Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors.

In the primate retina the small bistratified, "blue-yellow" color-opponent ganglion cell receives parallel ON-depolarizing and OFFhyperpolarizing inputs from short (S)-wavelength sensitive and combined long (L)-and middle (M)-wavelength sensitive cone photoreceptors, respectively. However, the synaptic pathways that create S versus LM cone-opponent receptive field structure remain controversial. Here, we show in the macaque monkey retina in vitro that at photopic light levels, when an identified rod input is excluded, the small bistratified cell displays a spatially coextensive receptive field in which the S-ON-input is in spatial, temporal, and chromatic balance with the LM-OFF-input. ON pathway block with L-AP-4, the mGluR6 receptor agonist, abolished the S-ON response but spared the LM-OFF response. The isolated LM component showed a center-surround receptive field structure consistent with an input from OFFcenter, ON-surround "diffuse" cone bipolar cells. Increasing retinal buffering capacity with HEPES attenuated the LM-ON surround component, consistent with a non-GABAergic outer retina feedback mechanism for the bipolar surround. The GABAa/c receptor antagonist picrotoxin and the glycine receptor antagonist strychnine did not affect chromatic balance or the basic coextensive receptive field structure, suggesting that the LM-OFF field is not generated by an inner retinal inhibitory pathway. We conclude that the opponent S-ON and LM-OFF responses originate from the excitatory receptive field centers of S-ON and LM-OFF cone bipolar cells, and that the LM-OFFand ON-surrounds of these parallel bipolar inputs largely cancel, explaining the small, spatially coextensive but spectrally antagonistic receptive field structure of the blue-ON ganglion cell.

SUMMARY Exogenously-expressed opsins are valuable tools for optogenetic control of neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here we develop a comprehensive optogenetic toolkit for controlling GABAA receptor-mediated inhibition in the brain. We synthesized a series of photoswitch ligands and the complementary genetically-modified GABAA receptor subunits. By conjugating the two components we generated light-sensitive versions of the entire GABAA receptor family. We validate these light-sensitive receptors for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, we generated a knock-in mouse in which the “photoswitch-ready” version of a GABAA receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABAA receptor function with high spatial, temporal, and biochemical precision.

Negative feedback from horizontal cells to cone photoreceptors is regarded as the critical pathway for the formation of the antagonistic surround of retinal neurons, yet the mechanism by which horizontal cells accomplish negative feedback has been difficult to determine. Recent evidence suggests that feedback uses a novel, non-GABAergic pathway that directly modulates the calcium current in cones. In non-mammalian vertebrates, enrichment of retinal pH buffering capacity attenuates horizontal cell feedback, supporting one model in which feedback occurs by horizontal cell modulation of the extracellular pH in the cone synaptic cleft. Here we test the effect of exogenous pH buffering on the response dynamics of H1 horizontal cells and the center-surround receptive field structure of parasol ganglion cells in the macaque monkey retina. Enrichment of the extracellular buffering capacity with HEPES selectively attenuates surround antagonism in parasol ganglion cells. The H1 horizontal cell light response includes a slow, depolarizing component that is attributed to negative feedback to cones. This part of the response is attenuated by HEPES and other pH buffers in a dose-dependent manner that is correlated with predicted buffering capacity. The selective effects of pH buffering on the parasol cell surround and H1 cell light response suggests that, in primate retina, horizontal cell feedback to cones is mediated via a pH-dependent mechanism and is a major determinant of the ganglion cell receptive field surround.

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Proteins of the major histocompatibility complex class I (MHCI) are known for their role in immunity and have recently been implicated in long-term plasticity of excitatory synaptic transmission. However, the mechanisms by which MHCI influences synaptic plasticity remain unknown. Here we show that endogenous MHCI regulates synaptic responses mediated by NMDA-type glutamate receptors (NMDARs) in the mammalian central nervous system (CNS). The AMPA/NMDA ratio is decreased at MHCI-deficient hippocampal synapses, reflecting an increase in NMDAR-mediated currents. This enhanced NMDAR response is not associated with changes in the levels, subunit composition, or gross subcellular distribution of NMDARs. Increased NMDAR-mediated currents in MHCI-deficient neurons are associated with characteristic changes in AMPA receptor trafficking in response to NMDAR activation. Thus, endogenous MHCI tonically inhibits NMDAR function and controls downstream NMDAR-induced AMPA receptor trafficking during the expression of plasticity.immune | GluR | hippocampus P roteins of the major histocompatibility complex class I (MHCI) are best known for their role in adaptive immunity, but several lines of evidence suggest they also have nonimmune functions in neurons (1, 2). MHCI is expressed by healthy neurons in the developing and adult CNS (3-7). Neuronal MHCI mRNA levels are dynamic during development and are regulated by electrical activity (3, 4) and by the cAMP-response element-binding protein (CREB) (8). MHCI protein is enriched in synaptic fractions (4) and is detected in hippocampal dendritic spines, where it colocalizes with PSD-95 (9).Studies in mice genetically deficient for cell-surface MHCI (β2m −/− TAP −/− mice) suggest a role for MHCI in activity-dependent plasticity. In MHCI-deficient mice, NMDA receptor (NMDAR)-dependent hippocampal long-term potentiation (LTP) is enhanced, whereas long-term depression (LTD) is abolished (4). Although the mechanisms by which MHCI mediates immune signaling have been relatively well characterized, nothing is known about how MHCI contributes to NMDAR-dependent plasticity in vitro or in vivo.In the adult hippocampus, plasticity induced by activation of NMDARs is expressed as changes in the trafficking and function of AMPA receptors (AMPARs) (10-13). In current models, the magnitude and kinetics of NMDAR activation determine whether potentiation or depression is induced, with large, transient NMDAR activation causing LTP and smaller, longer-lasting activation causing LTD (14, 15). Therefore, to better understand the role of endogenous MHCI in the induction or expression of synaptic plasticity, we examined the levels, distribution, trafficking, and function of AMPA-and NMDA-type receptors in MHCI-deficient hippocampal neurons.The current experiments reveal an unexpected role for postsynaptic MHCI in controlling NMDAR function. Loss of MHCI causes a drop in the AMPA/NMDA ratio and an enhancement of NMDAR-mediated responses at CA3-CA1 synapses. This enhancement cannot be attributed to changes in th...

Lateral inhibition at the first synapse in the retina is important for visual perception, enhancing image contrast, color discrimination, and light adaptation. Despite decades of research, the feedback signal from horizontal cells to photoreceptors that generates lateral inhibition remains uncertain. GABA, protons, or an ephaptic mechanism have all been suggested as the primary mediator of feedback. However, the complexity of the reciprocal cone to horizontal cell synapse has left the identity of the feedback signal an unsolved mystery.

Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.