Posttetanic Long-Term Potentiation in Rat Dentate Area Increases Postsynaptic 411B Immunoreactivity

411B is a monoclonal antibody raised to chick forebrain postsynaptic densities (PSDs) which also recognises an antigen in brain tissue from adult Wistar rats but not liver, heart, or lung. This antigen is enriched in the PSD fraction and appears to be a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. The aim of this study was to investigate whether 411B immunoreactivity is changed in various hippocampal subregions by post-tetanic long-term potentiation (LTP). LTP was elicited in freely moving rats by applying four trains of 300 square-wave pulses (frequency 200 Hz, pulse duration 0.2 ms, and intensity 300 mA) into the right perforant path; this included an increase in transmission efficacy at the ipsilateral perforant path-granular cell synapse of the dentate gyrus lasting several days. Eight hours after tetanisation, antigens recognised by monoclonal 411B and a polyclonal anti-actin antiserum were assayed in lysed homogenates of ipsi- and contralateral CA1. CA3, and CA4/dentate area hippocampal subfields as well as in visual cortex, cerebellum, and olfactory bulb dissected from LTP rats, and compared to passive controls. Under these experimental conditions, tetanisation of the perforant path resulted in a significant increase in the titre of 411B in the ipsilateral CA4/dentate area subfield (+34.0%; p less than 0.001) compared with passive controls, whereas in all other brain regions studied no differences between experimental and control rats were observed. In no region were anti-actin titres significantly different from controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of Acetylcholinesterase in Olfactory Bulb of the Pike <Emphasis Type="Italic">Esox lucius</Emphasis> and Its Role in Development of Long-Term Posttetanic Potentiation

A complex electrophysiological, biochemical, and histochemical study is carried out for determination of activity and distribution of acetylcholinesterase (AChE) in olfactory bulb (OB) of the pike during long-term posttetanic potentiation (PTP). Between the 30th and 60th min after tetanus, a stable increase of enzymatic activity in parallel with a rise of potentiation is observed. Sixty min after tetanus, at the point of maximal development of long-term PTP (the potentiation value is 170%), the specific activity of AChE rises by 89%. This increase was found to be due to synthesis of the enzyme de novo, with involvement of the majority of mitral cells and a significant part of granular cells.     

Alterations in Nitric Oxide-Stimulated Endogenous ADP-Ribosylation Associated with Long-Term Potentiation in Rat Hippocampus

Abstract: The present study examines the possible involvement of nitric oxide (NO)-stimulated endogenous ADP-ribosylation in long-term potentiation (LTP). LTP was induced in hippocampal slices by stimulation of Schaffer collateral inputs to the CA1 pyramidal neurons. Basal and sodium nitroprusside (SNP), which generates NO, stimulation of endogenous ADP-ribosylation was then studied in CA1 subfields isolated from the slices. Control slices received no treatment or were tetanized in the presence of aminophosphonovaleric acid, an NMDA receptor antagonist that blocks the development of LTP. SNP-stimulated ADP-ribosylation of endogenous proteins was reduced by 40–70% in LTP slices relative to control slices. LTP was also associated with a small but significant reduction in basal ADP-ribosylation activity. The results demonstrate that the induction of LTP is associated with regulation of endogenous ADP-ribosylation and suggest a role for this type of covalent modification in some aspect of LTP.     

On the Role of Extracellular ATP in the Induction of Long-Term Potentiation in the Hippocampus

Abstract: The involvement of a purinergic system in the mechanisms of ATP- and electrically induced long-term potentiation (LTP) has been investigated in mouse hippocampal slices. Extracellular ATP (500 n M ) and its slowly hydrolyzable analogue adenosine 5'- O -(3-thiotriphosphate) (ATP-γ-S; 2.5 µ M ) amplified permanently the magnitude of the population spike. This effect was antagonized by adenylimidodiphosphate (AMPPNP), a non-hydrolyzable analogue of ATP. AMPPNP, other ATP analogues [2-methylthioadenosine triphosphate (2-MeSATP) and α,β-methyleneadenosine 5'-triphosphate (α,β-methyleneATP)], or a purinergic receptor antagonist (Cibacron Blue 3G) tested in the concentration range of 3–40 µ M did not exert agonistic activity similar to that of ATP or ATP-γ-S, suggesting that ATP hydrolysis is required to exert this effect. All the tested nonhydrolyzable analogues reduced or prevented the establishment of stable, nondecremental LTP without blocking the short-lasting increase in the magnitude of the population spike immediately after electrical stimulation (short-term potentiation). These results indicate that ATP released by high-frequency stimulation contributes to the maintenance of stable LTP. The underlying mechanism operating in this process may involve a new type of ATP receptors or hydrolysis by ecto-ATPase. However, the findings that ATP-γ-S is less potent than ATP and that other ATP analogues known to act as agonists of purinergic receptors did not induce LTP but rather inhibited its maintenance are more consistent with the possibility that ecto-protein kinase, using extracellular ATP as a cosubstrate, plays a role in mechanisms underlying synaptic plasticity.     

Long-Term Potentiation in Isolated Dendritic Spines

Background

In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the expression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements.

Methodology/Principal Findings

We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [³H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation.

Conclusions/Significance

Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines.     

Characterisation of the Role of Calcium in AlF4-Induced Long-Term Potentiation in Area CA1 of Rat Hippocampus

We investigated calcium influx in the long lasting potentiation induced in area CA1 of rat hippocampus by brief bath application of the G-protein activator AlF₄ ^–(NaF/AlCl₃). Brief (10 min) bath application of AlF₄ ^– in standard saline (with 2mM Ca²⁺) consistently induced a long lasting potentiation which was not observed if AlF₄ ^– was bath-applied in nominally calcium free saline. Increasing the potential calcium influx, either by raising extracellular calcium concentration to 3.5 mM or by addition of the voltage operated calcium channel (VOCC) agonist BayK8644. failed to increase the number of slices exhibiting potentiation or the mean level of potentiation. Bath application of AlF₄ ^– in the presence of the VOCC antagonist failed to block the potentiation and AlF₄ ^– readily induced a long lasting potentiation under voltage clamp conditions, strongly suggesting that the calcium influx required for AlF₄-induced potentiation is not through NMDA receptors or VOCC channels. It is suggested that the calcium required may be provided by an ongoing recharging and emptying of IP3 sensitive intracellular Ca²⁺ stores.     

Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.Peptidomics is defined as the analysis of the peptide content within an organism, tissue, or cell (1–3). The proteome and peptidome have common features, but there are also prominent differences. Proteomics generally identifies proteins by using the information of biologically inactive peptides derived from tryptic digestion, whereas peptidomics tries to identify endogenous peptides using single peptide sequence information only (4). Endogenous neuropeptides are peptides used for intracellular signaling that can act as neurotransmitters or neuromodulators in the nervous system. These polypeptides of 3–100 amino acids can be abundantly produced in large neural populations or in trace levels from single neurons (5) and are often generated through the cleavage of precursor proteins. However, unwanted peptides can also be created through post-mortem induced proteolysis (6). The later aspect complicates the technical analysis of neuropeptides as post-mortem conditions increase the number of degradation peptides. The possibility to detect, identify, and quantify lowly expressed neuropeptides using label-free LC-MS techniques has improved with the development of new sample preparation techniques including rapid heating of the tissue, which prevents protein degradation and inhibition of post-mortem proteolytic activity (7, 8).It has been suggested by us (4, 5) and others (9) that comparing the peptidome between samples of e.g. diseased and normal tissue may lead to the discovery of biologically relevant peptides of certain pathological or pharmacological events. However, differences in relative peptide abundance measurements may not only originate from biological differences but also from systematic bias and noise. To reduce the effects of experimentally induced variability it is common to normalize the raw data. This is a concept well known in the area of genomics studies using gene expression microarrays (10–12). As a consequence, many methods developed for microarray data have also been adapted for normalizing peptide data produced with LC-MS techniques (10–16). Normally the underlying assumption for applying these techniques is that the total or mean/median peak abundances should be equal across different experiments, in this case between LC-MS analyses. Global normalization methods refer to cases where all peak abundances are used to determine a single normalization factor between experiments (13, 15, 16), a subset of peaks assumed to be similarly abundant between experiments (16) is used, or spiked-in peptides are used as internal standards. In a study by Callister et al. (14), normalization methods for tryptic LC-FTICR-MS peptide data were compared. The authors concluded that global or iterative linear regression works best in most cases but also recommended that the best procedure should be selected for each data set individually. Methods used for normalizing LC-MS data have been reviewed previously (14, 17, 18), but to our knowledge only Callister et al. (14) have used small data sets to systematically evaluate such methods. None of these studies have targeted data of endogenous peptides.In this study, the effects of 10 different normalization methods were evaluated on data produced by a nano-LC system coupled to an electrospray Q-TOF or linear trap quadrupole (LTQ)¹ mass spectrometer. Normalization methods that originally were developed for gene expression data were used, and one novel method, linear regression followed by analysis order normalization (RegrRun), is presented. The normalization methods were evaluated using three data sets of endogenous brain peptides originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses. Each data set contained both technical and biological replicates.     

Protected-Site Phosphorylation of Protein Kinase C in Hippocampal Long-Term Potentiation

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca²⁺, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

Hippocampal Long-Term Potentiation Attenuated by Lesions in the Marginal Division of Neostriatum

The marginal division (MrD) is a spindled-neurons consisted zone at the caudal border of the neostriatum in the mammalian brain and has been verified as contributing to associative learning and declarative memory in the rat and human with behavior and functional magnetic resonance imaging methods. It was proved to have functional connections with the limbic system. Whether the MrD has influence on the hippocampal long-term potentiation (LTP) was investigated in this study. LTP was induced from the dentate gyrus (DG) in the hippocampus by high-frequency stimulation (HFS) to the perforant path (PP). The amplitude of the population spike (PS) and the slope of the excitatory postsynaptic potential (EPSP) increased significantly to form LTP in the DG of the hippocampus after HFS of PP in normal and saline-injected control groups of rats. Lesions introduced in the MrD reduced significantly both the amplitude of PS and the slope of the EPSP following HFS of the PP. The results indicated that lesions in the MrD could attenuate LTP formation in the hippocampus. Our data suggest that the MrD might very possibly have excitatory functional influence on the hippocampus and therefore might influence the function of the hippocampus.     

The Role of Multiphosphorylated Peptides in Mineralized Tissue Regeneration

Several multiple phosphoseryl-containing proteins have been identified in mineralising tissue and body fluids where they have been proposed to act as nucleators and/or regulators of biomineralisation. In particular, the unique features of the multiphosphorylated caseins from milk and phosphophoryn from dentine enable them to have a role in mineralised tissue regeneration. In this review, three examples of therapeutic applications using multiphosphorylated peptides are described.     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

Increased Phosphorylation of Myelin Basic Protein During Hippocampal Long-Term Potentiation

Abstract: Hippocampal long-term potentiation (LTP) is a long-lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E-LTP). Using the back-phosphorylation method, we observed an increase in the phosphorylation of a 21-kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5-kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back-phosphorylated and then digested with either endoproteinase Lys-C or endoproteinase Asp-N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5-kDa isoform of MBP is increased during E-LTP.     

Failed Stabilization for Long-Term Potentiation in the Auditory Cortex of Fmr1 Knockout Mice

Fragile X syndrome is a developmental disorder that affects sensory systems. A null mutation of the Fragile X Mental Retardation protein 1 (Fmr1) gene in mice has varied effects on developmental plasticity in different sensory systems, including normal barrel cortical plasticity, altered ocular dominance plasticity and grossly impaired auditory frequency map plasticity. The mutation also has different effects on long-term synaptic plasticity in somatosensory and visual cortical neurons, providing insights on how it may differentially affect the sensory systems. Here we present evidence that long-term potentiation (LTP) is impaired in the developing auditory cortex of the Fmr1 knockout (KO) mice. This impairment of synaptic plasticity is consistent with impaired frequency map plasticity in the Fmr1 KO mouse. Together, these results suggest a potential role of LTP in sensory map plasticity during early sensory development.     

Disinhibition Mediates a Form of Hippocampal Long-Term Potentiation in Area CA1

The hippocampus plays a central role in memory formation in the mammalian brain. Its ability to encode information is thought to depend on the plasticity of synaptic connections between neurons. In the pyramidal neurons constituting the primary hippocampal output to the cortex, located in area CA1, firing of presynaptic CA3 pyramidal neurons produces monosynaptic excitatory postsynaptic potentials (EPSPs) followed rapidly by feedforward (disynaptic) inhibitory postsynaptic potentials (IPSPs). Long-term potentiation (LTP) of the monosynaptic glutamatergic inputs has become the leading model of synaptic plasticity, in part due to its dependence on NMDA receptors (NMDARs), required for spatial and temporal learning in intact animals. Using whole-cell recording in hippocampal slices from adult rats, we find that the efficacy of synaptic transmission from CA3 to CA1 can be enhanced without the induction of classic LTP at the glutamatergic inputs. Taking care not to directly stimulate inhibitory fibers, we show that the induction of GABAergic plasticity at feedforward inhibitory inputs results in the reduced shunting of excitatory currents, producing a long-term increase in the amplitude of Schaffer collateral-mediated postsynaptic potentials. Like classic LTP, disinhibition-mediated LTP requires NMDAR activation, suggesting a role in types of learning and memory attributed primarily to the former and raising the possibility of a previously unrecognized target for therapeutic intervention in disorders linked to memory deficits, as well as a potentially overlooked site of LTP expression in other areas of the brain.     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....