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Functional coupling between the amygdala and the dorsomedial prefrontal cortex (dmPFC) has been implicated in the generation of negative affective states; however, the mechanisms by which stress increases amygdala-dmPFC synaptic strength and generates anxiety-like behaviors are not well understood. Here, we show that the mouse basolateral amygdala (BLA)-prelimbic prefrontal cortex (plPFC) circuit is engaged by stress and activation of this pathway in anxiogenic. Furthermore, we demonstrate that acute stress exposure leads to a lasting increase in synaptic strength within a reciprocal BLA-plPFC-BLA subcircuit. Importantly, we identify 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling as a key mechanism limiting glutamate release at BLA-plPFC synapses and the functional collapse of multimodal 2-AG signaling as a molecular mechanism leading to persistent circuit-specific synaptic strengthening and anxiety-like behaviors after stress exposure. These data suggest that circuit-specific impairment in 2-AG signaling could facilitate functional coupling between the BLA and plPFC and the translation of environmental stress to affective pathology.
Copyright © 2019 Elsevier Inc. All rights reserved.
In the brain, AMPA-type glutamate receptors (AMPARs) form complexes with their auxiliary subunits and mediate the majority of fast excitatory neurotransmission. Signals transduced by these complexes are critical for synaptic plasticity, learning, and memory. The two major categories of AMPAR auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs); these subunits share little homology and play distinct roles in controlling ion channel gating and trafficking of AMPAR. Here, I report high-resolution cryo-electron microscopy structures of AMPAR in complex with CNIH3. Contrary to its predicted membrane topology, CNIH3 lacks an extracellular domain and instead contains four membrane-spanning helices. The protein-protein interaction interface that dictates channel modulation and the lipids surrounding the complex are revealed. These structures provide insights into the molecular mechanism for ion channel modulation and assembly of AMPAR/CNIH3 complexes.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Acquisition and extinction of learned fear responses utilize conserved but flexible neural circuits. Here we show that acquisition of conditioned freezing behavior is associated with dynamic remodeling of relative excitatory drive from the basolateral amygdala (BLA) away from corticotropin releasing factor-expressing (CRF) centrolateral amygdala neurons, and toward non-CRF (CRF) and somatostatin-expressing (SOM) neurons, while fear extinction training remodels this circuit back toward favoring CRF neurons. Importantly, BLA activity is required for this experience-dependent remodeling, while directed inhibition of the BLA-centrolateral amygdala circuit impairs both fear memory acquisition and extinction memory retrieval. Additionally, ectopic excitation of CRF neurons impairs fear memory acquisition and facilities extinction, whereas CRF neuron inhibition impairs extinction memory retrieval, supporting the notion that CRF neurons serve to inhibit learned freezing behavior. These data suggest that afferent-specific dynamic remodeling of relative excitatory drive to functionally distinct subcortical neuronal output populations represents an important mechanism underlying experience-dependent modification of behavioral selection.
Complex circuit interactions within the nucleus accumbens (NAc) facilitate goal-directed behavior. Medium spiny neurons (MSNs) mediate NAc output by projecting to functionally divergent brain regions, a property conferred, in part, by the differential projection patterns of D1- and D2 dopamine receptor-expressing MSNs. Glutamatergic afferents to the NAc direct MSN output by recruiting feedforward inhibitory microcircuits comprised of parvalbumin (PV)-expressing interneurons (INs). Furthermore, the GABA heteroreceptor (GABAR), a G-coupled G-protein-coupled receptor, is expressed at glutamatergic synapses throughout the mesolimbic network, yet its physiological context and synaptic mechanism within the NAc remains unknown. Here, we explored GABAR function at glutamatergic synapses within PV-IN-embedded microcircuits in the NAc core of male mice. We found that GABAR is expressed presynaptically and recruits a noncanonical signaling mechanism to reduce glutamatergic synaptic efficacy at D1(+) and D1(-) (putative D2) MSN subtypes. Furthermore, PV-INs, a robust source of neuronal GABA in the NAc, heterosynaptically target GABAR to selectively modulate glutamatergic transmission onto D1(+) MSNs. These findings elucidate a new mechanism of feedforward inhibition and refine mechanisms by which GABA heteroreceptors modulate mesolimbic circuit function. Glutamatergic transmission in the nucleus accumbens (NAc) critically contributes to goal-directed behaviors. However, intrinsic microcircuit mechanisms governing the integration of these synapses remain largely unknown. Here, we show that parvalbumin-expressing interneurons within feedforward microcircuits heterosynaptically target GABA heteroreceptors (GABAR) on glutamate terminals. Activation of presynaptically-expressed GABAR decreases glutamatergic synaptic strength by engaging a non-canonical signaling pathway that interferes with vesicular exocytotic release machinery. These findings offer mechanistic insight into the role of GABA heteroreceptors within reward circuitry, elucidate a novel arm to feedforward inhibitory networks, and inform the growing use of GABAR-selective pharmacotherapy for various motivational disorders, including addiction, major depressive disorder, and autism (Cousins et al., 2002; Kahn et al., 2009; Jacobson et al., 2018; Stoppel et al., 2018; Pisansky et al., 2019).
Copyright © 2019 the authors.
BACKGROUND - Relapse is a critical barrier to effective long-term treatment of alcoholism, and stress is often cited as a key trigger to relapse. Numerous studies suggest that stress-induced reinstatement to drug-seeking behaviors is mediated by norepinephrine (NE) and corticotropin-releasing factor (CRF) signaling interactions in the bed nucleus of the stria terminalis (BNST), a brain region critical to many behavioral and physiologic responses to stressors. Here, we sought to directly examine the effects of NE on BNST CRF neuron activity and determine whether these effects may be modulated by chronic intermittent EtOH (CIE) exposure or a single restraint stress.
METHODS - Adult male CRF-tomato reporter mice were treatment-naïve, or either exposed to CIE for 2 weeks or to a single 1-hour restraint stress. Effects of application of exogenous NE on BNST CRF neuron activity were assessed via whole-cell patch-clamp electrophysiological techniques.
RESULTS - We found that NE depolarized BNST CRF neurons in naïve mice in a β-adrenergic receptor (AR)-dependent mechanism. CRF neurons from CIE- or stress-exposed mice had significantly elevated basal resting membrane potential compared to naïve mice. Furthermore, CIE and stress individually disrupted the ability of NE to depolarize CRF neurons, suggesting that both stress and CIE utilize β-AR signaling to modulate BNST CRF neurons. Neither stress nor CIE altered the ability of exogenous NE to inhibit evoked glutamatergic transmission onto BNST CRF neurons as shown in naïve mice, a mechanism previously shown to be α-AR-dependent.
CONCLUSIONS - Altogether, these findings suggest that stress and CIE interact with β-AR signaling to modulate BNST CRF neuron activity, potentially disrupting the α/β-AR balance of BNST CRF neuronal excitability. Restoration of α/β-AR balance may lead to novel therapies for the alleviation of many stress-related disorders.
© 2019 by the Research Society on Alcoholism.
BACKGROUND - Cognitive impairment (CI) profoundly impacts quality of life for patients with multiple sclerosis (MS). Dysfunctional regulation of glutamate in gray matter (GM) has been implicated in the pathogenesis of MS by post-mortem pathological studies and in CI by in vivo magnetic resonance spectroscopy, yet GM pathology is subtle and difficult to detect using conventional T- and T-weighted magnetic resonance imaging (MRI). There is a need for high-resolution, clinically accessible imaging techniques that probe molecular changes in GM.
OBJECTIVE - To study cortical GM pathology related to CI in MS using glutamate-sensitive chemical exchange saturation transfer (GluCEST) MRI at 7.0 Tesla (7T).
METHODS - A total of 20 patients with relapsing-remitting MS and 20 healthy controls underwent cognitive testing, anatomical imaging, and GluCEST imaging. Glutamate-sensitive image contrast was quantified for cortical GM, compared between cohorts, and correlated with clinical measures of CI.
RESULTS AND CONCLUSION - Glutamate-sensitive contrast was significantly increased in the prefrontal cortex of MS patients with accumulated disability ( < 0.05). In addition, glutamate-sensitive contrast in the prefrontal cortex was significantly correlated with symbol digit modality test ( = -0.814) and choice reaction time ( = 0.772) scores in patients ( < 0.05), suggesting that GluCEST MRI may have utility as a marker for GM pathology and CI.
KEY POINTS - The ClC-3 2Cl /1H exchanger modulates endosome pH and Cl concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, I ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less "efficient", as reflected by a large Q/I . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Glu ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a "closed" inner gate conformation that maintains coupling during cycling.
ABSTRACT - We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (I ), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/I ratio, but an indistinguishable Cl /H coupling ratio. External SCN reduced H transport rate and uncoupled anion/H exchange by 80-90%. Removal of the external gating glutamate ("Glu ") (E224A mutation) reduced Q and abolished H transport. We hypothesized that Methionine 531 (M531) impedes "water wire" H transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/I ratio by 50% and enhanced H transport. External protons (pH 5.0) inhibited I and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl /H coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Glu adopting an outward/open position. Internal "anion gate" removal (Y572S) dramatically increased I and impaired coupling, without slowing H transport rate. Loss of both gates (Y572S/E224A) resulted in a large "open pore" conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/I must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H coupling as well as anion selectivity, and interacts directly with E224. Disruption of this "closed gate" conformation by internal protons may represent a critical step in the ClC-3 transport cycle.
© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.
Allosteric modulation of metabotropic glutamate receptor 2 (mGlu) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu allosteric modulators, we developed and characterized a novel mGlu positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu allosteric site labeled with the novel mGlu PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
Current clinical treatment of infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)--(PHLG-MHH), bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6--(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against , including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill in the stomach while not harming the commensal bacteria/normal tissues.
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 , the rate of the "fast" phase of the enzyme reduction by the redox partner NADPH-cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our data, along with those from previous cell culture and studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.