Raquel Revilla-Sanchez

Activation of estrogen receptor-beta regulates hippocampal synaptic plasticity and improves memory.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERα) and beta (ERβ) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERβ. Selective ERβ agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERβ knockout mice. In hippocampal slices, ERβ activation enhanced long-term potentiation, an effect that was absent in slices from ERβ knockout mice. ERβ activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERβ agonist, but not an ERα agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERβ can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

mGluR5 stimulates gliotransmission in the nucleus accumbens

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca2+ signaling in astrocytes and, as a consequence, the Ca2+-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca2+ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca2+ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca2+ elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca2+ oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function

Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein–protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1–TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1–TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.

Deficits in spatial memory correlate with modified {gamma}-aminobutyric acid type A receptor tyrosine phosphorylation in the hippocampus

Fast synaptic inhibition in the brain is largely mediated by γ-aminobutyric acid receptors (GABAAR). While the pharmacological manipulation of GABAAR function by therapeutic agents, such as benzodiazepines can have profound effects on neuronal excitation and behavior, the endogenous mechanisms neurons use to regulate the efficacy of synaptic inhibition and their impact on behavior remains poorly understood. To address this issue, we created a knock-in mouse in which tyrosine phosphorylation of the GABAARs γ2 subunit, a posttranslational modification that is critical for their functional modulation, has been ablated. These animals exhibited enhanced GABAAR accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the CA3 subdomain of the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. This enhanced inhibition correlated with a specific deficit in spatial object recognition, a behavioral paradigm dependent upon CA3. Thus, phospho-dependent regulation of GABAAR function involving just two tyrosine residues in the γ2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences.

The ability of BDNF to modify neurogenesis and depressive-like behaviors is dependent upon phosphorylation of tyrosine residues 365/367 in the GABA(A)-receptor γ2 subunit.

Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizing γ2 subunit-containing GABAA receptors (GABAARs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABAAR γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABAAR γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.

Multiphoton in vivo imaging of amyloid in animal models of Alzheimer's disease

Amyloid-beta (Abeta) deposition is a defining feature of Alzheimers disease (AD). The toxicity of Abeta aggregation is thought to contribute to clinical deficits including progressive memory loss and cognitive dysfunction. Therefore, Abeta peptide has become the focus of many therapeutic approaches for the treatment of AD due to its central role in the development of neuropathology of AD. In the past decade, taking the advantage of multiphoton microscopy and molecular probes for amyloid peptide labeling, the dynamic progression of Abeta aggregation in amyloid plaques and cerebral amyloid angiopathy has been monitored in real time in transgenic mouse models of AD. Moreover, amyloid plaque-associated alterations in the brain including dendritic and synaptic abnormalities, changes of neuronal and astrocytic calcium homeostasis, microglial activation and recruitment in the plaque location have been extensively studied. These studies provide remarkable insight to understand the pathogenesis and pathogenicity of amyloid plaques in the context of AD. The ability to longitudinally image plaques and related structures facilitates the evaluation of therapeutic approaches targeting toward the clearance of plaques.

Direct Interaction of GABAB Receptors with M2 Muscarinic Receptors Enhances Muscarinic Signaling

Downregulation of G-protein-coupled receptors (GPCRs) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M2 muscarinic receptors (M2Rs) causes internalization of M2R and G-protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that coexpression of GABAB R2 receptors (GBR2s) rescues both surface expression and function of M2R, including M2R-induced activation of GIRKs and inhibition of cAMP production. GBR2 showed significant association with M2R at the plasma membrane but not other GPCRs (M1R, μ-opioid receptor), as detected by fluorescence resonance energy transfer measured with total internal reflection fluorescence microscopy. Unique regions of the proximal C-terminal domains of GBR2 and M2R mediate specific binding between M2R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M2R and overlaps with M2R expression in cortical neurons. This novel heteromeric association between M2R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2.

Enhanced Tonic Inhibition Influences the Hypnotic and Amnestic Actions of the Intravenous Anesthetics Etomidate and Propofol

Intravenous anesthetics exert a component of their actions via potentiating inhibitory neurotransmission mediated by γ-aminobutyric type-A receptors (GABAARs). Phasic and tonic inhibition is mediated by distinct populations of GABAARs, with the majority of phasic inhibition by subtypes composed of α1–3βγ2 subunits, whereas tonic inhibition is dependent on subtypes assembled from α4–6βδ subunits. To explore the contribution that these distinct forms of inhibition play in mediating intravenous anesthesia, we have used mice in which tyrosine residues 365/7 within the γ2 subunit are mutated to phenyalanines (Y365/7F). Here we demonstrate that this mutation leads to increased accumulation of the α4 subunit containing GABAARs in the thalamus and dentate gyrus of female Y365/7F but not male Y365/7F mice. Y365/7F mice exhibited a gender-specific enhancement of tonic inhibition in the dentate gyrus that was more sensitive to modulation by the anesthetic etomidate, together with a deficit in long-term potentiation. Consistent with this, female Y365/7F, but not male Y365/7F, mice exhibited a dramatic increase in the duration of etomidate- and propofol-mediated hypnosis. Moreover, the amnestic actions of etomidate were selectively potentiated in female Y365/7F mice. Collectively, these observations suggest that potentiation of tonic inhibition mediated by α4 subunit containing GABAARs contributes to the hypnotic and amnestic actions of the intravenous anesthetics, etomidate and propofol.

Postsynaptic GABAB Receptor Activity Regulates Excitatory Neuronal Architecture and Spatial Memory

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

Tyrosine Phosphorylation of GABAA Receptor γ2-Subunit Regulates Tonic and Phasic Inhibition in the Thalamus

GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2Y365/367F+/− (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2Y365/367F +/− thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn2+, were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2Y356/367F +/− to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons.