Michael D. Browning

LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1

In the CA1 region of the rat hippocampus, long-term potentiation (LTP) requires the activation of NMDA receptors (NMDARs) and leads to an enhancement of AMPA receptor (AMPAR) function. In neonatal hippocampus, this increase in synaptic strength seems to be mediated by delivery of AMPARs to the synapse. Here we studied changes in surface expression of native AMPA and NMDA receptors following induction of LTP in the adult rat brain. In contrast to early postnatal rats, we find that LTP in the adult rat does not alter membrane association of AMPARs. Instead, LTP leads to rapid surface expression of NMDARs in a PKC- and Src-family-dependent manner. The present study suggests a developmental shift in the LTP-dependent trafficking of AMPA receptors. Moreover, our results indicate that insertion of NMDA receptors may be a key step in regulating synaptic plasticity.

Age-related working memory impairment is correlated with increases in the L-type calcium channel protein α1D (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)1.3) is selectively increased in area CA1 of aged rats. We and others have speculated that excessive Ca(2+) influx through L-VSCC may be detrimental to memory formation. Therefore, we investigated the relationship between age-related working memory decline and alpha(1D) protein expression in the hippocampus. In addition, we studied the effects of chronic treatment with the L-VSCC antagonist nimodipine (NIM) on age-related working memory deficits and alpha(1D) expression in the hippocampus. Here we report that age-related increases in alpha(1D) expression in area CA1 correlate with working memory impairment in Fischer 344 rats. Furthermore, we demonstrate that chronic NIM treatment ameliorates age-related working memory deficits and reduces expression of alpha(1D) protein in the hippocampus. The present results suggest that L-VSCCs participate in processes underlying memory formation and that increases in L-VSCC protein and currents observed with aging may play a role in age-related memory decline. Furthermore, the amelioration in age-related memory decline produced by NIM treatment may be mediated, at least in part, by reductions in the abnormally high levels of alpha(1D) protein in the aged hippocampus. These findings may have implications for patients with Alzheimers disease, who show increased L-VSCC protein expression in the hippocampus, and for patients receiving chronic treatment with L-VSCC antagonists.

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor.

The N‐methyl‐d‐aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non‐receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini‐slices with 500 nm phorbol 12‐myristate 13‐acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti‐NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 ± 11% above control) and NR2B (41 ± 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats.

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.

Evidence for a role for GABAA and NMDA receptors in ethanol inhibition of long-term potentiation

Abstract We have investigated the mechanisms by which acute ethanol inhibits the induction of long-term potentiation (LTP) in area CA1 of the rat hippocampal slice. In a previous report [Alcohol. Clin. Exp. Res. 21 (1997) 404] we demonstrated that ethanol produces only a modest inhibition of pharmacologically isolated N-methyl- d -aspartate receptors (NMDAR) in the CA1 region of the hippocampus. Moreover, this level of inhibition was not sufficient to account for ethanol’s complete inhibition of LTP induction in this brain region. One possible explanation of these results is that we may have underestimated ethanol’s ultimate effect on the NMDAR by focusing on pharmacologically isolated NMDAR responses. Ethanol might indirectly inhibit the NMDAR by, for example, potentiating the GABAAR. To explore this possibility, we first examined the effects of the GABAAR antagonist picrotoxin (PTX) and the allosteric GABAAR modulator flunitrazepam on NMDAR responses. We demonstrate that these modulators of GABAAR activity significantly affect the magnitude of synaptically evoked NMDAR responses. We next examined the effects of ethanol on NMDAR responses in the presence and absence of PTX. We see a significantly greater ethanol inhibition of the NMDAR when GABAARs are functional, i.e. in the absence of PTX. These data suggest that ethanol produces an inhibition of the NMDAR indirectly by affecting the GABAAR neurotransmission. Moreover, we found that ethanol inhibition of NMDAR activity, both directly through actions on the NMDAR, and indirectly, possibly through potentiation of GABAAR activity, is sufficient to account for ethanol’s complete blockade of LTP induction.

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ2 subunit of the GABAA receptor (GABAA‐R) is alternatively spliced. The long variant (γ2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP‐dependent protein kinase A (PKA)‐stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin‐dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC‐and CAM kinase II‐stimulated phosphorylation of this peptide were 102 and 35 μM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight‐amino‐acid insert of the γ2L subunit has been shown to be necessary for ethanol potentiation of the GABAA‐R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.

Cyclic AMP-Dependent Protein Kinase Decreases γ-Aminobutyric AcidA Receptor-Mediated 36Q1− Uptake by Brain Microsacs

The effect of cyclic AMP (cAMP)‐dependent protein phosphorylation on γ‐aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol‐stimulated 36C1− uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP‐ dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol‐stimulated 36C1− uptake by ∼25%. Jn parallel experiments, PKA and [γ‐32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitatc the entire GABAA receptor complex, under nondenaturing conditions, using an anti‐α1‐subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66‐kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti‐α1 subunit antibody. Phosphopeptide mapping of the 66‐kDa polypeptide demonstrated a 14‐kDa fragment similar to that obtained with the purified, PKA‐phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain G ABAAreceptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.

Regionally selective alterations in expression of the α1D subunit (Cav1.3) 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. We have studied the protein expression of the two pore forming subunits, alpha(1C) (Ca(v)1.2) and alpha(1D), of L-VSCCs in the hippocampus of young and aged rats. Here we report selective age-related changes in expression of alpha(1D) in the hippocampus. Specifically, we find that alpha(1D) protein is increased in area CA1 of aged rats while it is decreased in area CA3. Our data suggest that the altered calcium currents seen in aged animals may be due, at least in part, to alterations in the expression of the alpha(1D) subunit of the L-type calcium channel. These findings contribute to our understanding of the mechanisms responsible for changes in calcium homeostasis during aging.

αCaMKII 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. We were surprised to find that alphaCaMKII in a PSD-enriched fraction prepared from recovered hippocampal CA1-minislices had a relatively low level of threonine 286 (T286) phosphorylation and a relatively high level of threonine 305 (T305) phosphorylation. Furthermore, when the minislices were subjected to a treatment that mimics ischemic conditions, there was a significant translocation of alphaCaMKII to the PSD-enriched fraction accompanied with a dramatic reduction in T286 phosphorylation levels throughout the neuron. These findings have important implications for our understanding of the role of autophosphorylation in the localization of CaMKII.

Synapsin II phosphorylation and catecholamine release in bovine adrenal chromaffin cells : additive effects of histamine and nicotine

Abstract: Primary cultures of bovine adrenal medullary chromaffin cells can be stimulated with nicotine, which mimics the cholinergic stimulus from the splanchnic nerve. Histamine also stimulates catecholamine release in a time‐and dose‐dependent manner. We have previously shown that nicotine stimulates incorporation of 32Pi into the vesicle‐associated phosphoprotein synapsin II. We report here that histamine, too, stimulates an increase in 32Pi incorporation into synapsin II, which is blocked by the H1‐histamine receptor‐specific antagonist pyrilamine. The time course of histamine‐stimulated synapsin II phosphorylation closely paralleled that of histamine‐stimulated catecholamine release. Interestingly, histamine and nicotine produced an additive increase in both catecholamine release and synapsin II phosphorylation, suggesting that these two secretogogues stimulate the phenomena via independent mechanisms. When we investigated the dependence of these two agonists on extracellular calcium, we found that nicotine‐stimulated release and synapsin II phosphorylation were reduced to basal levels at low calcium concentrations. However, the histamine‐stimulated effects remained significantly elevated. This suggests that calcium arising from two separate pools can stimulate catecholamine release and synapsin II phosphorylation in bovine chromaffin cells. Taken together, these data support the hypothesis that synapsin II phosphorylation is a component of the secretory response from these cells.