New Context Significantly Changes Expression of Irs2 Gene in Hippocampal Areas.

Memory formation is a complex process involving changes in the synaptic activity and gene expression encoding the insulin-like growth factors. We analyzed changes in the expression of genes encoding the insulin/insulin-like growth factors' proteins at the early period of learning in the CA1 region and dentate gyrus of the dorsal and ventral hippocampus in mice 1 hour after presentation of a new context (contextual fear conditioning) with and without negative reinforcement. It was found that in addition to changes in the expression of immediate early genes c-Fos (in all studied hippocampal fields) and Arc (in dorsal and ventral CA1, as well as in dorsal dentate gyrus), exposure to a new context significantly altered expression of the insulin receptor substrate 2 gene (Irs2) in dorsal CA1 and ventral dentate gyrus irrespectively of the negative reinforcement, which suggests participation of the insulin/IGF system in the early stages of neural activation during learning.

Amyloid Aß25-35 Aggregates Say 'NO' to Long-Term Potentiation in the Hippocampus through Activation of Stress-Induced Phosphatase 1 and Mitochondrial Na+/Ca2+ Exchanger.

The search for strategies for strengthening the synaptic efficiency in Aß25-35-treated slices is a challenge for the compensation of amyloidosis-related pathologies. Here, we used the recording of field excitatory postsynaptic potentials (fEPSPs), nitric oxide (NO) imaging, measurements of serine/threonine protein phosphatase (STPP) activity, and the detection of the functional mitochondrial parameters in suspension of brain mitochondria to study the Aß25-35-associated signaling in the hippocampus. Aß25-35 aggregates shifted the kinase-phosphatase balance during the long-term potentiation (LTP) induction in the enhancement of STPP activity. The PP1/PP2A inhibitor, okadaic acid, but not the PP2B blocker, cyclosporin A, prevented Aß25-35-dependent LTP suppression for both simultaneous and delayed enzyme blockade protocols. STPP activity in the Aß25-35-treated slices was upregulated, which is reverted relative to the control values in the presence of PP1/PP2A but not in the presence of the PP2B blocker. A selective inhibitor of stress-induced PP1α, sephin1, but not of the PP2A blocker, cantharidin, is crucial for Aß25-35-mediated LTP suppression prevention. A mitochondrial Na+/Ca2+ exchanger (mNCX) blocker, CGP37157, also attenuated the Aß25-35-induced LTP decline. Aß25-35 aggregates did not change the mitochondrial transmembrane potential or reactive oxygen species (ROS) production but affected the ion transport and Ca2+-dependent swelling of organelles. The staining of hippocampal slices with NO-sensitive fluorescence dye, DAF-FM, showed stimulation of the NO production in the Aß25-35-pretreated slices at the dendrite-containing regions of CA1 and CA3, in the dentate gyrus (DG), and in the CA1/DG somata. NO scavenger, PTIO, or nNOS blockade by selective inhibitor 3Br-7NI partly restored the Aß25-35-induced LTP decline. Thus, hippocampal NO production could be another marker for the impairment of synaptic plasticity in amyloidosis-related states, and kinase-phosphatase balance management could be a promising strategy for the compensation of Aß25-35-driven deteriorations.

Nitric Oxide Regulates GluA2-Lacking AMPAR Contribution to Synaptic Transmission of CA1 Apical but Not Basal Dendrites.

The mechanisms of synaptic plasticity differ in distinct local circuits. In the CA1 region of the hippocampus, the mechanisms of long-term potentiation (LTP) at apical dendrites in stratum radiatum and basal dendrites in stratum oriens involve different molecular cascades. For instance, participation of nitric oxide in LTP induction was shown to be necessary only for apical dendrites. This phenomenon may play a key role in information processing in CA1, and one of the reasons for this difference may be differing synaptic characteristics in these regions. Here, we compared the synaptic responses to stimulation of apical and basal dendrites of CA1 pyramidal neurons and found a difference in the current-voltage characteristics of these inputs, which is presumably due to a distinct contribution of GluA2-lacking AMPA receptors to synaptic transmission. In addition, we obtained data that indicate the presence of these receptors in pyramidal dendrites in both stratum radiatum and stratum oriens. We also demonstrated that inhibition of NO synthase reduced the contribution of GluA2-lacking AMPA receptors at apical but not basal dendrites, and inhibition of soluble guanylate cyclase did not affect this phenomenon.

Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Optimal hyperosmotic agents for tissue immersion optical clearing in terahertz biophotonics.

In this work, a thorough analysis of hyperosmotic agents for the immersion optical clearing (IOC) in terahertz (THz) range was performed. It was aimed at the selection of agents for the efficient enhancement of penetration depth of THz waves into biological tissues. Pulsed spectroscopy in the frequency range of 0.1 to 2.5 THz was applied for investigation of the optical properties of common IOC agents. Using the collimated transmission spectroscopy in visible range, binary diffusion coefficients of tissue water and agent in ex vivo rat brain tissue were measured. IOC agents were objectively compared using two-dimensional nomogram, accounting for their THz-wave absorption coefficients and binary diffusion coefficients. The results of this study demonstrate an interplay between the penetration depth enhancement and the diffusion rate and allow for pointing out glycerol as an optimal agent among the considered ones for particular applications in THz biophotonics.

Modulation of AMPA Receptors by Nitric Oxide in Nerve Cells.

Nitric oxide (NO) is a gaseous molecule with a large number of functions in living tissue. In the brain, NO participates in numerous intracellular mechanisms, including synaptic plasticity and cell homeostasis. NO elicits synaptic changes both through various multi-chain cascades and through direct nitrosylation of targeted proteins. Along with the N-methyl-d-aspartate (NMDA) glutamate receptors, one of the key components in synaptic functioning are α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors-the main target for long-term modifications of synaptic effectivity. AMPA receptors have been shown to participate in most of the functions important for neuronal activity, including memory formation. Interactions of NO and AMPA receptors were observed in important phenomena, such as glutamatergic excitotoxicity in retinal cells, synaptic plasticity, and neuropathologies. This review focuses on existing findings that concern pathways by which NO interacts with AMPA receptors, influences properties of different subunits of AMPA receptors, and regulates the receptors' surface expression.

LTP suppression by protein synthesis inhibitors is NO-dependent.

For several decades, the ability of protein synthesis inhibitors (PSI) to suppress the long-term potentiation (LTP) of hippocampal responses is known. It is considered that mechanisms of such impairment are related to a cessation of translation and a delayed depletion of the protein pool required for maintenance of synaptic plasticity. The present study demonstrates that cycloheximide or anisomycin applications reduce amplitudes of the field excitatory postsynaptic potentials as well as the presynaptically mediated form of plasticity, the paired-pulse facilitation after LTP induction in neurons of the CA1 area of hippocampus. We showed that nitric oxide signaling could be one of the pathways that cause the LTP decrease induced by cycloheximide or anisomycin. Inhibitor of the NO synthase, L-NNA or the NO scavenger, PTIO, rescued the late-phase LTP and restored the paired-pulse facilitation up to the control levels. For the first time we have directly measured the nitric oxide production induced by application of the translation blockers in hippocampal neurons using the NO-sensitive dye DAF-FM. Inhibitory analysis demonstrated that changes during protein synthesis blockade downstream the NO signaling cascade are cGMP-independent and apparently are implemented through degradation of target proteins. Prolonged application of the NO donor SNAP impaired the LTP maintenance in the same manner as PSI.

Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons.

The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials (APs). Experimental results support this idea, however mechanisms of fast onset of APs in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of AP generation and encoding. Here we characterize properties of AP encoding in neocortical neurons grown for 11-25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and AP generation. The onset of APs becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of APs as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of AP generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of AP generation and encoding, and their maturation.

Cued memory reconsolidation in rats requires nitric oxide.

It is well-known that the reactivation of consolidated fear memory under boundary conditions of novelty and protein synthesis blockade results in an impairment of memory, suggesting that the reactivated memory is destabilized and requires synthesis of new proteins for reconsolidation. We tested the hypothesis of nitric oxide (NO) involvement in memory destabilization during the reconsolidation process in rats using memory reactivation under different conditions. We report that administration of NO-synthase selective blockers 3-Br-7-NI or ARL in the conditions of reactivation of memory under a protein synthesis blockade prevented destabilization of fear memory to the conditioned stimulus. Obtained results support the role of NO signaling pathway in the destabilization of existing fear memory triggered by reactivation, and demonstrate that the disruption of this pathway during memory reconsolidation may prevent changes in long-term memory.

Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation.

For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5' untranslated region (5'UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKMζ translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKMζ leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKMζ 5'UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKMζ translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKMζ translation in activated neurons, important at early stages of LTP.