Top Five Considerations When Choosing an Antibody.

The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process.

Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.

Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed.

Phosphorylation of FMRP and alterations of FMRP complex underlie enhanced mLTD in adult rats triggered by early life seizures.

Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS).

Radiation induces acute alterations in neuronal function.

Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage.

Functional adaptation of the N-methyl-D-aspartate receptor to inhibition by ethanol is modulated by striatal-enriched protein tyrosine phosphatase and p38 mitogen-activated protein kinase.

The hippocampal N-methyl-D-aspartate receptor (NMDAR) activity plays important roles in cognition and is a major substrate for ethanol-induced memory dysfunction. This receptor is a glutamate-gated ion channel, which is composed of NR1 and NR2 subunits in various brain areas. Although homomeric NR1 subunits form an active ion channel that conducts Na⁺ and Ca²⁺ currents, the incorporation of NR2 subunits allows this channel to be modulated by the Src family of kinases, phosphatases, and by simple molecules such as ethanol.

Optimized protocol to make phospho-specific antibodies that work.

Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state.

Correlated changes in NMDA receptor phosphorylation, functional activity, and sedation by chronic ethanol consumption.

Alcohol abuse leads to tolerance, dependence, and memory impairments that involve excitatory glutamatergic NMDA synaptic transmission. The NMDA receptor (NMDAR) is known to undergo activity-dependent adaptive functional changes. Since we observed that acute ethanol inhibition of the NMDAR was regulated by protein tyrosine phosphorylation, we investigated the role of protein tyrosine kinases and phosphatases on the NMDAR functions by chronic ethanol treatment.

Blueberry-enriched diet ameliorates age-related declines in NMDA receptor-dependent LTP.

NMDA receptor-dependent long-term potentiation (LTP) in the hippocampus is widely accepted as a cellular substrate for memory formation. Age-related declines in the expression of both NMDAR-dependent LTP and NMDAR subunit proteins in the CA1 region of the hippocampus have been well characterized and likely underlie age-related memory impairment. In the current study, we examined NMDAR-dependent LTP in young Fischer 344 rats (4 months old) and aged rats (24 months old) given either a control diet or a diet supplemented with blueberry extract for 6-8 weeks.

Long term synaptic depression that is associated with GluR1 dephosphorylation but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization.

Long lasting changes in the strength of synaptic transmission in the hippocampus are thought to underlie certain forms of learning and memory. Accordingly, the molecular mechanisms that account for these changes are heavily studied. Postsynaptically, changes in synaptic strength can occur by altering the amount of neurotransmitter receptors at the synapse or by altering the functional properties of synaptic receptors.

NMDA receptor trafficking at recurrent synapses stabilizes the state of the CA3 network.

Metaplasticity describes the stabilization of synaptic strength such that strong synapses are likely to remain strong while weak synapses are likely to remain weak. A potential mechanism for metaplasticity is a correlated change in both N-methyl-D-aspartate (NMDA) receptor-mediated postsynaptic conductance and synaptic strength. Synchronous activation of CA3-CA3 synapses during spontaneous bursts of population activity caused long-term potentiation (LTP) of recurrent CA3-CA3 glutamatergic synapses under control conditions and depotentiation when NMDA receptors were partially blocked by competitive antagonists.

alphaCaMKII autophosphorylation levels differ depending on subcellular localization.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has important roles in many processes in the central nervous system. It is enriched at the post-synaptic density (PSD), a localization which is thought to be critical for many of its proposed neuronal functions. In order to better understand the mechanisms that regulate association of CaMKII with the PSD, we compared the levels of autophosphorylation between PSD-associated kinase and kinase in other parts of the neuron.

A single episode of neonatal seizures permanently alters glutamatergic synapses.

Objective: The contribution of seizures to cognitive changes remains controversial. We tested the hypothesis that a single episode of neonatal seizures (sNS) on rat postnatal day (P) 7 permanently impairs hippocampal-dependent function in mature (P60) rats because of long-lasting changes at the synaptic level.

Methods: sNS was induced with subcutaneously injected kainate on P7.

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor is enhanced in synaptic membrane fractions of the adult rat hippocampus.

Hippocampal N-methyl-D-aspartate receptors (NMDARs) contribute to the expression of certain types of synaptic plasticity, such as long-term potentiation (LTP). It is well documented that tyrosine kinases increase NMDAR phosphorylation and potentiate NMDAR function. However, it is unclear how these phosphorylation changes result in enhanced NMDAR activity.

Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat.

N-Methyl-d-aspartate (NMDA)-type glutamate receptors in the hippocampus are important mediators of both memory formation and excitotoxicity. It is thought that glutamatergic neurons of the CA1, CA3 and dentate gyrus regions of the hippocampus contribute differentially to memory formation and are differentially sensitive to excitotoxicity. The subunit and/or splice variant composition of the NMDA receptor controls many aspects of receptor function such as ligand affinity, calcium permeability and channel kinetics, as well as interactions with intracellular anchoring and regulatory proteins.

Rosiglitazone improves contextual fear conditioning in aged rats.

Experimental, clinical, and epidemiologic studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) are beneficial in Alzheimer's disease and other neuroinflammatory processes. One possible mechanism is an interaction with peroxisome proliferator-activated receptors (PPARs). We examined the effect of a specific PPARgamma agonist, rosiglitazone, on contextual fear conditioning in aged rats.

Sulindac improves memory and increases NMDA receptor subunits in aged Fischer 344 rats.

Inflammatory processes in the central nervous system are thought to contribute to Alzheimer's disease (AD). Chronic administration of nonsteroidal anti-inflammatory drugs (NSAIDs) decreases the incidence of Alzheimer's disease. There are very few studies, however, on the cognitive impact of chronic NSAID administration.

Age-related working memory impairment is correlated with increases in the L-type calcium channel protein alpha1D (Cav1.3) in area CA1 of the hippocampus and both are ameliorated by chronic nimodipine treatment.

The hippocampus is critical for spatial memory formation in rodents. Calcium currents through L-type voltage-sensitive calcium channels (L-VSCCs) are increased in CA1 neurons of the hippocampus of aged rats. We have recently shown that expression of the calcium conducting L-VSCC subunit alpha(1D) (Ca(v)1.

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function.

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W.

Regionally selective alterations in expression of the alpha(1D) subunit (Ca(v)1.3) of L-type calcium channels in the hippocampus of aged rats.

Calcium currents through the L-type voltage-sensitive calcium channel (L-VSCC) are increased in neurons of area CA1 of the hippocampus in aged rats and rabbits. Furthermore, increases in mRNA for the pore forming subunit alpha(1D) (Ca(v)1.3) have been observed in the hippocampus of aged rats.

A hippocampal NR2B deficit can mimic age-related changes in long-term potentiation and spatial learning in the Fischer 344 rat.

Aged rats are known to have deficits in spatial learning behavior in the Morris water maze. We have found that aged rats also have deficits in NR2B protein expression and that the protein expression deficit is correlated with their performance in the Morris water maze. To test whether this NR2B deficit was sufficient to account for the behavioral deficit, we used antisense oligonucleotides to specifically knock down NR2B subunit expression in the hippocampus of young rats.

Aging and surface expression of hippocampal NMDA receptors.

Aging is known to alter many physiological processes within the brain including synaptic responses, long-term potentiation, learning, and memory. Aging has also been shown to alter the expression and distribution of N-methyl-d-aspartate (NMDA) receptors in many different brain regions, including the hippocampus. Additionally, we have recently reported that young adult rats show an activity-dependent increase in the surface expression of NMDA receptors.