Long-term plasticity in interneurons of the dentate gyrus

Single interneurons influence thousands of postsynaptic principal cells, and the control of interneuronal excitability is an important regulator of the computational properties of the hippocampus. However, the mechanisms underlying long-term alterations in the input–output functions of interneurons are not fully understood. We report a mechanism of interneuronal plasticity that leads to the functional enhancement of the gain of glutamatergic inputs in the absence of long-term potentiation of the excitatory synaptic currents. Interneurons in the dentate gyrus exhibit a characteristic, limited (≈8 mV) depolarization of their resting membrane potential after high-frequency stimulation of the perforant path. The depolarization can be observed with either whole-cell or perforated patch electrodes, and it lasts in excess of 3 h. The long-term depolarization is specific to interneurons, because granule cells do not show it. The depolarization requires the activation of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the rise of intracellular Ca2+, but not N-methyl-d-aspartate (NMDA) receptor activation. Data on the maintenance of the depolarization point to a major role for a long-term change in the rate of electrogenic Na+/K+-ATPase pump function in interneurons. As a result of the depolarization, interneurons after the tetanus respond with action potential discharges to previously subthreshold excitatory postsynaptic potentials (EPSPs), even though the EPSPs are not potentiated. These results demonstrate that the plastic nature of the interneuronal resting membrane potential underlies a unique form of long-term regulation of the gain of excitatory inputs to γ-aminobutyric acid (GABA)ergic neurons.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.

Expression of the human protooncogene bcl-2 protects neural cells from death induced by many forms of stress, including conditions that greatly elevate intracellular Ca2+. Considering that Bcl-2 is partially localized to mitochondrial membranes and that excessive mitochondrial Ca2+ uptake can impair electron transport and oxidative phosphorylation, the present study tested the hypothesis that mitochondria from Bcl-2-expressing cells have a higher capacity for energy-dependent Ca2+ uptake and a greater resistance to Ca(2+)-induced respiratory injury than mitochondria from cells that do not express this protein. The overexpression of bcl-2 enhanced the mitochondrial Ca2+ uptake capacity using either digitonin-permeabilized GT1-7 neural cells or isolated GT1-7 mitochondria by 1.7 and 3.9 fold, respectively, when glutamate and malate were used as respiratory substrates. This difference was less apparent when respiration was driven by the oxidation of succinate in the presence of the respiratory complex I inhibitor rotenone. Mitochondria from Bcl-2 expressors were also much more resistant to inhibition of NADH-dependent respiration caused by sequestration of large Ca2+ loads. The enhanced ability of mitochondria within Bcl-2-expressing cells to sequester large quantities of Ca2+ without undergoing profound respiratory impairment provides a plausible mechanism by which Bcl-2 inhibits certain forms of delayed cell death, including neuronal death associated with ischemia and excitotoxicity.

Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

The timing of synaptic vesicle endocytosis.

Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Cone photoreceptors respond to their own glutamate release in the tiger salamander.

Pulse-like currents resembling miniature postsynaptic currents were recorded in patch-clamped isolated cones from the tiger salamander retina. The events were absent in isolated cones without synaptic terminals. The frequency of events was increased by either raising the osmotic pressure or depolarizing the cell. It was decreased by the application of either glutamate or the glutamate-transport blockers dihydrokainate and D,L-threo-3-hydroxyaspartate. The events required external Na+ for which Li+ could not substitute. The reversal potential of these currents followed the equilibrium potential for Cl- when internal Cl- concentration was changed. Thus, these miniature currents appear to represent the presynaptic activation of the glutamate receptor with glutamate transporter-like pharmacology, caused by the photoreceptor's own vesicular glutamate release. Using a noninvasive method to preserve the intracellular Cl- concentration, we showed that glutamate elicits an outward current in isolated cones. Fluorescence of the membrane-permeable form of fura-2 was used to monitor Ca2+ entry at the cone terminal as a measure of membrane depolarization. The increase in intracellular Ca2+ concentration, elicited by puff application of 30 mM KCl, was completely suppressed in the presence of 100 microM glutamate. Puff application of glutamate alone had no measurable depolarizing effect. These results suggest that the equilibrium potential for Cl-, ECl, was more negative than the activation range for Ca2+ channels and that glutamate elicited an outward current, hyperpolarizing the cones.

Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors are ligand-gated channels that release intracellular Ca2+ stores in response to the second messenger, IP3. We investigated the potential role of IP3 receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca2+ mobilization is required for nuclear vesicle fusion and implicated IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibited IP3-stimulated CA2+ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP3. Because nuclear vesicle fusion was inhibited by antibodies that block Ca2+ flux, but not by control and preimmune antibodies, we concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

Extrapituitary expression of the rat V1b vasopressin receptor gene.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production

Introduction: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Materials and Methods: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappa B and activator protein-1 (AP-1) activity, Western blotting for phosphoextracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. Results: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappa B activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Conclusion: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.