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N -methyl- d -aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer’s disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration (“membrane-to-channel inhibition” (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism. Wilcox et al. (2022) show that NMDA receptor channel blockers, some of which are clinically important drugs, can access their binding site via 2 routes: a well-known path from the extracellular solution, and another path through the plasma membrane.

Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na and substrate binding. The activation of the anion channel by binding of Na alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na -dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or \"leak\" channel activity. Choline or N-methyl-D-glucamine replacement of external Na ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na -independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

To understand single neuron computation, it is necessary to know how specific physiological parameters affect neural spiking patterns that emerge in response to specific stimuli. Here we present a computational pipeline combining biophysical and statistical models that provides a link between variation in functional ion channel expression and changes in single neuron stimulus encoding. More specifically, we create a mapping from biophysical model parameters to stimulus encoding statistical model parameters. Biophysical models provide mechanistic insight, whereas statistical models can identify associations between spiking patterns and the stimuli they encode. We used public biophysical models of two morphologically and functionally distinct projection neuron cell types: mitral cells (MCs) of the main olfactory bulb, and layer V cortical pyramidal cells (PCs). We first simulated sequences of action potentials according to certain stimuli while scaling individual ion channel conductances. We then fitted point process generalized linear models (PP-GLMs), and we constructed a mapping between the parameters in the two types of models. This framework lets us detect effects on stimulus encoding of changing an ion channel conductance. The computational pipeline combines models across scales and can be applied as a screen of channels, in any cell type of interest, to identify ways that channel properties influence single neuron computation.

Summary Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5+ cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5+ amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Muller glia from Lgr5+ amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5+ amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age-related retinal degenerative diseases.

NMDA receptors (NMDARs), a subfamily of ionotropic glutamate receptors, have unique biophysical properties including high permeability to Ca2+. Activation of NMDARs increases the concentration of intracellular Ca2+ that can activate a vast array of signaling pathways. NMDARs are necessary for many processes including synaptic plasticity, dendritic integration, and cell survival. Aberrant NMDAR activation is implicated in many central nervous system disorders including neurodegenerative disorders, neuronal loss following ischemia, and neuropsychiatric disorders. Hope that NMDARs may serve as useful therapeutic targets is bolstered by the clinical success of two NMDAR antagonists, memantine and ketamine. Memantine and ketamine act as open channel blockers of the NMDAR-associated ion channel, and exhibit similar IC50 values and kinetics. Memantine is approved for treatment of Alzheimer's disease and shows promise in treatments of Huntington's disease, and ischemia. Ketamine was initially approved for use as a general anesthetic, but has recently shown efficacy in treatment of depression and of pain. Notably, memantine is not effective in treatment of depression or pain. In addition, memantine is well tolerated, whereas ketamine induces psychotomimetic side effects. The basis for the divergent clinical profiles of memantine and ketamine is not clear. One recently-proposed hypothesis is that memantine and ketamine inhibit overlapping but distinct subpopulations of NMDARs. However, mechanisms underlying inhibition of distinct NMDAR subpopulations by memantine or by ketamine are not fully understood. We therefore examined and compared mechanisms of inhibition by memantine and by ketamine. We also describe a novel fast perfusion system optimized for brief synaptic-like glutamate applications to lifted cells. We found that: (1) inhibition by memantine and ketamine exhibit differential dependence on duration of receptor activation and on NMDAR subtype; (2) the dependence of memantine inhibition on duration of NMDAR activation results from stabilization of a Ca2+-dependent desensitized state; (3) the endogenous NMDAR open channel blocker Mg2+ slows the binding kinetics of both memantine and ketamine, and, unexpectedly, speeds recovery from memantine inhibition; (4) although inhibition by memantine was thought to be mediated by only the charged form of memantine, the uncharged form of memantine also binds to and inhibits NMDARs, and exhibits surprisingly slow unbinding kinetics.