Calcium Channels in the Nuclear Envelope of Pyramidal Neurons of the Rat Hippocampus

As is known, an increase in the concentration of Са²⁺ in the nuclei of nerve cells leads to activation of genes responsible for the formation of long-lasting postsynaptic changes; mechanisms of memory and learning are based on such changes. The pathways necessary for the entry of calcium into the nuclei of hippocampal pyramidal neurons remained unstudied. Using a patch-clamp technique, we studied what types of calcium channels exist in the membranes of isolated nuclei of pyramidal neurons of the hippocampal СА1 area. In the inner nuclear membrane of these cells, we, for the first time, found inositol trisphosphate receptors (IP₃Rs) activated by inositol trisphosphate applied in the concentration of ≥0.1 μM. The conductivity of single channels of such receptors was, on average, 366 pS; these channels were permeable for both monovalent and bivalent cations. Our data indicate that the nuclear envelope of pyramidal neurons of the hippocampal СА1 area can play the role of the calcium store from which Са²⁺ enter the cell nucleus directly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 288–292, July–August, 2008.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

Puffs are local Ca²⁺ signals that arise by Ca²⁺ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP₃Rs). The locations of puff sites observed by Ca²⁺ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP₃Rs have shown that the majority of IP₃Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP₃Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP₃R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s⁻¹, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP₃Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP₃R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP₃Rs were apparent following activation of IP₃/Ca²⁺ signaling. We conclude that stable clusters of small numbers of immotile IP₃Rs may underlie local Ca²⁺ release sites, whereas the more numerous motile IP₃Rs appear to be functionally silent.     

Effects of Ginkgo Biloba Extract Constituents on Glycine-Activated Strychnine-Sensitive Receptors in Hippocampal Pyramidal Neurons of the Rat

Active compounds of Ginkgo biloba extract (ginkgolides A, B, C, and J) were tested on the main ligand-operated conductances in rat neurons using a patch-clamp technique in the whole-cell configuration and a concentration-clamp technique. It was found that all ginkgolides reduced glycine-activated currents in concentration- and use-dependent manners, whereas they did not affect other tested ligand-gated receptors (NMDA- and GABA-activated ones). The IC₅₀ values calculated from dose-response curves were as follows: 1.97, 0.273, 0.267, and 2.0 M for ginkgolides A, B, C, and J, respectively (200 M Gly ).The Hill coefficient in all cases was close to 1.0, which indicates a single site of the drug binding to the glycine-activated receptor.     

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP₃R) channel function. The residues selected were highly conserved in all three IP₃R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP₃R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

Determining the Roles of Inositol Trisphosphate Receptors in Neurodegeneration: Interdisciplinary Perspectives on a Complex Topic

It is well known that calcium (Ca²⁺) is involved in the triggering of neuronal death. Ca²⁺ cytosolic levels are regulated by Ca²⁺ release from internal stores located in organelles, such as the endoplasmic reticulum. Indeed, Ca²⁺ transit from distinct cell compartments follows complex dynamics that are mediated by specific receptors, notably inositol trisphosphate receptors (IP3Rs). Ca²⁺ release by IP3Rs plays essential roles in several neurological disorders; however, details of these processes are poorly understood. Moreover, recent studies have shown that subcellular location, molecular identity, and density of IP3Rs profoundly affect Ca²⁺ transit in neurons. Therefore, regulation of IP3R gene products in specific cellular vicinities seems to be crucial in a wide range of cellular processes from neuroprotection to neurodegeneration. In this regard, microRNAs seem to govern not only IP3Rs translation levels but also subcellular accumulation. Combining new data from molecular cell biology with mathematical modelling, we were able to summarize the state of the art on this topic. In addition to presenting how Ca²⁺ dynamics mediated by IP3R activation follow a stochastic regimen, we integrated a theoretical approach in an easy-to-apply, cell biology-coherent fashion. Following the presented premises and in contrast to previously tested hypotheses, Ca²⁺ released by IP3Rs may play different roles in specific neurological diseases, including Alzheimer’s disease and Parkinson’s disease.     

The Inositol Trisphosphate Receptor in the Control of Autophagy

The second messenger myo-inositol-1,4,5-trisphosphate (IP₃) acts on the IP₃ receptor (IP₃R), an IP3-activated Ca²⁺ channel of the endoplasmic reticulum (ER). The IP₃R agonist IP3 inhibits starvation-induced autophagy. The IP₃R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-X_L, two proteins that physically interact with IP₃R. Autophagy can also be induced by depletion of the IP₃R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca²⁺ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP₃R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Addendum to:

Regulation of Autophagy by the Inositol Trisphosphate Receptor

A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. Kroemer

Cell Death Differ 2007; In press     

Endocannabinoids Differentially Modulate Synaptic Plasticity in Rat Hippocampal CA1 Pyramidal Neurons

Background

Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.

Methodology/Principal Findings

By employing somatic and dendritic patch-clamp recordings, Ca²⁺ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca²⁺-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.

Conclusions/Significance

Our results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.     

Initial Characterization of the Nicotinic Acetylcholine Receptors in Rat Hippocampal Neurons

Abstract

The properties of the neuronal nicotinic acetylcholine receptor in primary cultures of hippocampal cells from fetal rats (17–18 days gestation) were studied using the whole-cell patch-clamp technique in Na⁺-external, Cs⁺-internal and nominally Mg²⁺-free solutions. The nicotinic agonists acetylcholine, (+)anatoxin-a, and (-) and (+)nicotine all evoked inward whole-cell currents in hippocampal neurons that were voltage clamped near their resting potentials. Sensitivity to (+)anatoxin-a was first detected at around day 6, and thereafter the magnitude of the response increased as a function of number of days in culture up to about 40 days. The whole-cell current waveforms consisted of more than one peak whose relative amplitude depended on the agonist concentration. These currents were reversibly blocked by micromolar concentrations of d-tubocurarine, mecamylamine, and dihydro-β-erythroidine. At nanomolar concentrations, neuronal bungarotoxin, α-bungarotoxin and α-cobratoxin caused an irreversible blockade of the currents but they were unaffected by tetrodotoxin, atropine, DL-2-amino-5-phosphonovaleric acid, Mg²⁺, and 6,7-dinitroquinoxaline-2,3-dione. In addition, the currents were also blocked in a reversible manner by methyllycaconitine at picomolar concentration. The current-voltage plots elicited by both (+)anatoxin-a and acetylcholine revealed larger inward currents and smaller or no outward currents. The present results demonstrate the existence of an inwardly rectifying, snake neurotoxin-sensitive functional nicotinic acetylcholine receptor ion channel in rat hippocampal neurons.     

Ablation of NMDA Receptors Enhances the Excitability of Hippocampal CA3 Neurons

Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.     

Minireview: Nicotinic Acetylcholine Receptors on Hippocampal Neurons: Distribution on the Neuronal Surface and Modulation of Receptor Activity

Abstract

The recent development of a technique that uses infrared microscopy for the visualization of well-defined areas on the surface of neurons, and a computerized system of micromanipulators led to the discovery that functional nicotinic acetylcholine receptors (nAChRs) are expressed at higher density on the dendrites than on the soma of rat hippocampal neurons. The finding that the expression of α-bungarotoxin-sensitive, α7-bearing, nAChRs and dihydro-β-erythroidine-sensitive,α4β2 nAChRs tends to increase along the dendritic length suggests that these receptors may be highly involved in the integration of synaptic functions in hippocampal neurons. The present report also discusses the finding that ligands such as the anticholinesterase galanthamine can modulate the nAChR activity by binding to a novel receptor site, and that 5-hydroxytryptamine (5-HT) may serve as an endogenous ligand for this site. The ability of 5-HT to modulate the nAChR function in vivo supports the concept that the overall CNS function is determined not only by the neuronal network established by the neuronal wiring, but also by a chemical network established by the ability of a single substance to act as the primary neurotransmitter in one system and as a co-transmitter in another system.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.