“Simplicity is prerequisite for reliability.”EW Dijkstra [1] Presynaptic action potentials trigger the fusion of vesicles to release neurotransmitter onto postsynaptic neurons. Each release site was originally thought to liberate at most one vesicle per action potential in a probabilistic fashion, rendering synaptic transmission unreliable. However, the simultaneous release of several vesicles, or multivesicular release (MVR), represents a simple mechanism to overcome the intrinsic unreliability of synaptic transmission. MVR was initially identified at specialized synapses but is now known to be common throughout the brain. MVR determines the temporal and spatial dispersion of transmitter, controls the extent of receptor activation, and contributes to adapting synaptic strength during plasticity and neuromodulation. MVR consequently represents a widespread mechanism that extends the dynamic range of synaptic processing.

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.

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In many brain regions, Ca(2+) influx through presynaptic P2X receptors influences GABA release from interneurones. In patch-clamp recordings of Purkinje cells (PCs) in rat cerebellar slices, broad spectrum P2 receptor antagonists, PPADS (30microM) or suramin (12microM), result in a decreased amplitude and increased failure rate of minimal evoked GABAergic synaptic currents from basket cells. The effect is mimicked by desensitizing P2X1/3-containing receptors with alpha,beta-methylene ATP. This suggests presynaptic facilitation of GABA release via P2XR-mediated Ca(2+) influx activated by endogenously released ATP. In contrast, activation of P2Y4 receptors (using UTP, 30microM, but not P2Y1 or P2Y6 receptor ligands) results in inhibition of GABA release. Immunological studies reveal the presence of most known P2Rs in >or=20% of GABAergic terminals in the cerebellum. P2X3 receptors and P2Y4 receptors occur in approximately 60% and 50% of GABAergic synaptosomes respectively and are localized presynaptically. Previous studies report that PC output is also influenced by postsynaptic purinergic receptors located on both PCs and interneurones. The high Ca(2+) permeability of the P2X receptor and the ability of ATP to influence intracellular Ca(2+) levels via P2Y receptor-mediated intracellular pathways make ATP the ideal transmitter for the multisite bidirectional modulation of the cerebellar cortical neuronal network.

Parvalbumin-expressing interneurons (PVs) in the dentate gyrus provide activity-dependent regulation of adult neurogenesis as well as maintain inhibitory control of mature neurons. In mature neurons, PVs evoke GABAA postsynaptic currents (GPSCs) with fast rise and decay phases that allow precise control of spike timing, yet synaptic currents with fast kinetics do not appear in adult-born neurons until several weeks after cell birth. Here we used mouse hippocampal slices to address how PVs signal to newborn neurons prior to the appearance of fast GPSCs. Whereas PV-evoked currents in mature neurons exhibit hallmark fast rise and decay phases, newborn neurons display slow GPSCs with characteristics of spillover signaling. We also unmasked slow spillover currents in mature neurons in the absence of fast GPSCs. Our results suggest that PVs mediate slow spillover signaling in addition to conventional fast synaptic signaling, and that spillover transmission mediates activity-dependent regulation of early events in adult neurogenesis.

Previous studies showed that the serum-and glucocorticoid-inducible kinase (sgk) gene plays an important role in long-term memory formation. The present study further examined the role of SGK in long-term potentiation (LTP). The dominant-negative mutant of sgk, SGKS422A, was used to inactivate SGK. Results revealed a time-dependent increase in SGK phosphorylation after tetanization with a significant effect observed 3 h and 5 h later. Transfection of SGKS422A impaired the expression, but not the induction, of LTP. Furthermore, the constitutively active sgk, SGKS422D, up-regulated postsynaptic density-95 expression in the hippocampus. These results together support the role of SGK in neuronal plasticity.The serum-and glucocorticoid-inducible kinase (sgk) gene is a member of the serine/threonine protein kinase gene family that is transcriptionally induced by glucocorticoid and serum (Webster et al. 1993). The sgk gene was well known for its role in regulation of sodium transport (Naray-Fejes-Toth et al. 2000;Pearce et al. 2000) and cell volume (Waldegger et al. 1997) in renal epithelia. But its role in the central nervous system is less known. In our previous study, we have shown that the sgk gene plays an important role in memory formation of spatial learning in rats. The expression level of this gene is significantly higher in the dorsal hippocampus of fast learners than slow learners from the water maze learning task (Tsai et al. 2002). Furthermore, transient transfection of the dominant-negative mutant of sgk, SGKS422A, to hippocampal CA1 neurons markedly impaired, whereas transfection of the sgk wild-type DNA facilitated spatial memory formation in rats (Tsai et al. 2002).More recently, we have found that environmental enrichment training significantly enhanced sgk expression in the hippocampus. Enrichment training also reversed SGKS422A mutant DNA-induced impairment in spatial learning, fear-conditioning learning, and novel object-recognition learning in rats (Lee et al. 2003). These results together suggest that SGK may play an important role in neuronal plasticity. In the present study, we further examined this issue by studying the role of SGK in long-term potentiation (LTP), a synaptic model for long-term memory formation (Bliss and Collingridge 1993), in hippocampal neurons.The dominant-negative form of SGK (SGKS422A) and the constitutively active form of SGK (SGKS422D) (Kobayashi and Cohen 1999) were used as molecular tools to inactivate and activate SGK at Ser422, respectively. SGKS422A was transfected to the rat CA1 area, and these animals were sacrificed at different time points after DNA transfection. SGK phosphorylation at Ser422 was evaluated by Western blot using the specific phospho (p)422SGK antibody. Results revealed that SGKS422A transfection produced a significant decrease in SGK Ser422 phosphorylation 48 h and 72 h later (F 5,12 = 38.22, P < 0.01, tD = 4.56 and 9.5, respectively, P < 0.05 and P < 0.01) (Fig. 1A,B). This effect was recovered 96 h after DNA transfection (tD = 1.81, P > 0.05)...

We have previously demonstrated that serum- and glucocorticoid-inducible kinase (SGK) plays a causal role in facilitating memory formation of spatial learning in rats, but the SGK signaling pathway involved in spatial memory formation is not known. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) also plays an important role in memory formation. We therefore examined whether SGK is a downstream target of the MAPK/ERK signaling cascade and whether ERK signaling to SGK mediates spatial memory formation in rats. Results from an in vitro kinase assay revealed that ERK directly phosphorylates SGK at Ser78, but not at Thr256 and Ser422, whereas inhibition of ERK by PD98059 significantly decreased SGK phosphorylation at Ser78, Thr256 and Ser422 following spatial training. Prior administration of PD98059 also antagonized the enhancing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator that also causes ERK activation, on SGK phosphorylation and cAMP response element binding protein (CREB) phosphorylation. Moreover, TPA-induced SGK phosphorylation and CREB phosphorylation was abolished by prior SGKS78A mutant DNA transfection. By contrast, SGKS78A mutant DNA transfection to hippocampal area CA1 did not affect spatial memory formation, whereas SGKT256A mutant DNA transfection to area CA1 significantly impaired spatial memory formation. ERK was known to regulate sgk mRNA expression, but in the present study we have demonstrated that SGK is also a downstream target of the ERK signaling cascade; ERK directly phosphorylates SGK at Ser78 and indirectly activates SGK at Thr256 and Ser422 through unknown intermediate molecules. Furthermore, ERK activation of SGK is involved in spatial memory formation in rats.

In the central nervous system, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuro-glial signaling.