In Vivo Phosphorylation of the Postsynaptic Density Glycoprotein gp180

Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [gamma-32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)-agarose demonstrated that greater than 70-80% of the 32P present in the glycoproteins eluted from Con A-agarose with alpha-methyl-D-mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.     

Isolation of Postsynaptic Densities from Day-Old Chicken Brain

Synaptic plasma membranes from chicken brain were used to isolate a postsynaptic density (PSD) fraction using an aqueous two-phase polymer system and the detergent n-octyl glucoside. The protein and glycoprotein composition and the morphology of the day-old chicken brain PSD fraction were compared with a PSD fraction isolated from 12-week-old chicken brain. The PSD fraction from day-old chicken brain contained predominantly PSDs although, like the fraction from 12-week-old chicken, there was some membrane contamination. The major polypeptides in the day-old chicken fraction resolved by polyacrylamide gel electrophoresis comigrated with alpha- and beta-tubulin (Mr 57,000 and 55,000) and actin (Mr 45,000). The major PSD polypeptide (mPSDp) of 12-week-old chicken forebrain, which has a molecular weight of 52,000 was not a major component in day-old chicken. A polypeptide of molecular weight 63,000 was also far more prominent in the 12-week-old chicken PSD fraction whereas the reverse was true for a polypeptide of 31,000. Day-old chicken brain PSDs contained at least 14 concanavalin A-binding glycoproteins of high (greater than 85,000) molecular weight, the two most prominent having molecular weights of 170,000 and 180,000. In contrast to the polypeptide composition, the glycoprotein pattern of day-old chicken PSDs was very similar to that of the 12-week-old bird. Intraperitoneally injected [3H]fucose was incorporated into the glycoproteins of synaptic plasma membranes and PSDs from day-old chickens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Granular Particles Isolated from Postsynaptic Densities

Abstract: We describe here the isolation and biochemical characterization of a population of protein aggregates from the postsynaptic density (PSD) prepared from pig cerebral cortex. The protein constituents of these aggregates are linked together primarily by disulfide bonds. Negative staining electron microscopy revealed that the isolated protein aggregates were granular objects with an average outside diameter of ∼21 nm and with small protrusions on their surface. The major constituents of the isolated granular aggregates consist of tubulin and an unidentified protein of 70 kDa in size. Small amounts of the α subunit of calcium/calmodulin-dependent protein kinase II and subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and NMDA subtypes of glutamate receptors were also detected by immunoblotting. Actin, however, was not found in these granular aggregates. We propose that these granular protein aggregates correspond to the ∼20-nm-diameter granular particles of the PSD on the basis of their biochemical and morphological characteristics. The spatial arrangement of these granular aggregates relative to other components of the postsynaptic terminal is also postulated here.     

Characterization of Na+-Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.     

Purification and Properties of p 103, a Novel 103-kDa Component of Postsynaptic Densities

A 103-kDa protein present in membrane cytoskeletal preparations from bovine brain has been identified. We have purified this protein to greater than 95% homogeneity using gel filtration and ion-exchange chromatography. This protein, p103, is an asymmetric dimer in dilute solution and has two major variants that can be distinguished by isoelectric focussing, pI 5.60 and 5.75. Using subcellular fractionation, it is most enriched in postsynaptic densities. Immunolocalization with anti-p103-specific antibodies reveals that it is confined to the dendrites and perikarya; it is apparently absent from spinal cord axons. It coextracts from brain membrane-skeletal preparations with brain spectrin and actin, but in vitro, it does not interact with them.     

Inhibition of Endogenous Phosphatase in a Postsynaptic Density Fraction Allows Extensive Phosphorylation of the Major Postsynaptic Density Protein

Abstract: The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the a subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Phosphorylation of the Postsynaptic Density Glycoprotein gp 180 by Ca2+/Calmodulin-Dependent Protein Kinase

Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

411B: A Monoclonal Postsynaptic Marker for Modulations of Synaptic Connectivity in the Rat Brain

Using 411B, a monoclonal marker raised to chick forebrain postsynaptic densities (PSDs), we have been able to demonstrate by enzyme-linked immunosorbent assay that the antigen recognised by this monoclonal exists in brain tissue from adult Wistar rats but not in liver, heart, or lung. Moreover, 411B immunoreactivity estimated in various cortical and subcortical brain structures exhibited remarkable differences. The pattern of subcellular distribution of 411B antiserum titre in rats was found to be qualitatively similar to that in day-old chicks, indicating an enrichment of the antigen concentration in the PSD fraction by about 60 times over that observed in the lysed homogenates. One aim of this study was to investigate whether 411B is a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. Antigen was assayed in lysed homogenates from various brain regions dissected from dopaminergically supersensitive rats. Dopaminergic supersensitivity induced by treating animals with haloperidol (1 mg/kg i.p.) for 21 consecutive days resulted in a significant increase in the titre of 411B in corpus striatum (+21%) and hippocampus (+45%) whereas the titre of Q155, a monoclonal marker for an integral synaptic vesicle protein, was unchanged. Our results support the hypothesis that drug-induced dopaminergic supersensitivity is based on plastic changes at the postsynaptic site. In addition, monoclonal antibody 411B does appear to be a useful tool for further investigation of plastic changes occurring in postsynaptic brain components.     

Biochemical and Morphological Comparison of Postsynaptic Densities Prepared from Rat, Hamster, and Monkey Brains by Phase Partitioning

Abstract: A new procedure for the preparation of postsynaptic densities (PSDs) is described. A synaptic membrane fraction was homogenized in an aqueous two-phase polymer system containing Poly(ethylene glycol) 6000 (5% wt/wt) and Dextran T500 (6% wt/wt) containing 1% I- o - n -octyl-β-D-glucoside. Following a brief centrifugation to separate the phases, highly purified PSDs banded at the interface of the two phases. Using this procedure PSDs have been isolated from rat and hamster cerebral cortex and from the frontal cortex, cerebral cortex, hippocampus, and pooled caudateiputamen regions of Macaca mulatta Rhesus monkeys. The isolated PSDs appeared as curved bars when sectioned or as discs when viewed en face in the electron microscope. The hamster PSDs were associated with large numbers of small rod-like structures 4.5 nm thick and 28 nm long. Similar structures were present, although in fewer numbers, in the rat and monkey preparations. Polyacrylamide gel electrophoresis showed that the PSDs contained a complex population of proteins with major components having molecular weights of 180,000, 130,000, 110,000, 94,000, 65,000, 60,000, and 51,000. Reaction of polyacrylamide gels with ¹²⁵I- concanavalin A (Con A) identified two major (apparent M_r 180,000 and 130,000) and three minor (apparent M_r 230,000, 145,000, and 110,000) Con A-binding glycoproteins in the PSD fractions. Although some quantitative variation between species and brain regions was apparent, the overall protein and glycoprotein composition was similar for all fractions studied.     

Synaptic Protein Tyrosine Kinase: Partial Characterization and Identification of Endogenous Substrates

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.     

The Effects of Detergents on the Composition of Postsynaptic Densities

A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3-14 removed most of the beta-tubulin but not alpha-tubulin. Sodium N-lauroyl sarcosinate completely destroyed the structural integrity of the PSD when the in vitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.     

β-Dystroglycan: Subcellular Localisation in Rat Brain and Detection of a Novel Immunologically Related, Postsynaptic Density-Enriched Protein

Abstract: The distribution of a glycoprotein component of the muscle dystrophin complex, β-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that β-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle β-dystroglycan) abolished reactivity against both β-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of β-dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species.     

Identification and Comparison of Protein I in Chick and Rat Forebrain

Abstract: Protein I has been identified and compared in membranes prepared from chick and rat forebrain. Based upon five criteria known to characterize protein I, namely, (1) its ability to serve as a substrate for both the cyclic AMP-dependent protein kinase and (2) the Ca²⁺-dependent, calmodulin-requiring protein kinase, (3) its ability to be extracted from membranes at low pH, (4) its characteristic pattern of digestion by collagenase, and (5) its existence as a basic protein, we have determined that although protein I of rat brain consists of the usual doublet polypeptides la and Ib, only a single chick forebrain polypeptide is detectable which possesses protein Mike properties.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

Posttetanic Long-Term Potentiation in Rat Dentate Area Increases Postsynaptic 411B Immunoreactivity

411B is a monoclonal antibody raised to chick forebrain postsynaptic densities (PSDs) which also recognises an antigen in brain tissue from adult Wistar rats but not liver, heart, or lung. This antigen is enriched in the PSD fraction and appears to be a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. The aim of this study was to investigate whether 411B immunoreactivity is changed in various hippocampal subregions by post-tetanic long-term potentiation (LTP). LTP was elicited in freely moving rats by applying four trains of 300 square-wave pulses (frequency 200 Hz, pulse duration 0.2 ms, and intensity 300 mA) into the right perforant path; this included an increase in transmission efficacy at the ipsilateral perforant path-granular cell synapse of the dentate gyrus lasting several days. Eight hours after tetanisation, antigens recognised by monoclonal 411B and a polyclonal anti-actin antiserum were assayed in lysed homogenates of ipsi- and contralateral CA1. CA3, and CA4/dentate area hippocampal subfields as well as in visual cortex, cerebellum, and olfactory bulb dissected from LTP rats, and compared to passive controls. Under these experimental conditions, tetanisation of the perforant path resulted in a significant increase in the titre of 411B in the ipsilateral CA4/dentate area subfield (+34.0%; p less than 0.001) compared with passive controls, whereas in all other brain regions studied no differences between experimental and control rats were observed. In no region were anti-actin titres significantly different from controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and Characterisation of a Monoclonal Antibody to an Antigen Enriched in Chick Brain Postsynaptic Densities

Abstract: The preparation and characterisation of a monoclonal antibody to an antigen enriched in day-old chick brain postsynaptic densities (PSDs), with respect to other subcellular loci, are described. Immunolabelling with this antibody produced a dendritic immunoprecipitate that was markedly stronger in PSDs than in other subcellular loci. Thus, the antiserum could be used as a marker for PSDs during their purification by subcellular fractionation, as well as in the study of PSD assembly. Monoclonal antibody 411B has already been shown to be a useful tool in the chemical determination of changes in synapse density after various experimental manipulations in both the chick and rat. In the present study, we have used the antiserum to monitor the appearance and maintenance or redundancy of synaptic components in the developing chick forebrain.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.