Gyorgy Lur

Compartmentalization of GABAergic Inhibition by Dendritic Spines

Dendritic Precision Strikes The effects of excitatory synaptic inputs are considered to be highly compartmentalized because of the biophysical properties of dendritic spines. Individual inhibitory synapses, however, are thought to affect dendritic integration in a more extended spatial region. Combining optogenetic stimulation of dendrite-targeting γ-aminobutyric acid—mediated interneurons with two-photon calcium imaging in postsynaptic pyramidal cell dendrites, Chiu et al. (p. 759) challenge this latter view. The findings suggest that the effect of an inhibitory synapse can be as compartmentalized as that of an excitatory synapse, provided that the synapses are localized on spine heads. Inhibitory synapses can control individual dendritic spines independently from their neighbors. γ-aminobutyric acid–mediated (GABAergic) inhibition plays a critical role in shaping neuronal activity in the neocortex. Numerous experimental investigations have examined perisomatic inhibitory synapses, which control action potential output from pyramidal neurons. However, most inhibitory synapses in the neocortex are formed onto pyramidal cell dendrites, where theoretical studies suggest they may focally regulate cellular activity. The precision of GABAergic control over dendritic electrical and biochemical signaling is unknown. By using cell type-specific optical stimulation in combination with two-photon calcium (Ca2+) imaging, we show that somatostatin-expressing interneurons exert compartmentalized control over postsynaptic Ca2+ signals within individual dendritic spines. This highly focal inhibitory action is mediated by a subset of GABAergic synapses that directly target spine heads. GABAergic inhibition thus participates in localized control of dendritic electrical and biochemical signaling.

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Summary Store-operated Ca2+ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2–8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP3) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca2+ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5, 10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca2+ release and Ca2+ reloading) and that any potential interference between Ca2+ release and Ca2+ influx is minimized by the spatial separation of the two processes.

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca2+. This excessive Ca2+ liberation induces Ca2+-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca2+ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca2+ concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca2+ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca2+ release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP3Rs) of types 2 and 3. POAEE evoked very little Ca2+ release and trypsin activation when IP3Rs of both types 2 and 3 were knocked out. Antibodies against IP3Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca2+ release and trypsin activation. We conclude that Ca2+ release through IP3Rs of types 2 and 3 in the acid granular Ca2+ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.

EGR1 Is a Target for Cooperative Interactions between Cholecystokinin and Leptin, and Inhibition by Ghrelin, in Vagal Afferent Neurons

Food intake is regulated by signals from peripheral organs, but the way these are integrated remains uncertain. Cholecystokinin (CCK) from the intestine and leptin from adipocytes interact to inhibit food intake. Our aim was to examine the hypothesis that these interactions occur at the level of vagal afferent neurons via control of the immediate early gene EGR1. We now report that CCK stimulates redistribution to the nucleus of early growth response factor-1 (EGR1) in these neurons in vivo and in culture, and these effects are not dependent on EGR1 synthesis. Leptin stimulates EGR1 expression; leptin alone does not stimulate nuclear translocation, but it strongly potentiates the action of CCK. Ghrelin inhibits CCK-stimulated nuclear translocation of EGR1 and leptin-stimulated EGR1 expression. Expression of the gene encoding the satiety peptide cocaine- and amphetamine-regulated transcript (CARTp) is stimulated by CCK via an EGR1-dependent mechanism, and this is strongly potentiated by leptin. Leptin potentiated inhibition of food intake by endogenous CCK in the rat in conditions reflecting changes in EGR1 activation. The data indicate that by separately regulating EGR1 activation and synthesis, CCK and leptin interact cooperatively to define the capacity for satiety signaling by vagal afferent neurons; manipulation of these interactions may be therapeutically beneficial.

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus–secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1–ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta—structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1–ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca2+ influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1–ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P2 depletion.

Stress Impairs Prefrontal Cortical Function via D1 Dopamine Receptor Interactions With Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

BACKGROUND Psychiatric disorders such as schizophrenia are worsened by stress, and working memory deficits are often a central feature of illness. Working memory is mediated by the persistent firing of prefrontal cortical (PFC) pyramidal neurons. Stress impairs working memory via high levels of dopamine D1 receptor (D1R) activation of cyclic adenosine monophosphate signaling, which reduces PFC neuronal firing. The current study examined whether D1R-cyclic adenosine monophosphate signaling reduces neuronal firing and impairs working memory by increasing the open state of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, which are concentrated on dendritic spines where PFC pyramidal neurons interconnect. METHODS A variety of methods were employed to test this hypothesis: dual immunoelectron microscopy localized D1R and HCN channels, in vitro recordings tested for D1R actions on HCN channel current, while recordings in monkeys performing a working memory task tested for D1R-HCN channel interactions in vivo. Finally, cognitive assessments following intra-PFC infusions of drugs examined D1R-HCN channel interactions on working memory performance. RESULTS Immunoelectron microscopy confirmed D1R colocalization with HCN channels near excitatory-like synapses on dendritic spines in primate PFC. Mouse PFC slice recordings demonstrated that D1R stimulation increased HCN channel current, while local HCN channel blockade in primate PFC protected task-related firing from D1R-mediated suppression. D1R stimulation in rat or monkey PFC impaired working memory performance, while HCN channel blockade in PFC prevented this impairment in rats exposed to either stress or D1R stimulation. CONCLUSIONS These findings suggest that D1R stimulation or stress weakens PFC function via opening of HCN channels at network synapses.

InsP3 receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2+ entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP3Rs (InsP3 receptors). The co-localization between Orai1 and IP3Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2+ store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2+ store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP3Rs and a basolateral pool that interacts with STIM1 following the Ca2+ store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai1 localization does not require IP3Rs and that IP3Rs are not necessary for the activation of SOCE. However, the InsP3-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP3Rs could have an inhibitory effect on this Ca2+ entry mechanism.

Hippocampal (CA1) activities in Wistar rats from different vendors Fundamental differences in acute ischemia

Two-vessel occlusion, a frequently used model of global cerebral ischemia in rats, results in a dysfunction predominantly within the CA1 field of the hippocampus; it induces many processes with different time-scales. However, the great divergence in the results of the studies reported in the literature suggests valuable differences in response to hypoperfusion-induced ischemia among the laboratory rats used in these studies. In the present work, the acute effects of two-carotid occlusion-induced global ischemia (2VO) on the CA3 stimulation-evoked population spike activity in the CA1 region of Wistar rats from different suppliers (Charles-River and Harlan) were compared. In the acute electrophysiological experiments, the hippocampal CA1 responses revealed that the Charles-River rats immediately compensated the 2VO much better than did the Harlan rats. However, 3 days later, no difference could be observed between the CA1 activities of these rats. The presented data show that the Wistar rats from different vendors represent an important source of variability in the results of acute experiments on the hippocampal ischemia. These observations draw attention to the importance of the careful choice of the laboratory rats (both strains and breeds) used in such experiments.

Glutamate Receptor Modulation Is Restricted to Synaptic Microdomains

A diverse array of neuromodulators governs cellular function in the prefrontal cortex (PFC) via the activation of G-protein-coupled receptors (GPCRs). However, these functionally diverse signals are carried and amplified by a relatively small assortment of intracellular second messengers. Here, we examine whether two distinct Gαi-coupled neuromodulators (norepinephrine and GABA) act as redundant regulators of glutamatergic synaptic transmission. Our results reveal that, within single dendritic spines of layer 5 pyramidal neurons, alpha-2 adrenergic receptors (α2Rs) selectively inhibit excitatory transmission mediated by AMPA-type glutamate receptors, while type B GABA receptors (GABA(B)Rs) inhibit NMDA-type receptors. We show that both modulators act via the downregulation of cAMP and PKA. However, by restricting the lifetime of active Gαi, RGS4 promotes the independent control of these two distinct target proteins. Our findings highlight a mechanism by which neuromodulatory microdomains can be established in subcellular compartments such as dendritic spines.