Synaptic Efficacy as a Function of Ionotropic Receptor Distribution: A Computational Study

Glutamatergic synapses are the most prevalent functional elements of information processing in the brain. Changes in pre-synaptic activity and in the function of various post-synaptic elements contribute to generate a large variety of synaptic responses. Previous studies have explored postsynaptic factors responsible for regulating synaptic strength variations, but have given far less importance to synaptic geometry, and more specifically to the subcellular distribution of ionotropic receptors. We analyzed the functional effects resulting from changing the subsynaptic localization of ionotropic receptors by using a hippocampal synaptic computational framework. The present study was performed using the EONS (Elementary Objects of the Nervous System) synaptic modeling platform, which was specifically developed to explore the roles of subsynaptic elements as well as their interactions, and that of synaptic geometry. More specifically, we determined the effects of changing the localization of ionotropic receptors relative to the presynaptic glutamate release site, on synaptic efficacy and its variations following single pulse and paired-pulse stimulation protocols. The results indicate that changes in synaptic geometry do have consequences on synaptic efficacy and its dynamics.     

Relation between subsynaptic receptor blockade and response to quantal transmitter at the mouse neuromuscular junction

When a quantum of transmitter is released into a synaptic cleft, the magnitude of the subsynaptic response depends upon how much transmitter becomes bound to receptors. Theoretical considerations lead to the conclusion that if receptor density is normally high enough that most of the quantal transmitter is captured, subsynaptic quantal responses may be insensitive to receptor blockade. The effectiveness of receptor blockers in depressing the subsynaptic response should be diminished by interference with processes that normally dispose of transmitter, but increased if receptor density is reduced. In conformity with equations derived from a simple mathematical model, the apparent potency of (+)- tubocurarine (dTC) to depress the peak height of miniature end-plate currents (MEPCs) in mouse diaphragm was substantially reduced by poisoning of acetylcholinesterase (AChE) and increased by partial blockade of receptors by immunoglobulin G from patients with myasthenia gravis or alpha-bungarotoxin. We calculated from the data that normally capture of quantal acetylcholine (ACh) by receptors is approximately 75% of what it would be if there were no loss of ACh by hydrolysis or diffusion of ACh form the synaptic cleft. This fraction is increased to approximately 90% by poisoning of AChE. Conversely, it normally requires blockade of approximately 80% of receptors-and after AChE poisoning, approximately 90% of receptors-to reduce ACh capture (and MEPC height) by 50%. The apparent potency of dTC to alter MEPC time- course (after AChE poisoning) and to depress responses to superperfused carbachol was much greater than its apparent potency to depress MEPC height, but corresponded closely with the potency of dTC to block receptors as calculated from the action of dTC on MEPC height. These results indicate that the amplitude of the response to nerve-applied acetylcholine does not give a direct measure of receptor blockade; it is, in general, to be expected that an alteration of subsynaptic receptor density may not be equally manifest in responses to exogenous and endogenous neurotransmitter.     

Ultrastructure of the cerebral cortex in the rat after the effect of electromagnetic impulse

In mature rats an area on the head has been subjected to a single radiation for 1.5 sec with microwaves in the continuous regimen of generation, frequency 2.4 GHz level of the specific absorbed power 5 W/g, that is accompanied with appearance of convulsions. Under anesthesia specimens of the superficial layer of the cerebral superlateral part are taken and subjected to electron microscopical investigation. Immediately after radiation and in 2 h certain disorders in microcirculation and reactive changes of mitochondria in perikaryons, axons, dendrites, synapses of the neurons and in gliocytes are revealed. The mitochondrial changes are designated as "edematous". In 2 and 6 h in karyoplasm of some neurons membranous structures appear; they are interpreted as a result of heat denaturation of the nuclear proteins. In synapses, together with lesions of mitochondria, synaptic complexes undergo destruction and osmiophilic substance is accumulated in the subsynaptic zone along the whole length of the contact. In one day, essential destructive changes are revealed as severe lesions of some neurons, vacuolization and destruction of mitochondria, localized in all the structures. Pathogenesis of the neurological disturbances is based on disturbances of interneuronal interactions, connected with an immediate heat effect of the electromagnetic radiation on the structures responsible for the synaptic transmission and with a rapidly developing tissue hypoxia as a consequence of microcirculatory disturbance and a sharp inhibition of energetic metabolism.     

Localization of the membrane-anchored MMP-regulator RECK at the neuromuscular junctions

Nerve apposition on nicotinic acetylcholine receptor clusters and invagination of the post-synaptic membrane (i.e. secondary fold formation) occur by embryonic day 18.5 at the neuromuscular junctions (NMJs) in mouse skeletal muscles. Finding the molecules expressed at the NMJ at this stage of development may help elucidating how the strong linkage between a nerve terminal and a muscle fiber is established. Immunohistochemical analyses indicated that the membrane-anchored matrix metalloproteinase regulator RECK was enriched at the NMJ in adult skeletal muscles. Confocal and electron microscopy revealed the localization of RECK immunoreactivity in secondary folds and subsynaptic intracellular compartments in muscles. Time course studies indicated that RECK immunoreactivity becomes associated with the NMJ in the diaphragm at around embryonic day 18.5 and thereafter. These findings, together with known properties of RECK, support the hypothesis that RECK participates in NMJ formation and/or maintenance, possibly by protecting extracellular components, such as synaptic basal laminae, from proteolytic degradation.     

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.     

An ultrastructural study of the development of afferent and efferent synapses on outer hair cells of the guinea pig organ of Corti

The guinea pig organ of Corti was studied using transmission electron microscopy, the second turn of the cochlea being examined at various ages between 20 days before birth and 30 days postnatal. Outer hair cells were examined at each of these ages. At all ages studied, the efferent (presynaptic) terminals are large and are packed with synaptic vesicles, whereas the afferent (postsynaptic) terminals are generally smaller, with a relatively small number of vesicles. During development, the subsynaptic cistern changes from a fragmented, diffuse profile extending over 50-70% of the length of the efferent contact zones, to a continuous, compact structure spanning neighbouring synapses. Synaptic vesicles in the efferent terminals are predominantly rounded in early development, flattened vesicles appearing postnatally. The synaptic bodies at afferent synapses do not change noticeably during development. Quantitative analysis revealed that the area of efferent terminals and the length of their active zone increase with increasing age, the same parameters decreasing in afferent terminals. Synaptic vesicles in the efferent terminals decrease in diameter, but remain constant in afferent terminals, with increasing age. The number of hair cell membrane invaginations decreases as development proceeds.     

The effect of light stimulation in the pre- and postnatal periods on the synaptic ultrastructure in the chick hyperstriatum

Morphometric analysis of synapses in the medial parts of the ventral and accessorium hyperstriatum in the right and left hemispheres in chicks has been performed after rhythmic optic stimulation from the 18th day of incubation, flickering stimulation at the 11-12th hour after hatching, as well as in chicks reared under normal illumination and those kept in darkness within the first two days after hatching. Numeric density of synapses, mean dimensions of postsynaptic thickenings, the number of synaptic vesicles per active zone in presynapses and mean length of the whole axodendritic contact were determined in ultrathin sections. The data obtained indicate that early visual experience significantly affects the synaptic structures in both parts of the brain. A discussion is made of the plasticity of synapses during stimulation, morphological and functional relationships between hyperstriatal areas in relation to different aspects of processing and storage of visual information.     

Amyloid precursor proteins are constituents of the presynaptic active zone

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface‐localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock‐out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.

     

Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe

Immunocytochemical localization of actin in rat cerebral cortex embedded in the resin LR White was performed using 5 nm colloidal gold as a probe. Antigenicity is maintained throughout the embedding procedure and the low electron opacity of LR White permits fine filamentous structures to be visualized. Control experiments included incubating the sections with normal goat serum or mouse IgG instead of the primary antibody, preadsorbing the antibody with actin from bovine muscle or liver acetone powder, and heat treating the primary antibody. Immunoreactive actin was identified primarily in dendritic spines, particularly in the postsynaptic density (PSD), the subsynaptic web, and the spine apparatus and endothelial and smooth muscle cells of blood vessels. Within dendritic spines, actin which is labeled in the PSD is in continuity with the filaments of the subsynaptic web. These filaments, in turn, are in continuity with the spine apparatus and/or the spine membranes adjacent to the PSD. The PSD may therefore function like other submembranous filamentous arrays which communicate events occurring at the membrane, in this case, the postsynaptic membrane, to the underlying cytoskeletal network, i.e., the subsynaptic web of the spine. It is also suggested that the actin present in the spine may play a role in changes in spine shape and synaptic curvature. Some actin was also seen in the presynaptic process in association with synaptic vesicles, the filamentous network that is contiguous with the synaptic vesicle membrane, and the presynaptic dense projections. Actin may be involved in dynamic processes in the presynaptic ending which include vesicle translocation.     

A note on the structure of synapses in the ventral nerve cord of the onychophoran peripatoides leuckarti

Summary The fine structure of synapses, their distribution and arrangement in the ventral nerve cord of Peripatoides leuckarti (phylum Onychophora) is described. The asymmetric synaptic junctions show a well developed synaptic cleft (300Å) and pre- and subsynaptic electron dense apposition. They frequently show an array of presynaptic projections and a subsynaptic cisterna of endoplasmic reticulum. The onychophoran synapses differ from those of annelids and insects.Supported by the Deutsche Forschungsgemeinschaft, grant Schu 374/1     

Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.     

The dependence of calcium-activated potassium currents on membrane potential.

Previous experiments on cholinergic synapses in chick cochlear hair cells have shown that calcium entering through acetylcholine-activated synaptic channels in turn activates calcium-dependent potassium currents, resulting in synaptic inhibition. In voltage-clamp experiments such currents would be expected to increase with depolarization (as the driving force for potassium entry is increased) and then decrease towards zero as the membrane approaches the calcium equilibrium potential (when calcium entry is suppressed). In the hair cells, however, such currents approached zero at about +20 mV, more than 170 mV negative to the calcium equilibrium potential. Another feature of the synapse is its post-junctional morphology: a uniform 20 nm cleft is formed between the postsynaptic membrane and the outermost membrane of an underlying cisterna. Here we present a model in which synaptic activation results in calcium influx into the subsynaptic cleft and thence into the bulk of the cytoplasm. The model suggests that the voltage dependence of the calcium-activated potassium current can be accounted for by only two basic assumptions: (i) entry of calcium through the activated synaptic channels by simple diffusion; and (ii) activation of the potassium channels by the cooperative action of four calcium ions. In addition, the model suggests that during activation the calcium concentration in the restricted subsynaptic space can reach levels adequate to activate the potassium channels, without requiring additional, more complicated, considerations (for example, secondary calcium release from the cisterna).     

Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.     

Synaptic junctional glycoconjugates from chick brain

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins.     

Seasonal encoding by the circadian pacemaker of the SCN

The circadian pacemaker of the suprachiasmatic nucleus (SCN) functions as a seasonal clock through its ability to encode day length [1-6]. To investigate the mechanism by which SCN neurons code for day length, we housed mice under long (LD 16:8) and short (LD 8:16) photoperiods. Electrophysiological recordings of multiunit activity (MUA) in the SCN of freely moving mice revealed broad activity profiles in long days and compressed activity profiles in short days. The patterns remained consistent after release of the mice in constant darkness. Recordings of MUA in acutely prepared hypothalamic slices showed similar differences between the SCN electrical activity patterns in vitro in long and short days. In vitro recordings of neuronal subpopulations revealed that the width of the MUA activity profiles was determined by the distribution of phases of contributing units within the SCN. The subpopulation patterns displayed a significantly broader distribution in long days than in short days. Long-term recordings of single-unit activity revealed short durations of elevated activity in both short and long days (3.48 and 3.85 hr, respectively). The data indicate that coding for day length involves plasticity within SCN neuronal networks in which the phase distribution of oscillating neurons carries information on the photoperiod's duration.     

Acetylcholinesterase activity and type C synapses in the hypoglossal,facial and spinal-cord motor nuclei of rats

Summary Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptie cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple active zones. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.In honour of Prof. P. van Duijn     

Morphometry of the vesicular apparatus of the neuromuscular junction with different modes of transmitter release

Neuromuscular junctions in the diaphragm muscle of rats were studied, using a usual chemical fixation technique and quick prefreezing of the muscle. The number of synaptic vesicles in the vicinity of presynaptic membrane decreased significantly during synaptic function activation. Quantitative investigation of axon terminal vesicle apparatus revealed heterogeneity in the size of synaptic vesicle pool. The pattern of synaptic vesicle size distribution in different functional states of the synapse suggests that size heterogeneity reflects their functional peculiarities.     

Body water and electrolyte responses to acetazolamide in humans

Acetazolamide (ACZ), a potent carbonic anhydrase inhibitor, is a known diuretic and causal agent in metabolic acidosis. Its diuretic qualities are well established with respect to urine flow and electrolyte excretion. However, the impact of ACZ on body hydration status has not been adequately quantified. Thus, to establish the influence of ACZ treatment on body water, nine healthy males were evaluated for hydration status after clinically prescribed doses of ACZ. The drug was administered in three 250-mg oral doses 14, 8, and 2 h before determination of body water compartments. ACZ led to a significant 1.7-liter reduction in total body water (3.4%). A significant reduction in extracellular water of 3.3 liters is partitioned as the loss of total body water and a significant increase in intracellular water (1.6 liters). Venous blood pH and plasma HCO3- were significantly reduced 0.09 units and 5.9 mM, respectively, with ACZ. Plasma protein concentration was increased, but plasma osmolality did not change. Plasma Na+, K+, and Cl- concentrations were not different with ACZ, but total electrolyte content was significantly decreased 45.2, 1.17, and 44.1 meq, respectively, for all three. Urine K+, HCO3-, flow, and pH were elevated after ACZ treatment, whereas Na+ and Cl- were the same as placebo levels. In conclusion, acute clinical doses of ACZ reduce body fluid compartments, leading to a moderate isosmotic hypovolemia with an intracellular volume expansion as well as metabolic acidosis.