Synaptic Junctional Complexes Research Articles

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

δ-Catenin Is Required for the Maintenance of Neural Structure and Function in Mature Cortex In Vivo

Delta-catenin is a brain-specific member of the adherens junction complex that localizes to the postsynaptic and dendritic compartments. This protein is likely critical for normal cognitive function; its hemizygous loss is linked to the severe mental retardation syndrome Cri-du-Chat and it directly interacts with presenilin-1 (PS1), the protein most frequently mutated in familial Alzheimer's disease. Here we examine dendritic structure and cortical function in vivo in mice lacking delta-catenin. We find that in cerebral cortex of 5-week-old mice, dendritic complexity, spine density, and cortical responsiveness are similar between mutant and littermate controls; thereafter, mutant mice experience progressive dendritic retraction, a reduction in spine density and stability, and concomitant reductions in cortical responsiveness. Our results indicate that delta-catenin regulates the maintenance of dendrites and dendritic spines in mature cortex but does not appear to be necessary for the initial establishment of these structures during development.

The CNS Synapse Revisited: Gaps, Adhesive Welds, and Borders

Although processes leading up to the point of synapse formation are fairly well understood, the precise sequence of events in which the membranes of two separate cells "lock in" to form a mature synaptic junctional complex is poorly understood. A careful study of the molecules operating at the synapse indicates that their roles are more multifarious than once imagined. In this review we posit that the synapse is a functional organelle with poorly defined boundaries and a complex biochemistry. The role of adhesion molecules, including the integration of their signaling and adhesive properties in the context of synaptic activity is discussed.

GRIP1 in GABAergic synapses

The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.

Protein Trafficking and Alzheimers Disease

Mutations in presenilin 1 (PS1) cause early-onset familial Alzheimer;s disease (FAD). Although FAD accounts for less than 5% of all cases of Alzheimer;s disease (AD), extensive analyses of PS1 function have elucidated an important neuronal mechanism underling AD pathogenesis. PS1 is considered to be an essential component of gamma-secretase, which cleaves amyloid precursor protein (APP) at the transmembrane region and releases amyloid beta (Abeta) peptide. In addition to this well-documented function, a growing amount of evidence suggests that PS1 is involved in the intracellular trafficking of selected membrane proteins (i.e. APP, nicastrin, trkB, telencephalin). Recently, we have also shown that PS1 is involved in the trafficking of N-cadherin from the endoplasmic reticulum to the plasma membrane via the microtubule network. N-cadherin is localized at the synaptic junctional complex, providing an adhesive force across the synaptic cleft, and the its regulation is crucial for the neuron to exert its specific function, i.e. synaptic activity. In a mature neuron, polarized targeting of proteins from the cell body to the axonal and dendritic processes is essential for its proper function, especially, for the maintenance of synaptic function. Alterations in protein transport caused by a dysfunction in PS1 could lead to a disturbance in synaptic transmission and finally to neurodegeneration. This article will review the current knowledge of PS1 function in protein trafficking and discuss its potential role in AD pathogenesis.

The successful aging of Carl Cotman: from proteins of the synapse, through sprouting, regeneration, and spinal cord injury to the mechanisms of brain aging.

I have a flash-bulb memory of the first time I spoketo Carl Cotman in May, 1970. I had applied to the gradu-ate program of the Department of Psychobiology at UCIrvine (my first choice for graduate school) and had notbeen accepted in the first round. I had resigned myself toattending graduate school elsewhere, but received a callfrom Carl one evening. Carl spoke with his characteris-tic candor, which can be either charming or intimidating(in this case, it was intimidating). He said that my cre-dentials weren’t all that great, but he liked the fact thatI had completed my undergraduate studies at the Uni-versity of Colorado in 3 years while supporting myselfthrough part-time jobs—hence the phone interview. Isweated bullets through the interview, and in the end, heoffered me a position in his lab. His decision to give mea chance highlights two features of his personality—hiswillingness to take risks and his personal ethic for hardwork. I don’t think that he had too many occasions toregret his decision, although he probably had some sec-ond thoughts during the early part of my graduate career.I joined Carl’s lab in the fall of 1970 at the start ofCarl’s third year at UCI. By that time, four other studentswere in the lab, included Gary Banker, William (Chip)Levy, Dee Ann Matthews, and Lynn Churchill. The cal-dron of Carl’s lab and the wonderfully intense graduateprogram in the department forged friendships and fos-tered collaborations that have lasted a lifetime. In fact,the University of Virginia later became an outpost ofCarl’s former trainees when I recruited Chip Levy to theDepartment of Neurosurgery and Gary Banker to thenewly established Department of Neuroscience.When I joined Carl’s lab, the main focus of researchwas on the chemistry of the synaptic junction. EachMonday morning started with the collection of rat brains(40 or so) for subcellular fractionation experiments.Everyone participated in this Monday morning “brainsand donuts” collection process. This was really nuts andbolts neurochemistry and led to fundamental, indeedlandmark, discoveries about the structure and proteincomposition of the synaptic junctional complex (1,2).The subcellular fractionation techniques he developed forisolating postsynaptic densities (psds) are still in usetoday, and the psd, which he identified as a definableand isolatable structure, has come to be recognized asa highly organized collection of interrelated signalingmolecules (3).At about the time that I joined the lab, Carl wastrying different approaches to begin to address broaderquestions of synaptic function, especially synaptic plas-ticity. It is remarkable how many important scientificthreads can be traced from his work during that period.My first project in the lab was to try to determinewhether synaptic activation altered protein synthesis inpostsynaptic cells (what we would now call synapticregulation of gene expression). Carl’s idea was to usethe abdominal ganglion of aplysia as a model for stud-ies of synaptic biochemistry. The experiment was toactivate connectives that contained nerves that synapsedon the large R60 neuron in the ganglion in the presenceof labeled amino acids, then dissect out the large R60neuron and assess incorporation into protein usingmicrobiochemical techniques. At that time, we collectedthe experimental animals ourselves in outings to the tidepools off Crystal Cove near Irvine. Fortunately, Carl didnot go on these outings and did not seem to be awareof the tide tables that limited collection times. Thus thecollection party (the graduate students in the lab)planned the day so as to collect the necessary specimens

A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites.

Long-lasting forms of activity-dependent synaptic plasticity involve molecular modifications that require gene expression. Here, we describe a cellular mechanism that mediates the targeting newly synthesized gene transcripts to individual synapses where they are locally translated. The features of this mechanism have been revealed through studies of the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein Arc. Arc is strongly induced by patterns of synaptic activity that also induce long-term potentiation, and Arc mRNA is then rapidly delivered into dendrites after episodes of neuronal activation. The newly synthesized Arc mRNA localizes selectively at synapses that recently have been activated, and the encoded protein is assembled into the synaptic junctional complex. The dynamics of trafficking of Arc mRNA reveal key features of the mechanism through which synaptic activity can both induce gene expression and target particular mRNA transcripts to the active synapses.

Neural (N)‐cadherin at developing thalamocortical synapses provides an adhesion mechanism for the formation of somatopically organized connections

Thalamic projections from the ventrobasal (VB) nucleus to rodent somatosensory cortex develop highly ordered terminations that form discrete, clustered patches localized to layer IV cellular aggregates that are termed “barrels.” The molecular signaling and adhesion events that occur at the synapse as barrel-clustered thalamic connections form are unknown. Here, we show that neural (N)-cadherin, a membrane glycoprotein mediating strong homophilic adhesion, is concentrated at the developing thalamocortical synaptic junctional complex and demarcates these synaptic junctions as they form their characteristic barrel clusters during the first postnatal week. Furthermore, experimentally altering the distribution of thalamocortical axon terminals by peripheral manipulation leads to an identically altered N-cadherin distribution pattern, which is significant in establishing that N-cadherin does not define region-specific patterns of synapse distribution proactively but, rather, conforms to patterning imposed by thalamic axons through instructional cues conveyed through several synaptic relays. At postnatal day 9, levels of N-cadherin expression rapidly decrease, leading to loss of N-cadherin labeling of the barrels and, at adulthood, elimination from VB thalamocortical synapses. However, αN- and β-catenin, which are critical binding partners of the classic cadherins, persist at the adult synapse, suggesting the presence of another classic cadherin as the thalamocortical synapse matures. This is the first evidence linking a synapse adhesion molecule with the establishment of patterned thalamocortical synapse distribution, suggesting strongly that N-cadherin performs a critical role in this process by adhering presynaptic and postsynaptic membranes as ingrowing thalamic axon terminals and postsynaptic thalamorecipient sites link and stabilize into mature synaptic junctional complexes distributed with precise topographic order. It is speculated that the developmental redistribution of N-cadherin may reflect dynamic regulation of synaptic membrane adhesion, which, in turn, might modulate plasticity of thalamocortical synaptic function. J. Comp. Neurol. 407:453–471, 1999. © 1999 Wiley-Liss, Inc.

Neural (N)-cadherin at developing thalamocortical synapses provides an adhesion mechanism for the formation of somatopically organized connections

Thalamic projections from the ventrobasal (VB) nucleus to rodent somatosensory cortex develop highly ordered terminations that form discrete, clustered patches localized to layer IV cellular aggregates that are termed "barrels." The molecular signaling and adhesion events that occur at the synapse as barrel-clustered thalamic connections form are unknown. Here, we show that neural (N)-cadherin, a membrane glycoprotein mediating strong homophilic adhesion, is concentrated at the developing thalamocortical synaptic junctional complex and demarcates these synaptic junctions as they form their characteristic barrel clusters during the first postnatal week. Furthermore, experimentally altering the distribution of thalamocortical axon terminals by peripheral manipulation leads to an identically altered N-cadherin distribution pattern, which is significant in establishing that N-cadherin does not define region-specific patterns of synapse distribution proactively but, rather, conforms to patterning imposed by thalamic axons through instructional cues conveyed through several synaptic relays. At postnatal day 9, levels of N-cadherin expression rapidly decrease, leading to loss of N-cadherin labeling of the barrels and, at adulthood, elimination from VB thalamocortical synapses. However, alphaN- and beta-catenin, which are critical binding partners of the classic cadherins, persist at the adult synapse, suggesting the presence of another classic cadherin as the thalamocortical synapse matures. This is the first evidence linking a synapse adhesion molecule with the establishment of patterned thalamocortical synapse distribution, suggesting strongly that N-cadherin performs a critical role in this process by adhering presynaptic and postsynaptic membranes as ingrowing thalamic axon terminals and postsynaptic thalamorecipient sites link and stabilize into mature synaptic junctional complexes distributed with precise topographic order. It is speculated that the developmental redistribution of N-cadherin may reflect dynamic regulation of synaptic membrane adhesion, which, in turn, might modulate plasticity of thalamocortical synaptic function.

A Model for Central Synaptic Junctional Complex Formation Based on the Differential Adhesive Specificities of the Cadherins

Cadherins control critical developmental events through well-documented homophilic interactions. In epithelia, they are hallmark constituents of junctions that mediate intercellular adhesion. Brain tissue expresses several cadherins, and we now show that two of these, neural (N)- and epithelial (E)-cadherin, are localized to synaptic complexes in mutually exclusive distributions. In cerebellum, N-cadherin is frequently found associated with synapses, some of which are perforated, and in hippocampus, N- and E-cadherin-containing synapses are found aligned along dendritic shafts within the stratum lucidum of CA3. We propose that the cadherins function as primary adhesive moieties between pre- and postsynaptic membranes in the synaptic complex. According to this model, once neurites have been guided to the vicinity of their cognate targets, it is the differential distribution of cadherins along the axonal and dendritic plasma membranes, and ultimately cadherin self-association, that “locks in” nascent synaptic connections.

Type I brain hexokinase: axonal transport and membrane associations within central nervous system presynaptic terminals.

While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.

Protein synthesis within dendrites: ionic and neurotransmitter modulation of synthesis of particular polypeptides characterized by gel electrophoresis.

This study evaluates whether physiological variables differentially affect the local synthesis of protein constituents of synapses in subcellular fractions containing pinched-off dendrites (synaptodendrosomes). Synaptodendrosomes were pulse-labeled in a medium containing 35S-methionine with 3 or 25 mM KCl and in the presence or absence of 0.5 mM EGTA or 10 microM glutamate. Synaptodendrosomes were then subfractionated to prepare synaptic plasma membranes and synaptic junctional complexes. The protein constituents of the synaptic plasma membrane and synaptic junctional complex fractions that were locally synthesized were identified using SDS-PAGE and two-dimensional gel electrophoresis and the extent of labeling of individual bands was analyzed using a Phosphorimager. Analysis of incorporation into individual bands resolved by SDS-PAGE revealed that depolarizing conditions (25 mM KCl) increased the extent of labeling of different bands to a different extent (ranging from 10-70% increases in labeling). Addition of 0.5 mM EGTA decreased the extent of labeling of the same group of bands in both 3 mM KCl and 25 mM KCl conditions. Addition of 10 microM glutamate reduced incorporation especially in the synaptodendrosomes incubated in 25 mM KCl. Two-dimensional gel electrophoresis analyses revealed that the labeled spots that showed differential labeling under the different conditions did not correspond to the most prominent Coomassie-stained spots. These results indicate that the proteins that are synthesized in synaptodendrosomes and regulated by physiological variables are not amongst the more abundant protein constituents of the fractions. Taken together, these results are consistent with the idea that protein synthesis within dendrites may be regulated by synaptic activity.

The rat brain postsynaptic density fraction contains a homolog of the drosophila discs-large tumor suppressor protein

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre-and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-I (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dig and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.

Localization of a 82 kDa protein in postsynaptic density and its association with cytoskeletons

A fraction of synaptic junctional complex (SJC) was prepared from rat synaptosomes and served as antigen material to produce monoclonal antibodies (Mab) for examining the component proteins of the SJC. An antibody, Mab SJ-8, was obtained, which recognized a protein with a molecular weight of 82 000 Da in the SJC preparation by immunoblot analysis. The immunohistochemical localization of the 82 kDa protein was studied with the rat cerebellum. Mab SJ-8 labeled the peripheral areas of the Purkinje and granule cells. Small punctate areas were also stained in the molecular layer with SJ-8. Intracellular localization of the protein was examined with rat brain synaptosomes. Immunoelectron microscopy demonstrated that Mab SJ-8 strongly labeled the postsynaptic density (PSD) and also a fibrous network spreading out of it. However, the antibody did not label the pre- or post-synaptic membrane or the cleft material.

Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: Time course of appearance of recently synthesized proteins in synaptic junctions

Previous studies have led to the hypothesis that some protein constituents of postsynaptic membrane specializations are locally synthesized near postsynaptic sites. The present study focuses on one prediction of this hypothesis, specifically, that if some proteins of the postsynaptic membrane specialization are locally synthesized, then the delay between synthesis and assembly into synaptic junctional membrane could be short. We evaluate the time course of appearance of recently synthesized protein in synaptic junctions by pulse-labeling hippocampal slices maintained in vitro with radiolabeled protein precursors, and then isolating subcellular fractions enriched in synaptic plasma membranes (SPM) and synaptic junctional complexes (SJC). We report that there is no evidence of a delay in the appearance of recently synthesized proteins in SPM and SJC fractions. Labeled proteins could be detected as early as 15 min after the initiation of the pulse-labeling period, and the extent of labeling increased monotonically thereafter. The labeling could not be accounted for by contamination of synaptic membrane fractions with other membranes, because the relative specific activity of the SPM and SJC fractions was the same or higher than that of the less pure fractions from which these synaptic fractions were derived. One-dimensional PAGE-fluorography was used to provide an initial characterization of which proteins were labeled in SJC fractions. We found that the most prominent labeled bands were at apparent molecular weights of approximately 43-44, 55-56, and 60 kd, with more lightly labeled bands at about 38 and 116 kd. In some preparations, there was a labeled doublet at about 36-38 kd. There were also other lightly labeled bands at other molecular weights. These bands were much less heavily labeled than the bands at 43-44, 55-56, and 60 kd, however. There was little labeling in the molecular weight range of the "major psd protein" (the alpha subunit of CAM-kinase), although there was diffuse labeling throughout the 45-52 kd region. These results are consistent with the hypothesis that some of the protein constituents of the postsynaptic junctional complex are synthesized by polyribosomes which are selectively localized beneath synaptic junctions.

Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes

Previous studies have led to the hypothesis that some proteins of the postsynaptic membrane are locally synthesized at postsynaptic sites. To evaluate this hypothesis, synaptosome fractions that included fragments of dendrites were allowed to incorporate labeled amino acid into protein. The labeled synaptosomes were then subfractionated to the level of the synaptic plasma membrane (SPM) and then the synaptic junctional complex (SJC). The specific activity (cpm/microgram protein) of the synaptosome fraction and its subfractions was assessed by scintillation counting and protein assay, and labeled polypeptides were characterized by SDS-PAGE and fluorography. The contribution of mitochondrial and eucaryotic protein synthesis to the overall incorporation was evaluated using cycloheximide (CYC), a eucaryotic protein synthesis inhibitor, and chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor. Both the SPM and the SJC subfractions obtained from labeled synaptosomes contained labeled polypeptides. The SPM from labeled synaptosomes had a specific activity approximately equal to that of other nonmitochondrial membrane components of the synaptosome. Thus, labeling of the SPM was not due to contamination by these other labeled membrane components. The mitochondrial fraction had the highest specific activity of the membrane components of the labeled synaptosome, but the specific activity was reduced by 47% in mitochondrial fractions from CAP-treated synaptosomes, while the specific activity of the SPM was not reduced by this treatment. Thus, SPM labeling is not due to mitochondrial contamination. The specific activity of the detergent-insoluble SJC was comparable to that of the SPM from which it was derived. The possibility of labeling of SPM and SJC by contamination with soluble proteins was assessed by adding labeled soluble proteins to a cold synaptosome preparation that was then subfractionated to obtain the SPM and SJC. There was no detectable binding of labeled soluble proteins to the SPM or SJC. These results support the hypothesis that some synaptic proteins are locally synthesized. Fluorographs of SDS gels of SPM from labeled synaptosomes revealed labeled bands at approximate molecular weights of 14, 18, 26, 28, 36, 38, 42, 45, 55, 60, and 116 kDa. Six of these labeled polypeptides at 38, 42, 45, 55, 60, and 116 kDa were still evident in fluorographs of the synaptic junctional complex from labeled synaptosomes. None of these labeled bands were seen in fluorographs of SPM and SJC from CYC-treated synaptosomes, whereas they were still present in fluorographs of CAP-treated synaptosomes. These labeled polypeptides are therefore produced by eucaryotic ribosomal systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Solubilisation of a glutamate binding protein from rat brain.

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.

Effects of chronic alcohol administration on synaptic membrane Na +[sbnd]Ca 2+ exchange activity

We have recently reported that ethanol and other n-alkanols added to in vitro assays inhibit the activity of the Na + Ca 2+ exchange system in brain synaptic plasma membrane vesicles. The present studies were undertaken to determine whether in vivo chronic ethanol administration leads to alterations in this Na + Ca 2+ antiporter that might be indicative of an adaptive response to alcohol. The Na +-dependent Ca 2+ transport activity in the plasma-membrane fractions obtained was measuredd at various Ca 2+ concentrations. Results of these experiments revealed that a 3-week ethanol regimen brought about a significant increase in the Na +-dependent Ca 2+ transport activity only in the membrane fraction enriched in synaptic junctional complexes. These membranes showed a near doubling in the maximal transport activity of the antiporter in alcohol-treated compared with the control animals. Changes in the kinetic parameters were reversible as the Na + Ca 2+ exchange activity in these membranes from animals maintained on alcohol for 3 weeks and then withdrawn for 1 week was indistringuishable from that of membranes from control animals. Thus it appears that ethanol-treated animals make a reversible adaptation in their neuronal cell membranes to compensate for the acute effects of ethanol on the Na + Ca 2+ antiporter.

Interaction of isolated synaptic vesicles with synaptic junctional complexes isolated from the rat brain

Interaction of isolated synaptic vesicles with synaptic junctional complexes isolated from the rat brain

Compartmentalization of clathrin in synaptic terminals

Immunofixation of sodium lauryl sulphate (SDS)-acrylamide gels has been used to study the distribution of the major protein (clathrin) of coated vesicles in various compartments of synaptic terminals. Synaptosomal subcellular fractions were isolated and purified from pig brain homogenates by the procedure of Ueda et al. and lysed in 6 mM Tris-Cl buffer at pH 6.6, 7.8, and 8.1. The synaptosomal particulate and soluble fractions were separated by centrifugation. The synaptic junctional complex (SJC) and postsynaptic density (PSD) fractions were obtained by detergent treatment of the synaptic plasma membrane (SPM). The synaptosomal subcellular fractions and purified coated vesicle (PCV) fractions were subjected to SDS gel electrophoresis (7.5%). The resulting slabs were divided vertically into 4 segments which were stained with Coomassie blue dye, or immunofixed with preimmune and anti-clathrin serum, or affinity labeled with concanavalin A (Con A)-peroxidase. The Comassie blue stained gel indicated the presence of 180,000-molecular weight band in gels of most synaptosomal subcellular fractions. However, immunofixation of an identical gel revealed positive staining of the 180,000-molecular weight protein in PCV, synaptosomal (SF), SPM and synaptoplasmic (SS) fractions only. These findings not only support the contention that a pool of cytosolic-coated vesicle protein is localized at synaptic terminals, they also indicate that clathrin appears highly unlikely to contribute to the structural frameworks of the SJC and PSD of mature synapses.