AMPA Channels Research Articles

Differential regulation of tetramerization of the AMPA receptor glutamate–gated ion channel by auxiliary subunits

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

Two types of GLR channels cooperate differently in light and dark growth of Arabidopsis seedlings

BackgroundGLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark.ResultsWild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development.ConclusionsOur work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

Adenosine A1 receptor antagonist-induced facilitation of postsynaptic AMPA currents in pyramidal neurons of the rat hippocampal CA2 area.

Adenosine A1 receptors (A1R) are widely expressed in hippocampal pyramidal neurons and their presynaptic terminals. It is well known that endogenous adenosine regulates hippocampal function through the activation of A1R in hippocampal pyramidal neurons and has been reported that blockade of A1R induces stronger potentiation of excitatory synaptic transmission in CA2 pyramidal neurons than in CA1 pyramidal neurons. This strong potentiation of CA2 neurons is thought to be caused by the specific modulation of excitatory synaptic transmission through postsynaptic A1R. However, the direct effects of A1R on postsynaptic AMPA channels remain unknown because of the technical difficulties of patch-clamp recording from mature hippocampal CA2 neurons. We recorded synaptic currents from pyramidal neurons in CA1 and CA2 and analyzed the effects of an A1R antagonist on stimulation-evoked synaptic transmission and local application-induced postsynaptic AMPA currents. The antagonist increased the amplitude of evoked synaptic transmission in neurons in both CA1 and CA2. This facilitation was larger in pyramidal neurons in CA2 than in CA1. The antagonist also increased postsynaptic AMPA currents in neurons in CA2 but not in CA1. This facilitation of CA2 AMPA currents was occluded by the intracellular application of a G-protein blocker. Even with the blockade of postsynaptic G-protein signaling, the A1R antagonist increased evoked synaptic transmission in neurons in CA2. These results suggest that synaptic transmission in pyramidal neurons in CA2 is regulated by both presynaptic and postsynaptic A1R. Moreover, A1R regulate excitatory synaptic transmission in pyramidal neurons in CA2 through the characteristic postsynaptic modulation of AMPA currents.

HGG-21. MALIGNANT SYNAPTIC PLASTICITY IN PEDIATRIC HIGH-GRADE GLIOMAS

Pediatric high-grade gliomas (pHGG) are a devastating group of diseases that urgently require novel therapeutic options. We have previously demonstrated that pHGGs directly synapse onto neurons and the subsequent tumor cell depolarization, mediated by calcium-permeable AMPA channels, promotes their proliferation. The regulatory mechanisms governing these postsynaptic connections are unknown. Here, we investigated the role of BDNF-TrkB signaling in modulating the plasticity of the malignant synapse. BDNF ligand activation of its canonical receptor, TrkB (which is encoded for by the gene NTRK2), has been shown to be one important modulator of synaptic regulation in the normal setting. Electrophysiological recordings of glioma cell membrane properties, in response to acute neurotransmitter stimulation, demonstrate in an inward current resembling AMPA receptor (AMPAR) mediated excitatory neurotransmission. Extracellular BDNF increases the amplitude of this glutamate-induced tumor cell depolarization and this effect is abrogated in NTRK2 knockout glioma cells. Upon examining tumor cell excitability using in situ calcium imaging, we found that BDNF increases the intensity of glutamate-evoked calcium transients in GCaMP6s expressing glioma cells. Western blot analysis indicates the tumors AMPAR properties are altered downstream of BDNF induced TrkB activation in glioma. We find that BDNF-TrkB signaling promotes neuron-to-glioma synaptogenesis as measured by high-resolution confocal and electron microscopy in culture and tumor xenografts. Our analysis of published pHGG transcriptomic datasets, together with brain slice conditioned medium experiments in culture, indicate the tumor microenvironment as the chief source of BDNF ligand. Disruption of the BDNF-TrkB pathway in patient-derived orthotopic glioma xenograft models, both genetically and pharmacologically, results in an increased overall survival and reduced tumor proliferation rate. These findings suggest that gliomas leverage mechanisms of plasticity to modulate the excitatory channels involved in synaptic neurotransmission and they reveal the potential to target the regulatory components of glioma circuit dynamics as a therapeutic strategy for these lethal cancers.

Biophysical mechanism of the interaction between default mode network and working memory network

Default mode network (DMN) is a functional brain network with a unique neural activity pattern that shows high activity in resting states but low activity in task states. This unique pattern has been proved to relate with higher cognitions such as learning, memory and decision-making. But neural mechanisms of interactions between the default network and the task-related network are still poorly understood. In this paper, a theoretical model of coupling the DMN and working memory network (WMN) is proposed. The WMN and DMN both consist of excitatory and inhibitory neurons connected by AMPA, NMDA, GABA synapses, and are coupled with each other only by excitatory synapses. This model is implemented to demonstrate dynamical processes in a working memory task containing encoding, maintenance and retrieval phases. Simulated results have shown that: (1) AMPA channels could produce significant synchronous oscillations in population neurons, which is beneficial to change oscillation patterns in the WMN and DMN. (2) Different NMDA conductance between the networks could generate multiple neural activity modes in the whole network, which may be an important mechanism to switch states of the networks between three different phases of working memory. (3) The number of sequentially memorized stimuli was related to the energy consumption determined by the network's internal parameters, and the DMN contributed to a more stable working memory process. (4) Finally, this model demonstrated that, in three phases of working memory, different memory phases corresponded to different functional connections between the DMN and WMN. Coupling strengths that measured these functional connections differed in terms of phase synchronization. Phase synchronization characteristics of the contained energy were consistent with the observations of negative and positive correlations between the WMN and DMN reported in referenced fMRI experiments. The results suggested that the coupled interaction between the WMN and DMN played important roles in working memory.

Stargazin and \u03b34 slow the channel opening and closing rates of GluA4 AMPA receptors

As auxiliary subunits, transmembrane AMPA receptor regulatory proteins (TARPs) are known to enhance macroscopic current amplitude and alter kinetic properties of AMPA receptors on slow time scale, such as desensitization rate. Whether TARPs affect the rate of AMPA channel opening and closing, however, remains elusive. Using a laser-pulse photolysis technique, we investigated the effect of γ-2 (stargazin, a type 1a TARP) and γ-4 (a type 1b TARP) on the channel-opening and channel-closing rate constants (i.e., kop and kcl) of GluA4 homomeric channels. We found both TARPs slow the kop and kcl by 4-fold and 3-fold, respectively, without appreciable change of channel-opening probability, as compared with GluA4 channel alone. On the other hand, γ-4 has a stronger effect on slowing the channel desensitization rate than γ-2; yet, γ-2 causes a much more pronounced left shift of the dose-response relationship by increasing its affinity towards glutamate than γ-4. Our study shows that on the faster time scale, the major impact of TARP association with GluA4 is to lengthen the lifetime of the open channel, which is slow to form, to allow a larger charge transfer through the open channel that closes more slowly, without appreciable change of channel opening probability.

Regenerative glutamate release in the hippocampus of Rett syndrome model mice.

Excess glutamate during intense neuronal activity is not instantly cleared and may accumulate in the extracellular space. This has various long-term consequences such as ectopic signaling, modulation of synaptic efficacy and excitotoxicity; the latter implicated in various neurodevelopmental and neurodegenerative diseases. In this study, the quantitative imaging of glutamate homeostasis of hippocampal slices from methyl-CpG binding protein 2 knock-out (Mecp2-/y) mice, a model of Rett syndrome (RTT), revealed unusual repetitive glutamate transients. They appeared in phase with bursts of action potentials in the CA1 neurons. Both glutamate transients and bursting activity were suppressed by the blockade of sodium, AMPA and voltage-gated calcium channels (T- and R-type), and enhanced after the inhibition of HCN channels. HCN and calcium channels in RTT and wild-type (WT) CA1 neurons displayed different voltage-dependencies and kinetics. Both channels modulated postsynaptic integration and modified the pattern of glutamate spikes in the RTT hippocampus. Spontaneous glutamate transients were much less abundant in the WT preparations, and, when observed, had smaller amplitude and frequency. The basal ambient glutamate levels in RTT were higher and transient glutamate increases (spontaneous and evoked by stimulation of Schaffer collaterals) decayed slower. Both features indicate less efficient glutamate uptake in RTT. To explain the generation of repetitive glutamate spikes, we designed a novel model of glutamate-induced glutamate release. The simulations correctly predicted the patterns of spontaneous glutamate spikes observed under different experimental conditions. We propose that pervasive spontaneous glutamate release is a hallmark of Mecp2-/y hippocampus, stemming from and modulating the hyperexcitability of neurons.

Design, synthesis, in vivo and in silico evaluation of phenacyl triazole hydrazones as new anticonvulsant agents

A series of phenacyl triazole hydrazones 3 have been designed based on the hybridization of (arylalkly)triazole and aroyl hydrazone scaffolds as new anticonvulsant agents. The target compounds 3 were easily synthesized from appropriate phenacyl triazoles and aryl acid hydrazides and characterized by IR, NMR and Mass spectroscopy. The in vivo anticonvulsant evaluation of synthesized compounds by using MES and PTZ tests revealed that they are more effective in MES model respect to PTZ test. All compounds showed 33–100% protection against MES-induced seizures at the dose of 100 mg/kg. However, the isonicotinic acid hydrazide derivative 3h showed the best profile of activity in both models. Molecular docking studies of compound 3h with different targets (NMDA, AMPA, GABAA and sodium channel), postulated that the compound acts mainly via GABAA receptors. In silico molecular properties predictions indicated that all compounds have favourable oral bioavailability and BBB permeability.

Zn2+-induced disruption of neuronal mitochondrial function: Synergism with Ca2+, critical dependence upon cytosolic Zn2+ buffering, and contributions to neuronal injury

Excitotoxic Zn2+ and Ca2+ accumulation contributes to neuronal injury after ischemia or prolonged seizures. Synaptically released Zn2+ can enter postsynaptic neurons via routes including voltage sensitive Ca2+ channels (VSCC), and, more rapidly, through Ca2+ permeable AMPA channels. There are also intracellular Zn2+ binding proteins which can either buffer neuronal Zn2+ influx or release bound Zn2+ into the cytosol during pathologic conditions. Studies in culture highlight mitochondria as possible targets of Zn2+; cytosolic Zn2+ can enter mitochondria and induce effects including loss of mitochondrial membrane potential (ΔΨm), mitochondrial swelling, and reactive oxygen species (ROS) generation. While brief (5 min) neuronal depolarization (to activate VSCC) in the presence of 300 μM Zn2+ causes substantial delayed neurodegeneration, it only mildly impacts acute mitochondrial function, raising questions as to contributions of Zn2+-induced mitochondrial dysfunction to neuronal injury.Using brief high (90 mM) K+/Zn2+ exposures to mimic neuronal depolarization and extracellular Zn2+ accumulation as may accompany ischemia in vivo, we examined effects of disrupted cytosolic Zn2+ buffering and/or the presence of Ca2+, and made several observations: 1. Mild disruption of cytosolic Zn2+ buffering—while having little effects alone—markedly enhanced mitochondrial Zn2+ accumulation and dysfunction (including loss of ∆Ψm, ROS generation, swelling and respiratory inhibition) caused by relatively low (10–50 μM) Zn2+ with high K+. 2. The presence of Ca2+ during the Zn2+ exposure decreased cytosolic and mitochondrial Zn2+ accumulation, but markedly exacerbated the consequent dysfunction. 3. Paralleling effects on mitochondria, disruption of buffering and presence of Ca2+ enhanced Zn2+-induced neurodegeneration. 4. Zn2+ chelation after the high K+/Zn2+ exposure attenuated both ROS production and neurodegeneration, supporting the potential utility of delayed interventions. Taken together, these data lend credence to the idea that in pathologic states that impair cytosolic Zn2+ buffering, slow uptake of Zn2+ along with Ca2+ into neurons via VSCC can disrupt the mitochondria and induce neurodegeneration.

Comparison of NMDA and AMPA Channel Expression and Function between Embryonic and Adult Neurons Utilizing Microelectrode Array Systems.

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke.

R/G editing in GluA2Rflop modulates the functional difference between GluA1 flip and flop variants in GluA1/2R heteromeric channels

In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors, RNA editing and alternative splicing generate sequence variants, and those variants, as in GluA2-4 AMPA receptor subunits, generally show different properties. Yet, earlier studies have shown that the alternatively spliced, flip and flop variants of GluA1 AMPA receptor subunit exhibit no functional difference in homomeric channel form. Using a laser-pulse photolysis technique, combined with whole-cell recording, we measured the rate of channel opening, among other kinetic properties, for a series of AMPA channels with different arginine/glycine (R/G) editing and flip/flop status. We find that R/G editing in the GluA2 subunit modulates the channel properties in both homomeric (GluA2Q) and complex (GluA2Q/2R and GluA1/2R) channel forms. However, R/G editing is only effective in flop channels. Specifically, editing at the R/G site on the GluA2R flop isoform accelerates the rate of channel opening and desensitization for GluA1/2R channels more pronouncedly with the GluA1 being in the flop form than in the flip form; yet R/G editing has no effect on either channel-closing rate or EC50. Our results suggest R/G editing via GluA2R serve as a regulatory mechanism to modulate the function of GluA2R-containing, native receptors involved in fast excitatory synaptic transmission.

AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability.

Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability.

Differential Vulnerability of CA1 versus CA3 Pyramidal Neurons After Ischemia: Possible Relationship to Sources of Zn2+ Accumulation and Its Entry into and Prolonged Effects on Mitochondria.

The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca2+ and Zn2+ contributing more in CA3, but with delayed and long-lasting accumulation of Zn2+ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn2+ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.

Differential expression of immediate early genes Zif268 and c-Fos in the hippocampus and prefrontal cortex following spatial learning and glutamate receptor antagonism

The objective of this study was to examine the effects of NMDAR and AMPAR antagonism on the expression of Zif268 and c-Fos in the hippocampus and medial prefrontal cortex during spatial memory encoding in rats trained in the Morris water maze. NMDAR inhibition impaired navigation and significantly attenuated expression of Zif268, but not c-Fos, in area CA1. AMPAR channel blockade had little effect on learning or IEG expression. Overall, Zif268 and c-Fos displayed markedly different patterns of hippocampal and prefrontal expression, with Zif268 being more closely linked to spatial learning.

Poly(ADP-Ribose)Polymerase 1 (PARP-1) Activation and Ca(2+) Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) Channels in Post-Ischemic Brain Damage: New Therapeutic Opportunities?

A significant number of laboratories observed that poly (ADP-ribose) polymerase (PARP) inhibitors, administered a few hours after ischemic or traumatic brain injury, may drastically reduce the subsequent neurological damage. It has also been shown that PARP inhibitors, administered for 24 hours to rats with permanent middle cerebral artery occlusion (MCAO), may reduce the number of dying neurons for a long period after surgery, thus suggesting that these agents could reduce the delayed brain damage and the neurological and cognitive impairment (dementia) frequently observed a few months after a stroke. In organotypic hippocampal slices exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), an alkylating agent able to activate PARP, a selective and delayed degeneration of the CA1 pyramidal cells which was anatomically similar to that observed after a short period of oxygen and glucose deprivation (OGD) has been described. Biochemical and electrophysiological approaches showed that MNNG exposure caused an increased expression and function of the calcium permeable α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels in the CA1 but not in the CA3 hippocampal region. PARP inhibitors prevented this increase and reduced CA1 cell death. The AMPA receptor antagonist 2,3-dihydroxy-6- nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione or the selective Ca(2+) permeable AMPA channel blocker 1-Naphthyl acetyl spermine (NASPM), also reduced the MNNG-induced CA1 pyramidal cell death. Since activation of PARP-1 facilitate the expression of Ca(2+) permeable channels and the subsequent delayed cell death, PARP inhibitors administered a few hours after a stroke may not only reduce the early post-ischemic brain damage but also the late neuronal death frequently occurring after severe stroke.

Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.

Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.

Catalytically Dead αCaMKII K42M Mutant Acts as a Dominant Negative in the Control of Synaptic Strength.

During long-term potentiation (LTP) of excitatory synapses, Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by Ca2+ influx through NMDA receptors that potentiate AMPA receptor currents by insertion of additional GluR1-containing receptors at the synapse and by increasing AMPA channel conductance, as well as by stimulating structural changes. CaMKII is also involved in the maintenance of LTP and contributes to maintenance of behavioral sensitization by cocaine or amphetamine. Recent studies show that transient expression of catalytically dead αCaMKII K42M mutant after exposure to amphetamine persistently reverses the behavioral effects of the addiction. A suggested interpretation is that this mutant acts as a dominant negative in the control of synaptic strength, but this interpretation has not been physiologically tested. Here we investigate the effect of αCaMKII K42M mutant expressed in single CA1 pyramidal neurons on basal excitatory neurotransmission in cultured rat hippocampal organotypic slices. The mutant caused nearly 50% reduction in the basal CA3–CA1 transmission, while overexpression of the wild-type αCaMKII had no effect. This result is consistent with the dominant negative hypothesis, but there are complexities. We found that the decrease in basal transmission did not occur when activity in the slices was suppressed after transfection by TTX or when NMDA receptors were blocked by APV. Thus, the dominant negative effect requires neural activity for its expression.

Functional consequences of age-related morphologic changes to pyramidal neurons of the rhesus monkey prefrontal cortex.

Layer 3 (L3) pyramidal neurons in the lateral prefrontal cortex (LPFC) of rhesus monkeys exhibit dendritic regression, spine loss and increased action potential (AP) firing rates during normal aging. The relationship between these structural and functional alterations, if any, is unknown. To address this issue, morphological and electrophysiological properties of L3 LPFC pyramidal neurons from young and aged rhesus monkeys were characterized using in vitro whole-cell patch-clamp recordings and high-resolution digital reconstruction of neurons. Consistent with our previous studies, aged neurons exhibited significantly reduced dendritic arbor length and spine density, as well as increased input resistance and firing rates. Computational models using the digital reconstructions with Hodgkin-Huxley and AMPA channels allowed us to assess relationships between demonstrated age-related changes and to predict physiological changes that have not yet been tested empirically. For example, the models predict that in both backpropagating APs and excitatory postsynaptic currents (EPSCs), attenuation is lower in aged versus young neurons. Importantly, when identical densities of passive parameters and voltage- and calcium-gated conductances were used in young and aged model neurons, neither input resistance nor firing rates differed between the two age groups. Tuning passive parameters for each model predicted significantly higher membrane resistance (R m ) in aged versus young neurons. This R m increase alone did not account for increased firing rates in aged models, but coupling these R m values with subtle differences in morphology and membrane capacitance did. The predicted differences in passive parameters (or parameters with similar effects) are mathematically plausible, but must be tested empirically.

Pathophysiology and principles of management of the many faces of the acute vaso-occlusive crisis in patients with sickle cell disease.

Effective management of sickle cell pain entails a thorough understanding of its pathophysiology and the pharmacogenomics of the opioids used to manage it. In recent years, there has been significant progress along these two lines. At the pathophysiologic level, there is evidence that the severity and frequency of painful stimuli modulate their transmission at the level of the dorsal horn of the spinal cord. This modulation is achieved via two channels: the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors. Initially, the AMPA channel controls the transmission of stimuli of mild-moderate severity. Once the AMPA channel reaches its limit of membrane depolarization, the NMDA channel is activated and facilitates the transmission of painful stimuli in a progressive fashion leading to central sensitization and glial activation. At the level of pharmacogenomics, the metabolism of each opioid is patient-specific. Glucuronidation is unique for the metabolism of morphine, hydromorphone, and oxymorphone. The metabolism of all other opioids requires specific Cytochrome P450 (CYP) isoenzymes. The activity of each isoenzyme and the activity of the metabolites of each opioid vary among patients depending on their genetic makeup and coexistent environmental factors such as the use of other medications that may enhance or inhibit the CYP isoenzyme activity.

Lipid rafts: linking prion protein to zinc transport and amyloid-β toxicity in Alzheimer's disease.

Dysregulation of neuronal zinc homeostasis plays a major role in many processes related to brain aging and neurodegenerative diseases, including Alzheimer's disease (AD). Yet, despite the critical role of zinc in neuronal function, the cellular mechanisms underpinning its homeostatic control are far from clear. We reported that the cellular prion protein (PrPC) is involved in the uptake of zinc into neurons. This PrPC-mediated zinc influx required the metal-binding octapeptide repeats in PrPC and the presence of the zinc permeable AMPA channel with which PrPC directly interacted. Together with the observation that PrPC is evolutionarily related to the ZIP family of zinc transporters, these studies indicate that PrPC plays a key role in neuronal zinc homeostasis. Therefore, PrPC could contribute to cognitive health and protect against age-related zinc dyshomeostasis but PrPC has also been identified as a receptor for amyloid-β oligomers which accumulate in the brains of those with AD. We propose that the different roles that PrPC has are due to its interaction with different ligands and/or co-receptors in lipid raft-based signaling/transport complexes.