Synaptic and Subcellular Localization of A-Kinase Anchoring Protein 150 in Rat Hippocampal CA1 Pyramidal Cells: Co-Localization With Excitatory Synaptic Markers

Excitatory and inhibitory ionotropic receptors are regulated by protein kinases and phosphatases, which are localized to specific subcellular locations by one of several anchoring proteins. One of these is the A-kinase anchoring protein (AKAP150), which confers spatial specificity to protein kinase A and protein phosphatase 2B in the rat brain. The distribution of AKAP150 was examined at rat hippocampal CA1 pyramidal cell asymmetric and symmetric post-synaptic densities and with respect to the distribution of markers of excitatory (vesicular glutamate transporter 1, glutamate receptor subunit 1) and inhibitory receptors (vesicular GABA transporter, GABA receptor type A β2/3 subunits, gephyrin) and the Golgi marker, trans-Golgi network glycoprotein 38. AKAP150 was close to asymmetric synapses, consistent with numerous molecular and biochemical studies suggesting its interaction with components of the excitatory postsynaptic density. In contrast, we did not find AKAP150-immunoreactivity associated with inhibitory synapses in rat CA1 neurons, despite reports demonstrating an in vitro interaction between AKAP150 and GABA receptor type A receptor β subunits, and the reported co-localization of these proteins in rat hippocampal cultures. There was some overlap between AKAP150 and GABA receptor type A receptor β2/3-immunoreactivity intracellularly in perinuclear clusters. These findings support previous work indicating the integration of kinase and phosphatase activity at excitatory synapses by AKAP150, but do not support a role for selective targeting of AKAP150 and its accompanying proteins to inhibitory synapses

Synaptic and Subcellular Localization of A-Kinase Anchoring Protein 150 in Rat Hippocampal CA1 Pyramidal Cells: Co-Localization With Excitatory Synaptic Markers

Excitatory and inhibitory ionotropic receptors are regulated by protein kinases and phosphatases, which are localized to specific subcellular locations by one of several anchoring proteins. One of these is the A-kinase anchoring protein (AKAP150), which confers spatial specificity to protein kinase A and protein phosphatase 2B in the rat brain. The distribution of AKAP150 was examined at rat hippocampal CA1 pyramidal cell asymmetric and symmetric post-synaptic densities and with respect to the distribution of markers of excitatory (vesicular glutamate transporter 1, glutamate receptor subunit 1) and inhibitory receptors (vesicular GABA transporter, GABA receptor type A β2/3 subunits, gephyrin) and the Golgi marker, trans-Golgi network glycoprotein 38. AKAP150 was close to asymmetric synapses, consistent with numerous molecular and biochemical studies suggesting its interaction with components of the excitatory postsynaptic density. In contrast, we did not find AKAP150-immunoreactivity associated with inhibitory synapses in rat CA1 neurons, despite reports demonstrating an in vitro interaction between AKAP150 and GABA receptor type A receptor β subunits, and the reported co-localization of these proteins in rat hippocampal cultures. There was some overlap between AKAP150 and GABA receptor type A receptor β2/3-immunoreactivity intracellularly in perinuclear clusters. These findings support previous work indicating the integration of kinase and phosphatase activity at excitatory synapses by AKAP150, but do not support a role for selective targeting of AKAP150 and its accompanying proteins to inhibitory synapses

Immunogold Electron Microscopic Evidence of Differential Regulation of GluN1, GluN2A, and GluN2B, NMDA-Type Glutamate Receptor Subunits in Rat Hippocampal CA1 Synapses During Benzodiazepine Withdrawal

Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal-anxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP-withdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles associated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal

Immunogold Electron Microscopic Evidence of Differential Regulation of GluN1, GluN2A, and GluN2B, NMDA-Type Glutamate Receptor Subunits in Rat Hippocampal CA1 Synapses During Benzodiazepine Withdrawal

Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal-anxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP-withdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles associated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal

Increased AMPA Receptor Glur1 Subunit Incorporation in Rat Hippocampal CA1 Synapses During Benzodiazepine Withdrawal

Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% ± 2.2%) compared with controls (74.4% ± 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 ± 0.3 gold particles/μm; CON: 10.8 ± 0.4 gold particles/μm). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity. J. Comp. Neurol. 511:832–846, 2008. © 2008 Wiley-Liss, Inc

Increased AMPA Receptor Glur1 Subunit Incorporation in Rat Hippocampal CA1 Synapses During Benzodiazepine Withdrawal

Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% ± 2.2%) compared with controls (74.4% ± 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 ± 0.3 gold particles/μm; CON: 10.8 ± 0.4 gold particles/μm). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity. J. Comp. Neurol. 511:832–846, 2008. © 2008 Wiley-Liss, Inc