Results: RNA sequencing revealed altered transcriptional profiles of D1-MSNs lacking functional SIRT1 and showed specific changes in synaptic genes including glutamatergic and GABAergic (gamma-aminobutyric acidergic) receptors in D1-MSNs. These molecular changes may be associated with decreased excitatory and increased inhibitory neural activity in Sirt1 KO D1-MSNs, accompanied by morphological changes. Moreover, the D1-MSN–specific Sirt1 KO mice exhibited proresilient changes in anxiety- and depression-like behaviors. Conclusions: SIRT1 coordinates excitatory and inhibitory synaptic genes to regulate the GABAergic output tone of D1-MSNs. These findings reveal a novel signaling pathway that has potential for the development of innovative treatments for affective disorders.
• Ma, X., Chen, P., Wei, J., Zhang, J., Chen, C., Zhao, H., Ferguson, D., McGee, A., Dai, Z., & Qiu, S. (2024). Protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections. STAR Protocols, 5(4). doi:10.1016/j.xpro.2024.103420
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  Here, we present a protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections. We describe steps for intracardiac perfusion, cryosectioning, and floating section mounting of brain sections, which enable runs on the Xenium analyzer and data delivery. We demonstrate that, in addition to the 10× Genomics-validated formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections, fixed frozen thin brain sections are compatible with the Xenium platform and provide excellent imaging and quantification results for spatially resolved gene expression. For complete details on the use and execution of this protocol, please refer to Ma et al.1
• Chen, C., Wei, J., Ma, X., Xia, B., Shakir, N., Zhang, J., Zhang, L., Cui, Y., Ferguson, D., Qiu, S., & Bai, F. (2023). Disrupted Maturation of Prefrontal Layer 5 Neuronal Circuits in an Alzheimer’s Mouse Model of Amyloid Deposition. Neuroscience Bulletin, 39(6). doi:10.1007/s12264-022-00951-5
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  Mutations in genes encoding amyloid precursor protein (APP) and presenilins (PSs) cause familial forms of Alzheimer’s disease (AD), a neurodegenerative disorder strongly associated with aging. It is currently unknown whether and how AD risks affect early brain development, and to what extent subtle synaptic pathology may occur prior to overt hallmark AD pathology. Transgenic mutant APP/PS1 over-expression mouse lines are key tools for studying the molecular mechanisms of AD pathogenesis. Among these lines, the 5XFAD mice rapidly develop key features of AD pathology and have proven utility in studying amyloid plaque formation and amyloid β (Aβ)-induced neurodegeneration. We reasoned that transgenic mutant APP/PS1 over-expression in 5XFAD mice may lead to neurodevelopmental defects in early cortical neurons, and performed detailed synaptic physiological characterization of layer 5 (L5) neurons from the prefrontal cortex (PFC) of 5XFAD and wild-type littermate controls. L5 PFC neurons from 5XFAD mice show early APP/Aβ immunolabeling. Whole-cell patch-clamp recording at an early post-weaning age (P22–30) revealed functional impairments; although 5XFAD PFC-L5 neurons exhibited similar membrane properties, they were intrinsically less excitable. In addition, these neurons received smaller amplitude and frequency of miniature excitatory synaptic inputs. These functional disturbances were further corroborated by decreased dendritic spine density and spine head volumes that indicated impaired synapse maturation. Slice biotinylation followed by Western blot analysis of PFC-L5 tissue revealed that 5XFAD mice showed reduced synaptic AMPA receptor subunit GluA1 and decreased synaptic NMDA receptor subunit GluN2A. Consistent with this, patch-clamp recording of the evoked L23>L5 synaptic responses revealed a reduced AMPA/NMDA receptor current ratio, and an increased level of AMPAR-lacking silent synapses. These results suggest that transgenic mutant forms of APP/PS1 overexpression in 5XFAD mice leads to early developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.
• Ferguson, D., Qiu, S., Bai, F., Cui, Y., Nehme, A., Zhang, L., Zhang, J. K., Shakir, N., Wei, J., Ma, X., & Chen, C. (2022). Early impairment of cortical circuit plasticity and connectivity in the 5XFAD Alzheimer’s disease mouse model. Translational Psychiatry. doi:10.1038/s41398-022-02132-4
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  Abstract Genetic risk factors for neurodegenerative disorders, such as Alzheimer’s disease (AD), are expressed throughout the life span. How these risk factors affect early brain development and function remain largely unclear. Analysis of animal models with high constructive validity for AD, such as the 5xFAD mouse model, may provide insights on potential early neurodevelopmental effects that impinge on adult brain function and age-dependent degeneration. The 5XFAD mouse model over-expresses human amyloid precursor protein (APP) and presenilin 1 (PS1) harboring five familial AD mutations. It is unclear how the expression of these mutant proteins affects early developing brain circuits. We found that the prefrontal cortex (PFC) layer 5 (L5) neurons in 5XFAD mice exhibit transgenic APP overloading at an early post-weaning age. Impaired synaptic plasticity (long-term potentiation, LTP) was seen at 6–8 weeks age in L5 PFC circuit, which was correlated with increased intracellular APP. APP overloading was also seen in L5 pyramidal neurons in the primary visual cortex (V1) during the critical period of plasticity (4–5 weeks age). Whole-cell patch clamp recording in V1 brain slices revealed reduced intrinsic excitability of L5 neurons in 5XFAD mice, along with decreased spontaneous miniature excitatory and inhibitory inputs. Functional circuit mapping using laser scanning photostimulation (LSPS) combined with glutamate uncaging uncovered reduced excitatory synaptic connectivity onto L5 neurons in V1, and a more pronounced reduction in inhibitory connectivity, indicative of altered excitation and inhibition during VC critical period. Lastly, in vivo single-unit recording in V1 confirmed that monocular visual deprivation-induced ocular dominance plasticity during critical period was impaired in 5XFAD mice. Our study reveals plasticity deficits across multiple cortical regions and indicates altered early cortical circuit developmental trajectory as a result of mutant APP/PS1 over-expression.
• Ferguson, D., Qiu, S., Levitt, P., Zuo, Y., Nehme, A., Zhang, L., Xia, B., Cui, Y., Wei, J., & Ma, X. (2022). Disrupted Timing of MET Signaling Derails the Developmental Maturation of Cortical Circuits and Leads to Altered Behavior in Mice. Cerebral Cortex, 32(8), 1769-1786. doi:10.1093/cercor/bhab323
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  The molecular regulation of the temporal dynamics of circuit maturation is a key contributor to the emergence of normal structure-function relations. Developmental control of cortical MET receptor tyrosine kinase, expressed early postnatally in subpopulations of excitatory neurons, has a pronounced impact on the timing of glutamatergic synapse maturation and critical period plasticity. Here, we show that using a controllable overexpression (cto-Met) transgenic mouse, extending the duration of MET signaling after endogenous Met is switched off leads to altered molecular constitution of synaptic proteins, persistent activation of small GTPases Cdc42 and Rac1, and sustained inhibitory phosphorylation of cofilin. These molecular changes are accompanied by an increase in the density of immature dendritic spines, impaired cortical circuit maturation of prefrontal cortex layer 5 projection neurons, and altered laminar excitatory connectivity. Two photon in vivo imaging of dendritic spines reveals that cto-Met enhances de novo spine formation while inhibiting spine elimination. Extending MET signaling for two weeks in developing cortical circuits leads to pronounced repetitive activity and impaired social interactions in adult mice. Collectively, our data revealed that temporally controlled MET signaling as a critical mechanism for controlling cortical circuit development and emergence of normal behavior.
• Ferguson, D., Chen, C., Wei, J., Ma, X., Zhang, L., Nehme, A., Cui, Y., Qiu, S., & Bai, F. (2021). Early Disruption of Synaptic Function, Impairment of Plasticity, and Decreased of Cortical Circuit Connectivity in an Alzheimer’S Mouse Model of Amyloid Deposition. Translational Neuroscience. doi:10.21203/rs.3.rs-948437/v1
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  Abstract Mutations in genes encoding amyloid precursor proteins and presenilins lead to increased β-amyloid (Aβ) production and cause familial Alzheimer’s disease (AD), a neurodegenerative disorder often associates with aging and features synapse loss and impaired synaptic plasticity. Aβ deposition is a pathological hallmark of AD. It is currently unknown whether and how AD risk alleles affects development of brain circuit function, and whether subtle synaptic pathology occurs prior to overt Aβ deposition. Transgenic mutated APP/PS1 over-expression mice lines are key tools to study molecular mechanisms of AD pathogenesis. Among these mice lines, the 5XFAD mice rapidly recapitulate key features of AD pathology, and have proven utility in studying amyloid plaque formation and Aβ-induced neurodegeneration. We reason that transgenic mutant APP/PS1 over-expression may lead to neurodevelopmental defects in early cortical neurons as a result of continuous APP/Aβ expression from early life. We first explored age-dependent plasticity changes in prefrontal cortical circuits in the 5XFAD mice, and found that at an early age (6-8 wks old) that does not produce overt impairment of hippocampus LTP, layer (L) 5 neuron circuit plasticity, measured by long term potentiation (LTP), is impaired. In addition, L5 neurons, which are reportedly vulnerable cortical neuron populations, show reduced mEPSC amplitude and frequency in early postweaning ages, indicating impaired synaptic transmission as a result of transgenic APP/Aβ overloading during early postnatal development. These functional changes were corroborated by the morphological findings of smaller, sparser dendritic spines on L5 pyramidal neurons, indicative of impaired prefrontal circuit function. Lastly, laser scanning photostimulation (LSPS) combined with glutamate uncaging revealed that L2/3 > L5 cortical connectivity was decreased at this early age. Our results suggest 5XFAD transgenic mutant APP/PS1 over expression causes developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.
• Kim, H. D., Wei, J., Call, T., Quintus, N. T., Summers, A. J., Carotenuto, S., Johnson, R., Ma, X., Xu, C., Park, J. G., Qiu, S., & Ferguson, D. (2021). Shisa6 mediates cell-type specific regulation of depression in the nucleus accumbens. Molecular psychiatry.
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  Depression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.
• Ma, X., Wei, J., Cui, Y., Xia, B., Zhang, L., Nehme, A., Zuo, Y., Ferguson, D., Levitt, P., & Qiu, S. (2021). Disrupted Timing of MET Signaling Derails the Developmental Maturation of Cortical Circuits and Leads to Altered Behavior in Mice. Cerebral cortex (New York, N.Y. : 1991).
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  The molecular regulation of the temporal dynamics of circuit maturation is a key contributor to the emergence of normal structure-function relations. Developmental control of cortical MET receptor tyrosine kinase, expressed early postnatally in subpopulations of excitatory neurons, has a pronounced impact on the timing of glutamatergic synapse maturation and critical period plasticity. Here, we show that using a controllable overexpression (cto-Met) transgenic mouse, extending the duration of MET signaling after endogenous Met is switched off leads to altered molecular constitution of synaptic proteins, persistent activation of small GTPases Cdc42 and Rac1, and sustained inhibitory phosphorylation of cofilin. These molecular changes are accompanied by an increase in the density of immature dendritic spines, impaired cortical circuit maturation of prefrontal cortex layer 5 projection neurons, and altered laminar excitatory connectivity. Two photon in vivo imaging of dendritic spines reveals that cto-Met enhances de novo spine formation while inhibiting spine elimination. Extending MET signaling for two weeks in developing cortical circuits leads to pronounced repetitive activity and impaired social interactions in adult mice. Collectively, our data revealed that temporally controlled MET signaling as a critical mechanism for controlling cortical circuit development and emergence of normal behavior.
• Xia, B., Wei, J., Ma, X., Nehme, A., Liong, K., Cui, Y., Chen, C., Gallitano, A., Ferguson, D., & Qiu, S. (2021). Conditional knockout of MET receptor tyrosine kinase in cortical excitatory neurons leads to enhanced learning and memory in young adult mice but early cognitive decline in older adult mice. Neurobiology of learning and memory, 179, 107397.
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  Human genetic studies established MET gene as a risk factor for autism spectrum disorders. We have previously shown that signaling mediated by MET receptor tyrosine kinase, expressed in early postnatal developing forebrain circuits, controls glutamatergic neuron morphological development, synapse maturation, and cortical critical period plasticity. Here we investigated how MET signaling affects synaptic plasticity, learning and memory behavior, and whether these effects are age-dependent. We found that in young adult (postnatal 2-3 months) Met conditional knockout (Met:emx1, cKO) mice, the hippocampus exhibits elevated plasticity, measured by increased magnitude of long-term potentiation (LTP) and depression (LTD) in hippocampal slices. Surprisingly, in older adult cKO mice (10-12 months), LTP and LTD magnitudes were diminished. We further conducted a battery of behavioral tests to assess learning and memory function in cKO mice and littermate controls. Consistent with age-dependent LTP/LTD findings, we observed enhanced spatial memory learning in 2-3 months old young adult mice, assessed by hippocampus-dependent Morris water maze test, but impaired spatial learning in 10-12 months mice. Contextual and cued learning were further assessed using a Pavlovian fear conditioning test, which also revealed enhanced associative fear acquisition and extinction in young adult mice, but impaired fear learning in older adult mice. Lastly, young cKO mice also exhibited enhanced motor learning. Our results suggest that a shift in the window of synaptic plasticity and an age-dependent early cognitive decline may be novel circuit pathophysiology for a well-established autism genetic risk factor.
• Chen, K., Ma, X., Nehme, A., Wei, J., Cui, Y., Cui, Y., Yao, D., Wu, J., Anderson, T., Ferguson, D., Levitt, P., & Qiu, S. (2020). Time-delimited signaling of MET receptor tyrosine kinase regulates cortical circuit development and critical period plasticity. Molecular psychiatry.
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  Normal development of cortical circuits, including experience-dependent cortical maturation and plasticity, requires precise temporal regulation of gene expression and molecular signaling. Such regulation, and the concomitant impact on plasticity and critical periods, is hypothesized to be disrupted in neurodevelopmental disorders. A protein that may serve such a function is the MET receptor tyrosine kinase, which is tightly regulated developmentally in rodents and primates, and exhibits reduced cortical expression in autism spectrum disorder and Rett Syndrome. We found that the peak of MET expression in developing mouse cortex coincides with the heightened period of synaptogenesis, but is precipitously downregulated prior to extensive synapse pruning and certain peak periods of cortical plasticity. These results reflect a potential on-off regulatory synaptic mechanism for specific glutamatergic cortical circuits in which MET is enriched. In order to address the functional significance of the 'off' component of the proposed mechanism, we created a controllable transgenic mouse line that sustains cortical MET signaling. Continued MET expression in cortical excitatory neurons disrupted synaptic protein profiles, altered neuronal morphology, and impaired visual cortex circuit maturation and connectivity. Remarkably, sustained MET signaling eliminates monocular deprivation-induced ocular dominance plasticity during the normal cortical critical period; while ablating MET signaling leads to early closure of critical period plasticity. The results demonstrate a novel mechanism in which temporal regulation of a pleiotropic signaling protein underlies cortical circuit maturation and timing of cortical critical period, features that may be disrupted in neurodevelopmental disorders.
• Ferguson, D. (2017). Cocaine Mediates the Cellular Mechanism of Satiation. Biological psychiatry, 81(7), e47-e48.
• Kim, H. D., Call, T., Carotenuto, S., Johnson, R., & Ferguson, D. (2017). Testing Depression in Mice: a Chronic Social Defeat Stress Model. Bio-protocol, 7(7), e2203.
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  A vast challenge within neuropsychiatric research has been the development of animal models that accurately reflect symptoms associated with affective disorders. An ethologically valid model that has been shown to be effective in studying depression is the chronic social defeat stress model. In this model, C57BL/6J mice are subjected to chronic social defeat stress induced by CD-1 aggressor mice for 10 consecutive days. Discussed here is a protocol describing the screening process of the CD-1 aggressor mice, the confrontations between the C57BL/6J and CD-1 aggressor mice, and analysis of social avoidance scores as an indication of depression-like behaviors.
• Kim, H. D., Call, T., Magazu, S., & Ferguson, D. (2017). Drug Addiction and Histone Code Alterations. Advances in experimental medicine and biology, 978, 127-143.
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  Acute and prolonged exposure to drugs of abuse induces changes in gene expression, synaptic function, and neural plasticity in brain regions involved in reward. Numerous genes are involved in this process, and persistent changes in gene expression coincide with epigenetic histone modifications and DNA methylation. Histone modifications are attractive regulatory mechanisms, which can encode complex environmental signals in the genome of postmitotic cells, like neurons. Recently, it has been demonstrated that specific histone modifications are involved in addiction-related gene regulatory mechanisms, by a diverse set of histone-modifying enzymes and readers. These histone modifiers and readers may prove to be valuable pharmacological targets for effective treatments for drug addiction.
• Kim, H. D., Hesterman, J., Call, T., Magazu, S., Keeley, E., Armenta, K., Kronman, H., Neve, R. L., Nestler, E. J., & Ferguson, D. (2016). SIRT1 Mediates Depression-Like Behaviors in the Nucleus Accumbens. The Journal of neuroscience : the official journal of the Society for Neuroscience, 36(32), 8441-52.
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  Depression is a recurring and life-threatening illness that affects up to 120 million people worldwide. In the present study, we show that chronic social defeat stress, an ethologically validated model of depression in mice, increases SIRT1 levels in the nucleus accumbens (NAc), a key brain reward region. Increases in SIRT1, a well characterized class III histone deacetylase, after chronic social defeat suggest a role for this enzyme in mediating depression-like behaviors. When resveratrol, a pharmacological activator of SIRT1, was directly infused bilaterally into the NAc, we observed an increase in depression- and anxiety-like behaviors. Conversely, intra-NAc infusions of EX-527, a SIRT1 antagonist, reduced these behaviors; EX-527 also reduced acute stress responses in stress-naive mice. Next, we increased SIRT1 levels directly in NAc by use of viral-mediated gene transfer and observed an increase in depressive- and anxiety-like behaviors when mice were assessed in the open-field, elevated-plus-maze, and forced swim tests. Using a Cre-inducible viral vector system to overexpress SIRT1 selectively in dopamine D1 or D2 subpopulations of medium spiny neurons (MSNs) in the NAc, we found that SIRT1 promotes depressive-like behaviors only when overexpressed in D1 MSNs, with no effect seen in D2 MSNs. Conversely, selective ablation of SIRT1 in the NAc using viral-Cre in floxed Sirt1 mice resulted in decreased depression- and anxiety-like behaviors. Together, these results demonstrate that SIRT1 plays an essential role in the NAc in regulating mood-related behavioral abnormalities and identifies a novel signaling pathway for the development of innovative antidepressants to treat major depressive disorders.
• Dias, C., Dietz, D., Mazei-Robison, M., Sun, H., Damez-Werno, D., Ferguson, D., Wilkinson, M., Magida, J., Gao, V., Neve, R., & Nestler, E. J. (2015). Dishevelled-2 regulates cocaine-induced structural plasticity and Rac1 activity in the nucleus accumbens. Neuroscience letters, 598, 23-8.
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  Chronic cocaine exposure increases the density of dendritic spines on medium spiny neurons (MSNs), the predominant neuronal cell type of the nucleus accumbens (NAc), a key brain reward region. We recently showed that suppression of Rac1, a small GTPase, is a critical mediator of this structural plasticity, but the upstream determinants of Rac1 activity in this context remain to be elucidated. In this study we examined whether isoforms of Dishevelled, a key hub protein of multiple branches of Wnt signaling, including Rac1, are regulated in the NAc by chronic cocaine, and whether these Dishevelled isoforms control Rac1 activity in this brain region in vivo. We found that chronic cocaine administration decreased expression of Dishevelled-2, and several other Wnt signaling components, in the NAc, and that overexpression of Dishevelled-2, but not Dishevelled-1, conversely upregulated Rac1 activity and prevented the cocaine induction of dendritic spines on NAc MSNs. We posit that the cocaine-induced downregulation of Dishevelled-2 in the NAc is an upstream regulator of Rac1 activity and plays an important role in the dynamic structural plasticity of NAc MSNs seen in response to chronic cocaine exposure.
• Feng, J., Shao, N., Shao, N., Szulwach, K. E., Szulwach, K., Vialou, V., Huynh, J., Zhong, C., Le, T., Le, T. T., Ferguson, D., Cahill, M. E., Cahill, M., Li, Y., Koo, J., Koo, J. W., Ribeiro, E., Labonte, B., Labonté, B., , Laitman, B. M., et al. (2015). Role of Tet1 and 5-hydroxymethylcytosine in cocaine action. Nature Neuroscience, 18(4). doi:10.1038/nn.3976
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  Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.
• Feng, J., Shao, N., Szulwach, K. E., Vialou, V., Huynh, J., Zhong, C., Le, T., Ferguson, D., Cahill, M. E., Li, Y., Koo, J. W., Ribeiro, E., Labonte, B., Laitman, B. M., Estey, D., Stockman, V., Kennedy, P., Couroussé, T., Mensah, I., , Turecki, G., et al. (2015). Role of Tet1 and 5-hydroxymethylcytosine in cocaine action. Nature neuroscience.
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  Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.
• Feng, J., Wilkinson, M., Liu, X., Purushothaman, I., Ferguson, D., Vialou, V., Maze, I., Shao, N., Kennedy, P., Koo, J., Dias, C., Laitman, B., Stockman, V., LaPlant, Q., Cahill, M. E., Nestler, E. J., & Shen, L. (2015). Erratum to: Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens. Genome biology, 16, 227.
• Feng, J., Wilkinson, M., Liu, X., Purushothaman, I., Ferguson, D., Vialou, V., Maze, I., Shao, N., Kennedy, P., Koo, J., Dias, C., Laitman, B., Stockman, V., LaPlant, Q., Cahill, M., Nestler, E., & Shen, L. (2015). Erratum to: Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens [Genome Biol, 15 (2014), R65]. Genome Biology, 16(1). doi:10.1186/s13059-015-0789-8
• Ferguson, D., Mazei-Robison, M. S., Nestler, E. J., Neve, R. L., Kennedy, P. J., Fallon, B., Kaska, S., & Heller, E. A. (2015). Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. Journal of Neurochemistry, 132(2), 243-253. doi:10.1111/jnc.12925
• Ferguson, D., Nestler, E. J., Zhang, S., Zhang, F., Knight, S. W., Pena, C. J., Sun, H., Cates, H. M., & Heller, E. A. (2015). Locus-specific epigenetic reprogramming for the study of reward pathology. Drug and Alcohol Dependence. doi:10.1016/j.drugalcdep.2014.09.297
• Ferguson, D., Shao, N., Heller, E., Feng, J., Neve, R., Kim, H. D., Call, T., Magazu, S., Shen, L., & Nestler, E. J. (2015). SIRT1-FOXO3a regulate cocaine actions in the nucleus accumbens. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(7), 3100-11.
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  Previous studies have shown that chronic cocaine administration induces SIRT1, a Class III histone deacetylase, in the nucleus accumbens (NAc), a key brain reward region, and that such induction influences the gene regulation and place conditioning effects of cocaine. To determine the mechanisms by which SIRT1 mediates cocaine-induced plasticity in NAc, we used chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 1 d after 7 daily cocaine (20 mg/kg) or saline injections, to map SIRT1 binding genome-wide in mouse NAc. Our unbiased results revealed two modes of SIRT1 action. First, despite its induction in NAc, chronic cocaine causes depletion of SIRT1 from most affected gene promoters in concert with enrichment of H4K16ac (itself a deacetylation target of SIRT1), which is associated with increased expression of these genes. Second, we deduced the forkhead transcription factor (FOXO) family to be a downstream mechanism through which SIRT1 regulates cocaine action. We proceeded to demonstrate that SIRT1 induction causes the deacetylation and activation of FOXO3a in NAc, which leads to the induction of several known FOXO3a gene targets in other systems. Finally, we directly establish a role for FOXO3a in promoting cocaine-elicited behavioral responses by use of viral-mediated gene transfer: we show that overexpressing FOXO3a in NAc enhances cocaine place conditioning. The discovery of these two actions of SIRT1 in NAc in the context of behavioral adaptations to cocaine represents an important step forward in advancing our understanding of the molecular adaptations underlying cocaine action.
• Ferguson, D., Shao, N., Heller, E., Feng, J., Neve, R., Kim, H., Call, T., Magazu, S., Shen, L., & Nestler, E. J. (2015). SIRT1-FOXO3a Regulate Cocaine Actions in the Nucleus Accumbens.. The Journal of Neuroscience.
• Heller, E., Kaska, S., Fallon, B., Ferguson, D., Kennedy, P., Neve, R., Nestler, E., & Mazei-Robison, M. (2015). Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. Journal of Neurochemistry, 132(2). doi:10.1111/jnc.12925
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  Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug-induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up-regulated by both drugs was serum- and glucocorticoid-inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N-myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug-related behaviors, we over-expressed constitutively active (CA) SGK1 in the VTA. SGK1-CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1-CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug-induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction.
• Koo, J. W., Mazei-Robison, M. S., LaPlant, Q., Egervari, G., Braunscheidel, K. M., Adank, D. N., Ferguson, D., Feng, J., Sun, H., Scobie, K. N., Damez-Werno, D. M., Ribeiro, E., Peña, C. J., Walker, D., Bagot, R. C., Cahill, M. E., Anderson, S. A., Labonté, B., Hodes, G. E., , Browne, H., et al. (2015). Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area. Nature neuroscience.
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  Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.
• Cates, H. M., Feng, J., Ferguson, D., Gerber, M. A., Golden, S. A., Han, M., Heller, E. A., Herman, J. P., Knight, S. W., Mazei-Robison, M. S., Nestler, E. J., Neve, R. L., Nievera, C., Pena, C. J., Russo, S. J., Shao, N., Shen, L., Sun, H., Tamminga, C. S., , Walsh, J. J., et al. (2014). Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors. PMC.
• Dias, C., Feng, J., Sun, H., Shao, N. Y., Mazei-Robison, M. S., Damez-Werno, D., Scobie, K., Bagot, R., LaBonté, B., Ribeiro, E., Liu, X., Kennedy, P., Vialou, V., Ferguson, D., Peña, C., Calipari, E. S., Koo, J. W., Mouzon, E., Ghose, S., , Tamminga, C., et al. (2014). β-catenin mediates stress resilience through Dicer1/microRNA regulation. Nature, 516(7529), 51-5.
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  β-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice β-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide β-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis--as a β-catenin target gene that mediates resilience. Small RNA profiling after excising β-catenin from nucleus accumbens in the context of chronic stress reveals β-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish β-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.
• Dias, C., Feng, J., Sun, H., Shao, N., Mazei-Robison, M., Damez-Werno, D., Scobie, K., Bagot, R., Ribeiro, E., Liu, X., Kennedy, P., Vialou, V., Ferguson, D., Calipari, E., Koo, J., Mouzon, E., Ghose, S., Tamminga, C., Neve, R., , Shen, L., et al. (2014). β-catenin mediates stress resilience through Dicer1/microRNA regulation. Nature, 516(729). doi:10.1038/nature13976
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  β-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice β-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide β-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis-as a β-catenin target gene that mediates resilience. Small RNA profiling after excising β-catenin from nucleus accumbens in the context of chronic stress reveals β-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish β-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.
• Feng, J., Wilkinson, M., Liu, X., Purushothaman, I., Ferguson, D., Vialou, V., Maze, I., Shao, N., Kennedy, P., Koo, J., Dias, C., Laitman, B., Stockman, V., LaPlant, Q., Cahill, M. E., Nestler, E. J., & Shen, L. (2014). Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens. Genome biology, 15(4), R65.
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  Increasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies that have intrinsic limitations.
• Feng, J., Wilkinson, M., Liu, X., Purushothaman, I., Ferguson, D., Vialou, V., Maze, I., Shao, N., Kennedy, P., Koo, J., Dias, C., Laitman, B., Stockman, V., LaPlant, Q., Cahill, M., Nestler, E., & Shen, L. (2014). Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens. Genome Biology, 15(4). doi:10.1186/gb-2014-15-4-r65
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  Background: Increasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies that have intrinsic limitations. Results: We use next generation sequencing methods, RNA-seq and ChIP-seq for RNA polymerase II and several histone methylation marks, to obtain a more complete view of cocaine-induced changes in gene expression and associated adaptations in numerous modes of chromatin regulation in the mouse nucleus accumbens, a key brain reward region. We demonstrate an unexpectedly large number of pre-mRNA splicing alterations in response to repeated cocaine treatment. In addition, we identify combinations of chromatin changes, or signatures, that correlate with cocaine-dependent regulation of gene expression, including those involving pre-mRNA alternative splicing. Through bioinformatic prediction and biological validation, we identify one particular splicing factor, A2BP1(Rbfox1/Fox-1), which is enriched at genes that display certain chromatin signatures and contributes to drug-induced behavioral abnormalities. Together, this delineation of the cocaine-induced epigenome in the nucleus accumbens reveals several novel modes of regulation by which cocaine alters the brain. Conclusions: We establish combinatorial chromatin and transcriptional profiles in mouse nucleus accumbens after repeated cocaine treatment. These results serve as an important resource for the field and provide a template for the analysis of other systems to reveal new transcriptional and epigenetic mechanisms of neuronal regulation.
• Gaspari, S., Papachatzaki, M. M., Koo, J. W., Carr, F. B., Tsimpanouli, M., Stergiou, E., Bagot, R. C., Ferguson, D., Mouzon, E., Chakravarty, S., Deisseroth, K., Lobo, M. K., & Zachariou, V. (2014). Nucleus accumbens-specific interventions in RGS9-2 activity modulate responses to morphine. Neuropsychopharmacology, 39(8), 1968-77.
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  Regulator of G protein signalling 9-2 (Rgs9-2) modulates the actions of a wide range of CNS-acting drugs by controlling signal transduction of several GPCRs in the striatum. RGS9-2 acts via a complex mechanism that involves interactions with Gα subunits, the Gβ5 protein, and the adaptor protein R7BP. Our recent work identified Rgs9-2 complexes in the striatum associated with acute or chronic exposures to mu opioid receptor (MOR) agonists. In this study we use several new genetic tools that allow manipulations of Rgs9-2 activity in particular brain regions of adult mice in order to better understand the mechanism via which this protein modulates opiate addiction and analgesia. We used adeno-associated viruses (AAVs) to express forms of Rgs9-2 in the dorsal and ventral striatum (nucleus accumbens, NAc) in order to examine the influence of this protein in morphine actions. Consistent with earlier behavioural findings from constitutive Rgs9 knockout mice, we show that Rgs9-2 actions in the NAc modulate morphine reward and dependence. Notably, Rgs9-2 in the NAc affects the analgesic actions of morphine as well as the development of analgesic tolerance. Using optogenetics we demonstrate that activation of Channelrhodopsin2 in Rgs9-2-expressing neurons, or in D1 dopamine receptor (Drd1)-enriched medium spiny neurons, accelerates the development of morphine tolerance, whereas activation of D2 dopamine receptor (Drd2)-enriched neurons does not significantly affect the development of tolerance. Together, these data provide new information on the signal transduction mechanisms underlying opiate actions in the NAc.
• Heller, E. A., Cates, H. M., Peña, C. J., Sun, H., Shao, N., Feng, J., Golden, S. A., Herman, J. P., Walsh, J. J., Mazei-Robison, M., Ferguson, D., Knight, S., Gerber, M. A., Nievera, C., Han, M., Russo, S. J., Tamminga, C. S., Neve, R. L., Shen, L., , Zhang, H. S., et al. (2014). Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors. Nature neuroscience, 17(12), 1720-7.
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  Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.
• Heller, E. A., Kaska, S., Fallon, B., Ferguson, D., Kennedy, P. J., Neve, R. L., Nestler, E. J., & Mazei-Robison, M. S. (2014). Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. Journal of neurochemistry.
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  Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug-induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up-regulated by both drugs was serum- and glucocorticoid-inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N-myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug-related behaviors, we over-expressed constitutively active (CA) SGK1 in the VTA. SGK1-CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1-CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug-induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction. We find that repeated, but not acute, morphine or cocaine administration induces an increase in serum- and glucocorticoid-inducible kinase (SGK1) gene expression and activity in the ventral tegmental area (VTA). This increase in SGK1 activity may play a role in drug-dependent behaviors and suggests a novel signaling cascade for potential intervention in drug dependence and addiction.
• Vialou, V., Bagot, R. C., Cahill, M. E., Ferguson, D., Robison, A. J., Dietz, D. M., Fallon, B., Mazei-Robison, M., Ku, S. M., Harrigan, E., Winstanley, C. A., Joshi, T., Feng, J., Berton, O., & Nestler, E. J. (2014). Prefrontal cortical circuit for depression- and anxiety-related behaviors mediated by cholecystokinin: role of ΔFosB. The Journal of Neuroscience, 34(11), 3878-87.
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  Decreased medial prefrontal cortex (mPFC) neuronal activity is associated with social defeat-induced depression- and anxiety-like behaviors in mice. However, the molecular mechanisms underlying the decreased mPFC activity and its prodepressant role remain unknown. We show here that induction of the transcription factor ΔFosB in mPFC, specifically in the prelimbic (PrL) area, mediates susceptibility to stress. ΔFosB induction in PrL occurred selectively in susceptible mice after chronic social defeat stress, and overexpression of ΔFosB in this region, but not in the nearby infralimbic (IL) area, enhanced stress susceptibility. ΔFosB produced these effects partly through induction of the cholecystokinin (CCK)-B receptor: CCKB blockade in mPFC induces a resilient phenotype, whereas CCK administration into mPFC mimics the anxiogenic- and depressant-like effects of social stress. We previously found that optogenetic stimulation of mPFC neurons in susceptible mice reverses several behavioral abnormalities seen after chronic social defeat stress. Therefore, we hypothesized that optogenetic stimulation of cortical projections would rescue the pathological effects of CCK in mPFC. After CCK infusion in mPFC, we optogenetically stimulated mPFC projections to basolateral amygdala or nucleus accumbens, two subcortical structures involved in mood regulation. Stimulation of corticoamygdala projections blocked the anxiogenic effect of CCK, although no effect was observed on other symptoms of social defeat. Conversely, stimulation of corticoaccumbens projections reversed CCK-induced social avoidance and sucrose preference deficits but not anxiogenic-like effects. Together, these results indicate that social stress-induced behavioral deficits are mediated partly by molecular adaptations in mPFC involving ΔFosB and CCK through cortical projections to distinct subcortical targets.
• Walsh, J. J., Friedman, A. K., Sun, H., Heller, E. A., Ku, S. M., Juarez, B., Burnham, V. L., Mazei-Robison, M. S., Ferguson, D., Golden, S. A., Koo, J. W., Chaudhury, D., Christoffel, D. J., Pomeranz, L., Friedman, J. M., Russo, S. J., Nestler, E. J., & Han, M. (2014). Stress and CRF gate neural activation of BDNF in the mesolimbic reward pathway. Nature neuroscience, 17(1), 27-9.
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  Mechanisms controlling release of brain-derived neurotrophic factor (BDNF) in the mesolimbic dopamine reward pathway remain unknown. We report that phasic optogenetic activation of this pathway increases BDNF amounts in the nucleus accumbens (NAc) of socially stressed mice but not of stress-naive mice. This stress gating of BDNF signaling is mediated by corticotrophin-releasing factor (CRF) acting in the NAc. These results unravel a stress context-detecting function of the brain's mesolimbic circuit.
• Chaudhury, D., Walsh, J. J., Friedman, A. K., Juarez, B., Ku, S. M., Koo, J. W., Ferguson, D., Tsai, H., Pomeranz, L., Christoffel, D. J., Nectow, A. R., Ekstrand, M., Domingos, A., Mazei-Robison, M. S., Mouzon, E., Lobo, M. K., Neve, R. L., Friedman, J. M., Russo, S. J., , Deisseroth, K., et al. (2013). Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature, 493(7433), 532-6.
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  Ventral tegmental area (VTA) dopamine neurons in the brain's reward circuit have a crucial role in mediating stress responses, including determining susceptibility versus resilience to social-stress-induced behavioural abnormalities. VTA dopamine neurons show two in vivo patterns of firing: low frequency tonic firing and high frequency phasic firing. Phasic firing of the neurons, which is well known to encode reward signals, is upregulated by repeated social-defeat stress, a highly validated mouse model of depression. Surprisingly, this pathophysiological effect is seen in susceptible mice only, with no apparent change in firing rate in resilient individuals. However, direct evidence--in real time--linking dopamine neuron phasic firing in promoting the susceptible (depression-like) phenotype is lacking. Here we took advantage of the temporal precision and cell-type and projection-pathway specificity of optogenetics to show that enhanced phasic firing of these neurons mediates susceptibility to social-defeat stress in freely behaving mice. We show that optogenetic induction of phasic, but not tonic, firing in VTA dopamine neurons of mice undergoing a subthreshold social-defeat paradigm rapidly induced a susceptible phenotype as measured by social avoidance and decreased sucrose preference. Optogenetic phasic stimulation of these neurons also quickly induced a susceptible phenotype in previously resilient mice that had been subjected to repeated social-defeat stress. Furthermore, we show differences in projection-pathway specificity in promoting stress susceptibility: phasic activation of VTA neurons projecting to the nucleus accumbens (NAc), but not to the medial prefrontal cortex (mPFC), induced susceptibility to social-defeat stress. Conversely, optogenetic inhibition of the VTA-NAc projection induced resilience, whereas inhibition of the VTA-mPFC projection promoted susceptibility. Overall, these studies reveal novel firing-pattern- and neural-circuit-specific mechanisms of depression.
• Chaudhury, D., Walsh, J., Friedman, A., Juarez, B., Ku, S., Koo, J., Ferguson, D., Tsai, H., Pomeranz, L., Christoffel, D., Nectow, A., Ekstrand, M., Domingos, A., Mazei-Robison, M., Mouzon, E., Lobo, M., Neve, R., Friedman, J., Russo, S., , Deisseroth, K., et al. (2013). Rapid control of depressive behaviors by firing pattern- and projection-specific optogenetic regulation of midbrain dopamine neurons.. Nature, 532-536.
• Ferguson, D., Koo, J. W., Feng, J., Heller, E., Rabkin, J., Heshmati, M., Renthal, W., Neve, R., Liu, X., Shao, N., Sartorelli, V., Shen, L., & Nestler, E. J. (2013). Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33(41), 16088-98.
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  Sirtuins (SIRTs), class III histone deacetylases, are well characterized for their control of cellular physiology in peripheral tissues, but their influence in brain under normal and pathological conditions remains poorly understood. Here, we establish an essential role for SIRT1 and SIRT2 in regulating behavioral responses to cocaine and morphine through actions in the nucleus accumbens (NAc), a key brain reward region. We show that chronic cocaine administration increases SIRT1 and SIRT2 expression in the mouse NAc, while chronic morphine administration induces SIRT1 expression alone, with no regulation of all other sirtuin family members observed. Drug induction of SIRT1 and SIRT2 is mediated in part at the transcriptional level via the drug-induced transcription factor ΔFosB and is associated with robust histone modifications at the Sirt1 and Sirt2 genes. Viral-mediated overexpression of SIRT1 or SIRT2 in the NAc enhances the rewarding effects of both cocaine and morphine. In contrast, the local knockdown of SIRT1 from the NAc of floxed Sirt1 mice decreases drug reward. Such behavioral effects of SIRT1 occur in concert with its regulation of numerous synaptic proteins in NAc as well as with SIRT1-mediated induction of dendritic spines on NAc medium spiny neurons. These studies establish sirtuins as key mediators of the molecular and cellular plasticity induced by drugs of abuse in NAc, and of the associated behavioral adaptations, and point toward novel signaling pathways involved in drug action.
• Dietz, D. M., Sun, H., Lobo, M. K., Cahill, M. E., Chadwick, B., Gao, V., Koo, J. W., Mazei-Robison, M. S., Dias, C., Maze, I., Damez-Werno, D., Dietz, K. C., Scobie, K. N., Ferguson, D., Christoffel, D., Ohnishi, Y., Hodes, G. E., Zheng, Y., Neve, R. L., , Hahn, K. M., et al. (2012). Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons. Nature neuroscience, 15(6), 891-6.
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  Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.
• Koo, J. W., Mazei-Robison, M. S., Chaudhury, D., Juarez, B., LaPlant, Q., Ferguson, D., Feng, J., Sun, H., Scobie, K. N., Damez-Werno, D., Crumiller, M., Ohnishi, Y. N., Ohnishi, Y. H., Mouzon, E., Dietz, D. M., Lobo, M. K., Neve, R. L., Russo, S. J., Han, M., & Nestler, E. J. (2012). BDNF is a negative modulator of morphine action. Science (New York, N.Y.), 338(6103), 124-8.
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  Brain-derived neurotrophic factor (BDNF) is a key positive regulator of neural plasticity, promoting, for example, the actions of stimulant drugs of abuse such as cocaine. We discovered a surprising opposite role for BDNF in countering responses to chronic morphine exposure. The suppression of BDNF in the ventral tegmental area (VTA) enhanced the ability of morphine to increase dopamine (DA) neuron excitability and promote reward. In contrast, optical stimulation of VTA DA terminals in nucleus accumbens (NAc) completely reversed the suppressive effect of BDNF on morphine reward. Furthermore, we identified numerous genes in the NAc, a major target region of VTA DA neurons, whose regulation by BDNF in the context of chronic morphine exposure mediated this counteractive function. These findings provide insight into the molecular basis of morphine-induced neuroadaptations in the brain's reward circuitry.
• Vialou, V., Feng, J., Robison, A. J., Ku, S. M., Ferguson, D., Scobie, K. N., Mazei-Robison, M. S., Mouzon, E., & Nestler, E. J. (2012). Serum response factor and cAMP response element binding protein are both required for cocaine induction of ΔFosB. The Journal of neuroscience : the official journal of the Society for Neuroscience, 32(22), 7577-84.
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  The molecular mechanism underlying induction by cocaine of ΔFosB, a transcription factor important for addiction, remains unknown. Here, we demonstrate a necessary role for two transcription factors, cAMP response element binding protein (CREB) and serum response factor (SRF), in mediating this induction within the mouse nucleus accumbens (NAc), a key brain reward region. CREB and SRF are both activated in NAc by cocaine and bind to the fosB gene promoter. Using viral-mediated Cre recombinase expression in the NAc of single- or double-floxed mice, we show that deletion of both transcription factors from this brain region completely blocks cocaine induction of ΔFosB in NAc, whereas deletion of either factor alone has no effect. Furthermore, deletion of both SRF and CREB from NAc renders animals less sensitive to the rewarding effects of moderate doses of cocaine when tested in the conditioned place preference (CPP) procedure and also blocks locomotor sensitization to higher doses of cocaine. Deletion of CREB alone has the opposite effect and enhances both cocaine CPP and locomotor sensitization. In contrast to ΔFosB induction by cocaine, ΔFosB induction in NAc by chronic social stress, which we have shown previously requires activation of SRF, is unaffected by the deletion of CREB alone. These surprising findings demonstrate the involvement of distinct transcriptional mechanisms in mediating ΔFosB induction within this same brain region by cocaine versus stress. Our results also establish a complex mode of regulation of ΔFosB induction in response to cocaine, which requires the concerted activities of both SRF and CREB.
• Brown, T. E., Lee, B. R., Mu, P., Ferguson, D., Dietz, D., Ohnishi, Y. N., Lin, Y., Suska, A., Ishikawa, M., Huang, Y. H., Shen, H., Kalivas, P. W., Sorg, B. A., Zukin, R. S., Nestler, E. J., Dong, Y., & Schlüter, O. M. (2011). A silent synapse-based mechanism for cocaine-induced locomotor sensitization. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(22), 8163-74.
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  Locomotor sensitization is a common and robust behavioral alteration in rodents whereby following exposure to abused drugs such as cocaine, the animal becomes significantly more hyperactive in response to an acute drug challenge. Here, we further analyzed the role of cocaine-induced silent synapses in the nucleus accumbens (NAc) shell and their contribution to the development of locomotor sensitization. Using a combination of viral vector-mediated genetic manipulations, biochemistry, and electrophysiology in a locomotor sensitization paradigm with repeated, daily, noncontingent cocaine (15 mg/kg) injections, we show that dominant-negative cAMP-element binding protein (CREB) prevents cocaine-induced generation of silent synapses of young (30 d old) rats, whereas constitutively active CREB is sufficient to increase the number of NR2B-containing NMDA receptors (NMDARs) at synapses and to generate silent synapses. We further show that occupancy of CREB at the NR2B promoter increases and is causally related to the increase in synaptic NR2B levels. Blockade of NR2B-containing NMDARs by administration of the NR2B-selective antagonist Ro256981 directly into the NAc, under conditions that inhibit cocaine-induced silent synapses, prevents the development of cocaine-elicited locomotor sensitization. Our data are consistent with a cellular cascade whereby cocaine-induced activation of CREB promotes CREB-dependent transcription of NR2B and synaptic incorporation of NR2B-containing NMDARs, which generates new silent synapses within the NAc. We propose that cocaine-induced activation of CREB and generation of new silent synapses may serve as key cellular events mediating cocaine-induced locomotor sensitization. These findings provide a novel cellular mechanism that may contribute to cocaine-induced behavioral alterations.
• Christoffel, D. J., Golden, S. A., Dumitriu, D., Robison, A. J., Janssen, W. G., Ahn, H. F., Krishnan, V., Reyes, C. M., Han, M., Ables, J. L., Eisch, A. J., Dietz, D. M., Ferguson, D., Neve, R. L., Greengard, P., Kim, Y., Morrison, J. H., & Russo, S. J. (2011). IκB kinase regulates social defeat stress-induced synaptic and behavioral plasticity. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(1), 314-21.
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  The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.
• Knoll, A. T., Muschamp, J. W., Sillivan, S. E., Ferguson, D., Dietz, D. M., Meloni, E. G., Carroll, F. I., Nestler, E. J., Konradi, C., & Carlezon, W. A. (2011). Kappa opioid receptor signaling in the basolateral amygdala regulates conditioned fear and anxiety in rats. Biological psychiatry, 70(5), 425-33.
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  The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats.
• Knoll, A., Muschamp, J., Sillivan, S., Ferguson, D., Dietz, D., Meloni, E., Carroll, F., Nestler, E., Konradi, C., & Carlezon, W. (2011). Kappa Opioid Receptor Signaling in the Basolateral Amygdala Regulates Conditioned Fear and Anxiety in Rats.. Biological Psychiatry.
• Psifogeorgou, K., Psigfogeorgou, K., Terzi, D., Papachatzaki, M. M., Varidaki, A., Ferguson, D., Gold, S. J., & Zachariou, V. (2011). A unique role of RGS9-2 in the striatum as a positive or negative regulator of opiate analgesia. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(15), 5617-24.
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  The signaling molecule RGS9-2 is a potent modulator of G-protein-coupled receptor function in striatum. Our earlier work revealed a critical role for RGS9-2 in the actions of the μ-opioid receptor (MOR) agonist morphine. In this study, we demonstrate that RGS9-2 may act as a positive or negative modulator of MOR-mediated behavioral responses in mice depending on the agonist administered. Paralleling these findings we use coimmunoprecipitation assays to show that the signaling complexes formed between RGS9-2 and Gα subunits in striatum are determined by the MOR agonist, and we identify RGS9-2 containing complexes associated with analgesic tolerance. In striatum, MOR activation promotes the formation of complexes between RGS9-2 and several Gα subunits, but morphine uniquely promotes an association between RGS9-2 and Gαi3. In contrast, RGS9-2/Gαq complexes assemble after acute application of several MOR agonists but not after morphine application. Repeated morphine administration leads to the formation of distinct complexes, which contain RGS9-2, Gβ5, and Gαq. Finally, we use simple pharmacological manipulations to disrupt RGS9-2 complexes formed during repeated MOR activation to delay the development of analgesic tolerance to morphine. Our data provide a better understanding of the brain-region-specific signaling events associated with opiate analgesia and tolerance and point to pharmacological approaches that can be readily tested for improving chronic analgesic responsiveness.
• Dumas, T. C., Gillette, T., Ferguson, D., Hamilton, K., & Sapolsky, R. M. (2010). Anti-glucocorticoid gene therapy reverses the impairing effects of elevated corticosterone on spatial memory, hippocampal neuronal excitability, and synaptic plasticity. The Journal of neuroscience : the official journal of the Society for Neuroscience, 30(5), 1712-20.
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  Moderate release of the major stress hormones, glucocorticoids (GCs), improves hippocampal function and memory. In contrast, excessive or prolonged elevations produce impairments. Enzymatic degradation and reformation of GCs help to maintain optimal levels within target tissues, including the brain. We hypothesized that expressing a GC-degrading enzyme in hippocampal neurons would attenuate the negative impact of an excessive elevation in GC levels on synaptic physiology and spatial memory. We tested this by expressing 11-beta-hydroxysteroid dehydrogenase (type II) in dentate gyrus granule cells during a 3 d GC treatment followed by examination of synaptic responses in hippocampal slices or spatial performance in the Morris water maze. In adrenalectomized rats with basal GC replacement, additional GC treatments for 3 d reduced synaptic strength and promoted the expression of long-term depression at medial perforant path synapses, increased granule cell and CA1 pyramidal cell excitability, and impaired spatial reference memory (without influencing learning). Expression of 11-beta-hydroxysteroid dehydrogenase (type II), mostly in mature dentate gyrus granule cells, reversed the effects of high GC levels on granule cell and pyramidal cell excitability, perforant path synaptic plasticity, and spatial memory. These data demonstrate the ability of neuroprotective gene expression limited to a specific cell population to both locally and trans-synaptically offset neurophysiological disruptions produced by prolonged increases in circulating stress hormones. This report supplies the first physiological explanation for previously demonstrated cognitive sparing by anti-stress gene therapy approaches and lends additional insight into the hippocampal processes that are important for memory.
• Koo, J. W., Russo, S. J., Ferguson, D., Nestler, E. J., & Duman, R. S. (2010). Nuclear factor-kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior. Proceedings of the National Academy of Sciences of the United States of America, 107(6), 2669-74.
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  Proinflammatory cytokines, such as IL-1beta, have been implicated in the cellular and behavioral effects of stress and in mood disorders, although the downstream signaling pathways underlying these effects have not been determined. In the present study, we demonstrate a critical role for NF-kappaB signaling in the actions of IL-1beta and stress. Stress inhibition of neurogenesis in the adult hippocampus, which has been implicated in the prodepressive effects of stress, is blocked by administration of an inhibitor of NF-kappaB. Further analysis reveals that stress activates NF-kappaB signaling and decreases proliferation of neural stem-like cells but not early neural progenitor cells in the adult hippocampus. We also find that depressive-like behaviors caused by exposure to chronic stress are mediated by NF-kappaB signaling. Together, these data identify NF-kappaB signaling as a critical mediator of the antineurogenic and behavioral actions of stress and suggest previously undescribed therapeutical targets for depression.
• Ferguson, D., Sapolsky, R. M., & Mitra, R. (2009). Tracing of a basolateral amygdala neuron overlaid on an SK2-βgal-expressing neuron. Molecular Psychiatry. doi:10.1038/mp.2009.83
• Mitra, R., Ferguson, D., & Sapolsky, R. (2009). SK2 potassium channel overexpression in basolateral amygdala reduces anxiety, stress-induced corticosterone secretion and dendritic arborization.. Mol Psychiatry, 9(14), 847-855.
• Mitra, R., Ferguson, D., & Sapolsky, R. M. (2009). Mineralocorticoid receptor overexpression in basolateral amygdala reduces corticosterone secretion and anxiety. Biological psychiatry, 66(7), 686-90.
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  The amygdala plays a critical role in the development of anxiety and the regulation of stress hormone secretion. Reciprocally, stress and stress hormones can induce amygdala hypertrophy, a phenomenon related to enhanced anxiety. As such, modulating amygdaloid function can potentially reduce maladaptive features of the stress response. The amygdala contains two kind of receptor for corticosteroids, the adrenal steroid hormone released during stress: glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs). Although high-affinity MRs are heavily occupied during basal conditions, low-affinity GRs are heavily occupied only by stress levels of glucocorticoids. Prolonged and heavy occupancy of GRs tends to mediate the deleterious effects of glucocorticoids on neurons, whereas MR occupancy tends to mediate beneficial effects.
• Ferguson, D., & Sapolsky, R. (2008). Overexpression of mineralocorticoid and transdominant glucocorticoid receptor blocks the impairing effects of glucocorticoids on memory. Hippocampus, 18(11), 1103-11.
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  It is well established that mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) influence hippocampal-dependent spatial memory. MRs are saturated in the presence of low corticosterone (CORT) levels; consequently receptor protein levels play a rate-limiting role in regulating the positive effects of MR-mediated gene transcription. In this study, viral vector-mediated transgene expression was used to simultaneously manipulate both MR and GR signaling. This approach allowed us to investigate the effects of spatially restricted overexpression of MR and a negative transdominant GR (TD) in the dentate gyrus (DG) subfield of the hippocampus, on short term and long term spatial memory in animals overexpressing one copy of MR or TD, two copies of MR ("MR/MR"), or one copy of each ("MR/TD"). Expression of transgenes did not influence the acquisition (learning) phase of the Morris water maze task. However, we found an overall enhancing effect of MR/MR expression on short term memory performance. In addition, rats expressing TD and MR/TD blocked the high CORT-induced impairments on long term spatial memory retrieval. These findings illustrate the potential beneficial effects of increasing MR signaling or decreasing GR signaling to enhance specific aspects of cognitive function.
• Ferguson, D., Lin, S., & Sapolsky, R. (2008). Viral vector-mediated blockade of the endocrine stress-response modulates non-spatial memory. Neuroscience letters, 437(1), 1-4.
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  Stress results in the release of glucocorticoids (GCs) which at high levels, impair performance on hippocampus-dependent tasks. Estrogen is neurotrophic and can rescue stress-induced memory impairments. Here we report the use of a viral vector to overexpress a chimeric gene (ER/GR) that converts the deleterious effects of glucocorticoids into beneficial estrogenic effects. A short immobilization stress regimen was sufficient to impair non-spatial memory. In contrast, viral vector-mediated overexpression of ER/GR in the dentate gyrus of the hippocampus protected against stress-induced impairments of non-spatial memory. These data add to the growing evidence that increasing estrogenic signaling can protect against the impairing effects of stress on non-spatial memory.
• Ferguson, D., & Sapolsky, R. (2007). Mineralocorticoid receptor overexpression differentially modulates specific phases of spatial and nonspatial memory. The Journal of neuroscience : the official journal of the Society for Neuroscience, 27(30), 8046-52.
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  Glucocorticoids (GCs) and stress modulate specific phases of information processing. The modulatory affects of GCs on hippocampal function are thought to be mediated by the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The GR plays a critical role in mediating the impairing effects of GCs on hippocampal function. Conversely, activation of MR facilitates hippocampal function. The high affinity of MR for GCs suggests that the receptor protein levels play a key role in regulating the beneficial effects of MR-mediated gene transcription. Using herpes simplex vectors, we transiently increased MR levels in dentate gyrus granule cells, which in turn enhanced MR signaling. We then examined its effects on spatial and nonspatial memory consolidation and retrieval using the object placement and object recognition task. Additionally, we assessed whether an increased MR signal could block the impairing effects of high GCs on memory retrieval. Rats overexpressing MR displayed an enhancement in the consolidation of nonspatial memory relative to rats expressing green fluorescent protein and suggest the potential for gene transfer techniques for enhancing cognition during stress. Moreover, rats overexpressing MR were spared from the disruptive effects of high GCs on the retrieval of nonspatial memory. Thus, this study illustrates the critical role of MR in mediating the retrieval and consolidation of nonspatial memory.
• Nicholas, A., Munhoz, C. D., Ferguson, D., Campbell, L., & Sapolsky, R. (2006). Enhancing cognition after stress with gene therapy. The Journal of neuroscience : the official journal of the Society for Neuroscience, 26(45), 11637-43.
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  Hippocampal function is essential for the acquisition, consolidation, and retrieval of spatial memory. High circulating levels of glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, have been shown to impair both acquisition and retrieval and can either impair or enhance consolidation, depending on experimental conditions. In contrast, estrogen can enhance spatial memory performance and can block the deleterious effects of GCs on such performance. We therefore constructed a chimeric gene ("ER/GR") containing the hormone-binding domain of the GC receptor and the DNA binding domain of the estrogen receptor; as a result, ER/GR transduces deleterious GC signals into beneficial estrogenic ones. We show here that acute immobilization stress, before acquisition and retrieval phases, increases latencies for male rats in a hidden platform version of the Morris water maze. This impairment is blocked by hippocampal expression of the ER/GR transgene. ER/GR expression also blocks decreases in platform crossings caused by acute stress, either after acquisition or before retrieval. Three days of stress before acquisition produces an estrogen-like enhancement of performance in ER/GR-treated rats. Moreover, ER/GR blocks the suppressive effects of GCs on expression of brain-derived neurotrophic factor (BDNF), a growth factor central to hippocampal-dependent cognition and plasticity, instead producing an estrogenic increase in BDNF expression. Thus, ER/GR expression enhances spatial memory performance and blocks the impairing effects of GCs on such performance.
• Altemus, M., Fong, J., Yang, R., Damast, S., Luine, V., & Ferguson, D. (2004). Changes in cerebrospinal fluid neurochemistry during pregnancy. Biological psychiatry, 56(6), 386-92.
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  Little is known about changes in brain function that may occur during pregnancy. Studies in rodents and sheep suggest that several brain neurotransmitter and neurohormonal systems known to modulate anxiety may be altered during pregnancy.
• Kaufer, D., Ogle, W., Pincus, Z., Clark, K., Nicholas, A., Dinkel, K., Dumas, T., Ferguson, D., Lee, A., Winters, M., & Sapolsky, R. (2004). Restructuring the neuronal stress response with anti-glucocorticoid gene delivery.. Nat Neurosci, 9, 947-953.
• Bisagno, V., Ferguson, D., & Luine, V. N. (2003). Chronic D-amphetamine induces sexually dimorphic effects on locomotion, recognition memory, and brain monoamines. Pharmacology, biochemistry, and behavior, 74(4), 859-67.
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  While acute and chronic D-amphetamine (AMPH) treatments produce greater scores for locomotor activity in female rats in comparison with male rats, little is known about AMPH-induced gender differences on cognition. The objectives of the present study were to (1) investigate during a withdrawal period following chronic AMPH treatment whether performance of two memory tasks, object recognition (OR) and object placement (OP) are altered, and (2) determine if an AMPH challenge dose after a withdrawal period amplifies previously reported gender differences in locomotor activity and neurochemistry. Sprague-Dawley male and female adult rats were included in a chronic AMPH treatment (10 injections, 1 every other day; males: 3 mg/kg, females 2.6 mg/kg). Locomotor activity was quantified (acute, chronic, and after a 16-day withdrawal period). Neurotransmitter levels in brain areas were evaluated after an AMPH challenge dose on the 16th withdrawal day. During the withdrawal period, OR (2- and 4-h delays) was impaired in AMPH-treated males but they did not show any impairment in OP; AMPH females also showed impairments in OR (only 4-h delay). AMPH females showed more locomotion after acute and chronic treatment but AMPH-induced hyperactivity was comparable for females and males after a challenge dose. Following a challenge dose of AMPH after a withdrawal period, gender differences in dopaminergic and serotonergic neurotransmission in the striatum were found. These gender differences elicited by AMPH in monoaminergic pathways may be related to sex differences on behavioral components involved in locomotion and OR memory.
• Alves, S. E., Hoskin, E., Lee, S. J., Brake, W. G., Luine, V., Ferguson, D., Allen, P. B., Greengard, P., & McEwen, B. (2002). Serotonin Mediates CA1 Spine Density But is Not Crucial For Ovarian Steroid Regulation of Synapotgenesis in the Female Rat Hippocampus.. Synapse, 2(45), 143-151.
• Bisagno, V., Ferguson, D., & Luine, V. (2002). Short toxic Methamphetamine schedule impairs object recognition task in male rats.. Brain Res., 1-2(940), 95-101.
• Bowman, R., Ferguson, D., & Luine, V. (2002). Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats.. Neuroscience, 2(113), 401-410.