AMPA receptor phosphorylation during synaptic plasticity.

A widely studied example of vertebrate plasticity is LTP (long-term potentiation), the persistent synaptic enhancement that follows a brief period of coinciding pre- and post-synaptic activity. During LTP, different kinases, including CaMKII (calcium/calmodulin-dependent protein kinase II) and protein kinase A, become activated and play critical roles in induction and maintenance of enhanced transmission. Biochemical analyses have revealed several regulated phosphorylation sites in the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunits, GluR1 and GluR4. The regulated insertion of these receptors is a key event in the induction of LTP. Here, we discuss the phosphorylation of GluR1 and GluR4 and its role in receptor delivery and neuronal plasticity.

Transient oxygen-glucose deprivation induces rapid morphological changes in rat hippocampal dendrites.

Previous studies have revealed that pyramidal neurons in the CA1 region of the hippocampus are extremely susceptible to ischemia-induced cell damage and undergo selective degeneration 2-4 days after the insult. Little is known about early morphological changes in neurons occurring immediately after ischemic insult. Using two-photon laser scanning microscopy we monitored dendritic morphology of cells expressing enhanced green fluorescent protein in response to a transient hypoxic-ischemic episode in organotypic hippocampal slice preparations. This type of vital imaging provides direct evidence of dendritic rearrangements in rat CA1 pyramidal neurons occurring as soon as 20 min after oxygen-glucose deprivation. We propose that dendritic reorganization, resembling that occurring after tetanic stimulation, may be an early stage response to compensate the loss of synapses caused by ischemia-induced neuronal injury.

Postsynaptic conversion of silent synapses during LTP affects synaptic gain and transmission dynamics.

Synaptic transmission relies on both the gain and the dynamics of synapses. Activity-dependent changes in synaptic gain are well-documented at excitatory synapses and may represent a substrate for information storage in the brain. Here we examine the mechanisms of changes in transmission dynamics at excitatory synapses. We show that paired-pulse ratios (PPRs) of AMPAR and NMDAR EPSCs onto dentate gyrus granule cells are often different; this difference is reduced during LTP, reflecting PPR changes of AMPAR but not NMDAR EPSCs. Presynaptic manipulations, however, produce parallel changes in AMPAR and NMDAR EPSCs. LTP at these synapses reflects a reduction in the proportion of silent synapses lacking functional AMPARs. Changes in PPR during LTP therefore reflect the initial difference between PPRs of silent and functional synapses. Functional conversion of silent synapses permits postsynaptic sampling from additional release sites and thereby affects the dynamics and gain of signals conveyed between neurons.

Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.

AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. Here we show that these AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.

Genetic manipulation of the odor-evoked distributed neural activity in the Drosophila mushroom body.

Odor-induced neural activity was recorded by Ca2+ imaging in the cell body region of the Drosophila mushroom body (MB), which is the second relay of the olfactory central nervous system. The signals recorded are mainly from the cell layers on the brain surface because of the limited penetration of Ca2+-sensitive dyes. The densely packed cell bodies and their accessibility allow visualization of odor-induced population neural activity. It is revealed that odors evoke diffused neural activities in the MB. Although the signals cannot be attributed to individual neurons, patterns of the population neural activity can be analyzed. The activity pattern, but not the amplitude, of an odor-induced population response is specific for the chemical identity of an odor and its concentration. The distribution pattern of neural activity can be altered specifically by genetic manipulation of an odor binding protein and this alteration is closely associated with a behavioral defect of odor preference. These results suggest that the spatial pattern of the distributed neural activity may contribute to coding of odor information at the second relay of the olfactory system.

Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity.

To examine how functional circuits are established in the brain, we studied excitatory transmission in early postnatal hippocampus. Spontaneous neural activity was sufficient to selectively deliver GluR4-containing AMPA receptors (AMPA-Rs) into synapses. This delivery allowed non-functional connections to transmit at resting potentials and required NMDA receptors (NMDA-Rs) but not CaMKII activation. Subsequently, these delivered receptors were exchanged with non-synaptic GluR2-containing AMPA-Rs in a manner requiring little neuronal activity. The enhanced transmission resulting from this delivery and subsequent exchange was maintained for at least several days and required an interaction between GluR2 and NSF. Thus, this sequence of subunit-specific trafficking events triggered by spontaneous activity in early postnatal development may be crucial for initial establishment of long-lasting functional circuitry.

[Lack of correlation between plasma homocysteine levels and cerebral microangiopathy in patients wtih transient ischemic attack]. / Ausencia de correlación entre homocisteína plasmática y microangiopatía cerebral en pacientes con ataque isquémico transitorio.

OBJECTIVE: The aim of this study is to evaluate cerebral MRI findings in patients with atherothrombotic transient ischemic attacks (TIA) and its correlation with plasma homocysteine (Hcy) levels. PATIENTS AND METHODS: A total of 62 consecutive patients with the diagnosis of TIA of atherothrombotic origin were studied. MRI examinations were performed in all patients for the evaluation of the presence of infarct and/or white matter hyperintensities (WMHI). Plasma Hcy levels were determined according to the method described by Smolin and Schneider modified. RESULTS: Plasma Hcy levels were significantly (p < 0.036) higher in patients with MRI-detected infarcts (9.69 +/- 2.06 mumol/l) compared with patients without infarcts (8.65 +/- 1.7 mumol/l. There was no correlation (p < 0.33) between plasma Hcy levels and the presence or absence of WMHI seen on MRI. CONCLUSIONS: In TIA patients, plasma Hcy levels were significantly higher in patients with cerebral infarcts, but did not correlate with the presence of WMHI. Our results suggest that mild hyperhomocysteinemia would be associated with large-medium vessel rather than with small vessel disease.

LTP mechanisms: from silence to four-lane traffic.

Brief periods of strong neuronal activity induce long-lasting changes in synaptic function. This synaptic plasticity is thought to play important roles in learning and memory. One example--long-term potentation in the CA1 region of the hippocampus--has been studied extensively, and conflicting views regarding the underlying mechanisms have emerged. Recent findings, regarding basic properties of synaptic transmission, appear to reconcile these diverging views.

Ausencia de correlación entre homocisteína plasmática y microangiopatía cerebral en pacientes con ataque isquémico transitorio / Lack of correlation between plasma homocysteine levels and cerebral microangiopathy in patients with transient ischemic attacks

Objetivo. Evaluar los hallazgos de la resonancia magnética (RM) craneal en pacientes con ataque isquémico transitorio (AIT) de origen aterotrombótico y su relación con los niveles de homocisteína (Hcy) plasmática. Pacientes y métodos. Se estudiaron 62 pacientes consecutivos con el diagnóstico de AIT de origen aterotrombótico. Se realizaron RM en todos los enfermos para valorar la existencia de infarto cerebral o hiperintensidades de la sustancia blanca (HISB).Se determinaron los niveles de homocisteína plasmática según el método descrito por Smolin y Schneider modificado. Resultados. Los niveles de Hcy plasmática eran significativamente mayores (p< 0,036) en los pacientes con AIT e infarto detectados mediante RM (9,69 ñ 2,06 mmol/l), en comparación con los pacientes sin infarto (8,65 ñ 1,7 mmol/l). No hubo correlación (p< 0,33) entre los niveles de homocisteína plasmática y la presencia o ausencia de HISB detectada mediante RM. Conclusiones. En pacientes con AIT los niveles de Hcy eran significativamente mayores que en enfermos con infartos cerebrales, pero no se correlacionaban con la presencia de HISB.Nuestros resultados sugieren que la hiperhomocistinemia moderada estaría más asociada con la arteriopatía de mediano-gran vaso, que con la arteriopatía cerebral de pequeño vaso (AU)

Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction.

To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.

Enhanced synaptic potentiation in transgenic mice expressing presenilin 1 familial Alzheimer's disease mutation is normalized with a benzodiazepine.

Mutations in presenilin 1 (PS1) are the most common causes of familial Alzheimer's disease (FAD). We examined synaptic physiology in hippocampal brain slices of transgenic mice expressing the FAD-linked PS1 deletion of exon 9 variant. Basal excitatory transmission and paired-pulse facilitation in PS1 mutant mice were unchanged. Short- and long-term potentiation of excitatory transmission following high-frequency stimulation were greater in transgenic mice expressing mutant PS1. Mutants had enhanced synaptic inhibition, which may be a compensatory change offsetting an abnormally sensitized plasticity of excitatory transmission. Increasing inhibitory transmission in mutant animals even more with a benzodiazepine reverted synaptic potentiation to the levels of controls. These results support the potential use of benzodiazepines in the treatment of familial Alzheimer's disease.

Distinct sites of opiate reward and aversion within the midbrain identified using a herpes simplex virus vector expressing GluR1.

Repeated administration of morphine increases expression of GluR1 (an AMPA glutamate receptor subunit) in the ventral tegmental area (VTA) of the midbrain, an important neural substrate for the rewarding actions of morphine. Microinjections of a herpes simplex virus (HSV) vector that causes local overexpression of GluR1 (HSV-GluR1) into the VTA can enhance the ability of morphine to establish conditioned place preferences, suggesting that altered GluR1 expression in this region is directly associated with changes in the rewarding efficacy of morphine. We now report that in rats given HSV-GluR1 directly into the VTA, morphine is most rewarding when maximal transgene expression is in the rostral VTA, whereas morphine is aversive when maximal transgene expression is in the caudal VTA. Dual-labeling immunohistochemistry shows that this difference cannot be explained by a different fraction of dopaminergic neurons infected in the rostral versus caudal VTA. No such anatomical specificity is seen in rats given VTA microinjections of HSV-LacZ, a vector expressing a control protein (-galactosidase). These results suggest that distinct substrates within the VTA itself differentially contribute to the rewarding and aversive properties of opiates.

[Plasma homocysteine levels in patients with transient ischemic attacks]. / Homocisteína plasmática en pacientes con ataques isquémicos transitorios.

OBJECTIVE: To evaluate plasma homocysteine (Hcy) levels in patients with atherotrombotic transient ischemic attacks (TIA), its temporal profile and response to folic acid (FA) treatment. PATIENTS AND METHODS: Hcy was determined in 62 patients and in 69 controls. RESULTS: There were no differences (p < 0.87) of baseline Hcy in TIA patients vs controls. Hcy levels were higher 4-6 weeks after TIA (p = 0.02). FA treatment decreases the Hcy levels (p < 0.00001). CONCLUSION: Hyperhomocisteinemia is a possible risk factor for atherothrombotic TIA and should be measured between 4-6 weeks after TIA. Treatment with FA normalizes Hcy levels.

Two-photon imaging in living brain slices.

Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its key advantages can be easily implemented as a modification of commercial laser scanning microscopes.

Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.

To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.

Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses.

Early in postnatal development, glutamatergic synapses transmit primarily through NMDA receptors. As development progresses, synapses acquire AMPA receptor function. The molecular basis of these physiological observations is not known. Here we examined single excitatory synapses with immunogold electron-microscopic analysis of AMPA and NMDA receptors along with electrophysiological measurements. Early in postnatal development, a significant fraction of excitatory synapses had NMDA receptors and lacked AMPA receptors. As development progressed, synapses acquired AMPA receptors with little change in NMDA receptor number. Thus, synapses with NMDA receptors but no AMPA receptors can account for the electrophysiologically observed 'silent synapse'.

Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity.

Activity shapes the structure of neurons and their circuits. Two-photon imaging of CA1 neurons expressing enhanced green fluorescent protein in developing hippocampal slices from rat brains was used to characterize dendritic morphogenesis in response to synaptic activity. High-frequency focal synaptic stimulation induced a period (longer than 30 minutes) of enhanced growth of small filopodia-like protrusions (typically less than 5 micrometers long). Synaptically evoked growth was long-lasting and localized to dendritic regions close (less than 50 micrometers) to the stimulating electrode and was prevented by blockade of N-methyl-D-aspartate receptors. Thus, synaptic activation can produce rapid input-specific changes in dendritic structure. Such persistent structural changes could contribute to the development of neural circuitry.

No change in NMDA receptor-mediated response rise-time during development: evidence against transmitter spillover.

Glutamatergic transmission was examined in tadpole optic tectum to test the possibility that transmitter concentration reaching N-methyl-D-aspartate (NMDA) receptors increases over development. Pharmacologically isolated NMDA receptor-mediated transmission was monitored with whole-cell recordings. Synaptic responses were recorded from cells at different locations in the optic tectum, corresponding to different stages of development. Rise-times and decay-times of NMDA currents were analyzed. We found no significant correlation between rise-time and developmental stage. As NMDA rise-times can correlate with concentration for glutamate concentrations below 200 microM, these results argue that, if there is developmental variation in transmitter concentration, this occurs for values greater than 200 microM. Furthermore, we found a correlation between rise-times and decay-times, consistent with a model in which transmitter concentration is high and rise-time is controlled by channel closings. These results argue against synaptic models in which low concentrations of transmitter (as by spillover from nearby release sites) selectively activates NMDA receptors.

Calcium-evoked dendritic exocytosis in cultured hippocampal neurons. Part I: trans-Golgi network-derived organelles undergo regulated exocytosis.

Exocytosis is a widely observed cellular mechanism for delivering transmembrane proteins to the cell surface and releasing signaling molecules into the extracellular space. Calcium-evoked exocytosis, traditionally thought to be restricted to presynaptic specializations in neurons, has been described recently in many cells. Here, calcium-evoked dendritic exocytosis (CEDE) is visualized in living cultured hippocampal neurons. Organelles that undergo CEDE are in somata, dendrites, and perisynaptic regions, identified by using immunocytochemistry and correlative light and electron microscopy. CEDE is regulated developmentally: neurons <9 d in vitro do not show CEDE. In addition, CEDE is blocked by tetanus toxin, an inhibitor of regulated exocytosis, and nocodazole, an inhibitor of microtubule polymerization. Organelles that undergo CEDE often are found on the base of spines, putative sites of synaptic plasticity. CEDE therefore could be involved in structural and functional modification of spines and could play a role in synaptic plasticity, where it might involve changes in receptor/channel density, release of active compounds having effect on pre- and postsynaptic function, and/or growth of synaptic structures.