γ-Aminobutyric acid receptors in brain postsynaptic densities

Rat brain synaptic plasma membranes contain two receptorlike binding sites for the inhibitory transmitter γ-aminobutyric acid. Postsynaptic junctional structures (postsynaptic densities) isolated from these membranes contain only the higher affinity site enriched more than sixfold compared to the membranes. The results provide the first direct evidence for the association of transmitter receptors with postsynaptic junctional sites in the brain.     

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-1 (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 dlg 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.     

The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones

Molecular mechanisms linking pre- and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, alpha N- and beta-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and catenin-based adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/ catenin complex may play a critical role in the formation or maintenance of synaptic junctions.     

Morphology and molecular composition of isolated postsynaptic junctional structures.

The solubilization of isolated brain synaptosomal plasma membranes by various detergents was studied and in each case found to depend upon detergent concentration. By using conditions sufficient to extract maximally protein and phospholipid from the membranes, postsynaptic junctional particles were isolated with each of four detergents and their ultrastructural appearances and protein contents compared. Two basic structural forms were identified. One, isolated with Triton X-100, consists of a planar array of dense-staining particles ca. 20 nm in diameter. It resembles the postsynaptic density seen in undigested synaptosomal plasma membranes. The other, isolated with sodium deoxycholate, contains less protein. It has the same overall shape and dimensions as the postsynaptic density, but consists of a branching network of short 5 nm fibres (the postsynaptic junctional lattice) making up an array of contiguous polygons, each ca. 20 nm across. The interior of these polygonal elements seems to be hydrophobic since it cannot be penetrated by metallic salts used for negative staining. It is suggested that the dense-staining 20 nm subunits observed at the postsynaptic junctional site may be composed of hydrophobic proteins inserted into the hollow cores of the lattice polygons. Electrophoretic analysis of the proteins present in the various postsynaptic junctional preparations identified two major common components with molecular masses of 275000 and 47500. The latter is tentatively identified as actin. Components comigrating respectively with alpha- and beta-tubulin are present, and the relation of the lattice structure to subjacent microtubules is discussed.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Pre- Versus Postsynaptic Localization of Benzodiazepine and β-Carboline Binding Sites

[3H]Flunitrazepam (FNP) and [3H]methyl beta-carboline-3-carboxylate (MCC) binding was examined in soluble and particulate fractions from membranes solubilized with Triton X-100 or in subfractions of synaptosomal membranes obtained by a physical separation technique. Results using both methods demonstrate that benzodiazepine and beta-carboline sites reside on both pre- and postsynaptic membranes. Further, subfractionation experiments indicate that the binding sites for both ligands are unequally distributed within the synapse and among brain regions. For example, in cerebral cortical presynaptic membranes there are twice as many FNP as MCC sites whereas in postsynaptic membranes this ratio is reversed. The number of FNP and MCC sites are equal in the presynaptic fraction from cerebellum. The postsynaptic membranes derived from cerebellum have three times the number of FNP compared to MCC sites. In hippocampus this ratio varies between 1.5 and 2.8 in each subfraction. These results support the idea that benzodiazepine and beta-carboline binding sites represent different recognition sites.     

Inhibitory synaptic currents in voltage-clamped locust muscle fibres desensitized to their excitatory transmitter

Inhibitory junctional currents (i.j.c.s) have been examined in locust muscle fibres to give properties of GABA-channels activated by the neurally released transmitter. A nerve-muscle preparation is described which has proved suitable for voltage-clamp analysis of inhibitory transmission. I.j.c.s were recorded from fibres in which excitatory synapses had been desensitized with glutamate, to abolish excitatory junctional currents. This procedure had no apparent effect on inhibitory channel properties. The time constant of decay of the i.j.c. was 7.7 +/- 0.3 ms, slightly exceeding the time constant of the membrane noise induced by externally applied GABA. Peak i.j.c. conductance decreased with hyperpolarization. I.j.c.s showed measurable fluctuations permitting an estimate of the mean size of the quantal events composing the i.j.c. Their mean size coincided with the spontaneously occurring miniature inhibitory junctional currents that could be directly recorded in some fibres. The inhibitory nerve-impulse released an average of 35 transmitter packets at sites distributed along the muscle fibre length. Since each m.i.j.c. produced a current of about 0.6 nA (at Vm = -80 mV, ECl = -40 mV) the single quantum of inhibitory transmitter opens 600-1000 postsynaptic chloride channels. This is roughly three to four times the number of channels opened by the excitatory transmitter packet at glutamate synapses in the same fibres.     

Glutamate and γ-Aminobutyric Acid Neurotransmitter Systems in the Acute Phase of Maple Syrup Urine Disease and Citrullinemia Encephalopathies in Newborn Calves

Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na(+)-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to less than 50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fine structure of a rectifying electrotonic synapse

The synapses between the lateral giant axon and the giant motor axon found in the abdominal ganglia of the ventral nerve cord of the crayfish Procambarus clarkii are electronic. The junctional membrane rectifies, favoring impulse transmission from lateral giant fiber to giant motor fiber. This rectifying electronic junction consists of closely apposed membranes indistinguishable from ordinary arthropod gap junctions. The apposed membranes contain intramembrane particles that are approximately 12.5 nm in width. These particles have a central depression and are arranged in a loosely ordered array with a center-to- center spacing of about 20 nm. The only obvious morphological evidence of asymmetry is the presence of vesicles (about 80 nm in diameter) in the cytoplasm adjacent to the junctional region of the presynaptic lateral giant fiber. Vesicles are not present in the adjacent cytoplasm of the postsynaptic giant motor fiber; however, mitochondria and smooth tubular endoplasmic reticulum are more frequent in the cytoplasm of the giant motor fiber.     

Brain postsynaptic densities: the relationship to glial and neuronal filaments

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214- 225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.     

Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes.

A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3',5'-cyclic nucleotide phosphodiesterase, and (Na+ + K+)-ATPase. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A2 attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A2 was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3',5'-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500-4000A), which do not form vesicles.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

Presynaptic uptake blockade hypothesis for LSD action at the lateral inhibitory synapse in Limulus

We investigated the action of LSD at the putative indoleaminergic lateral inhibitory synapse in the lateral eye of Limulus polyphemus. We recorded extracellular and intracellular voltage responses from eccentric cells while producing inhibition either by light or by antidromic stimulation of the optic nerve in the presence of LSD, serotonin (5-HT), chlorimipramine, or a bathing medium whose high Mg++ and low Ca++ concentrations partially or completely blocked synaptic transmission. We found (a) light-evoked and antidromically stimulated lateral inhibition is enhanced during superfusion of low (1-5 microM) concentrations of LSD and suppressed by higher (5-20 microM) concentrations; (b) these actions of LSD are markedly reduced by bathing the retina in a medium high in Mg++ and low in Ca++; (c) very low concentrations of chlorimipramine, a putative uptake blocker of serotonin, appear to mimic actions of LSD both on eccentric cell firing rate and on lateral inhibition; (d) superfused 5-HT depresses lateral inhibition at all superthreshold concentrations (0.1-25 microM). These results suggest that LSD's action may require an intact inhibitory transmitter release and postsynaptic response mechanism, whereas serotonin exerts a direct postsynaptic effect. We propose that LSD blocks presynaptic uptake of transmitter at the lateral inhibitory synapse. The concentration dependence of LSD's action can be accounted for as follows: low concentrations partially restrict transmitter reuptake, thereby prolonging the lifetime of the transmitter in the synaptic cleft and thus increasing the magnitude and duration of postsynaptic inhibition. Higher concentrations cause more presynaptic uptake sites to be blocked; this causes accumulation of transmitter in the synaptic cleft, which causes a functional blockade of the synapse because of postsynaptic desensitization. As an alternative, we propose a hypothesis based on LSD action at presynaptic autoreceptors. Similar hypotheses can account for many aspects of LSD's action in mammalian brain.     

The oscillatory responses of skate electroreceptors to small voltage stimuli

Tonic nerve activity in skate electroreceptors is thought to result from spontaneous activity of the lumenal membranes of the receptor cells which is modulated by applied stimuli. When physiological conditions are simulated in vitro, the receptor epithelium produces a current which flows inward across the lumenal surface. This epithelial current exhibits small spontaneous sinusoidal fluctuations about the mean that are associated with corresponding but delayed fluctuations in postsynaptic response. Small voltage stimuli produce damped oscillations in the epithelial current similar in time-course to the spontaneous fluctuations. For lumen-negative, excitatory stimuli, these responses are predominantly an increase over the mean inward current. For inhibitory stimuli they are predominantly a decrease. Increased inward current across the lumenal membranes of the receptor cells increases depolarization of the presynaptic membranes in the basal faces leading to increased release of transmitter and an excitatory postsynaptic response. Decreased inward current decreases depolarization of the presynaptic membranes leading to a reduction in transmitter release and an inhibitory postsynaptic response. Clear changes in postsynaptic response are detectable during stimuli as small as 5 microV with saturation occurring at +/- 400 microV. The evoked oscillations in epithelial current are damped and the postsynaptic responses decline during maintained stimuli with large off-responses occurring at stimulus termination. The initial peak of the off-response is similar to the response produced by onset of an oppositely directed stimulus. These observations substantiate the role of receptor cell excitability in the detection of small voltage changes.     

Interaction of brain somatostatin receptors with the PDZ domains of PSD-95

Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95kDa (PSD-95) and PSD-93. The existence of the SSTR4/PSD-95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD-95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD-95, the association may serve to localize the receptor to postsynaptic sites.     

Neuromodulator octopamine attenuates extrajunctional glutamate sensitivity in insect muscle

Octopamine was found to decrease extrajunctional, but not junctional glutamate responses, in mealworm neuromuscular preparations. This action of octopamine was mimicked by forskolin, 8-(4-chlorophenylthio)-adenosine 3′:5′-cyclic monophosphate (CPT-cyclic AMP), and 8-bromoguanosine 3′:5′-cyclic monophosphate (8-bromo-cyclic GMP), but not by 1,2-oleoylacetylglycerol (OAG), a protein kinase C activator. We suggest that the octopamine-induced reduction in the glutamate sensitivity of extrajunctional membranes may enable the muscle to more closely follow its neuronal input by preventing a depolarization (and hence a conductance increase) due to the discharge of unsequestered transmitter molecules at nonsynaptic sites.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

Barbiturate competition for TRH receptors in mouse brain: neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.     

Transmitter concentration profiles in the synaptic cleft: an analytical model of release and diffusion.

A three-dimensional model for release and diffusion of glutamate in the synaptic cleft was developed and solved analytically. The model consists of a source function describing transmitter release from the vesicle and a diffusion function describing the spread of transmitter in the cleft. Concentration profiles of transmitter at the postsynaptic side were calculated for different transmitter concentrations in a vesicle, release scenarios, and diffusion coefficients. From the concentration profiles the receptor occupancy could be determined using alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor kinetics. It turned out that saturation of receptors and sufficiently fast currents could only be obtained if the diffusion coefficient was one order of magnitude lower than generally assumed, and if the postsynaptic receptors formed clusters with a diameter of roughly 100 nm directly opposite the release sites. Under these circumstances the gradient of the transmitter concentration at the postsynaptic membrane outside the receptor clusters was steep, with minimal cross-talk among neighboring receptor clusters. These findings suggest that for each release site a corresponding receptor aggregate exists, subdividing an individual synapse into independent functional subunits without the need for specific lateral diffusion barriers.     

Synapse proteomics: current status and quantitative applications

Chemical synapses are key organelles for neurotransmission. The coordinated actions of protein networks in diverse synaptic subdomains drive the sequential molecular events of transmitter release from the presynaptic bouton, activation of transmitter receptors located in the postsynaptic density and the changes of postsynaptic potential. Plastic change of synaptic efficacy is thought to be caused by the alteration of protein constituents and their interaction in the synapse. As a first step toward the understanding of the organization of synapse, several proteomics studies have been carried out to profile the protein constituents and the post-translational modifications in various rodent excitatory chemical synaptic subdomains, including postsynaptic density, synaptic vesicle and the synaptic phosphoproteome. Quantitative proteomics have been applied to examine the changes of synaptic proteins during brain development, in knockout mice model developed for studies of synapse physiology and in rodent models of brain disorders. These analyses generate testable hypotheses of synapse function and regulation both in health and disease.