Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.     

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3

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Estimation of the number of synaptic vesicles in asymmetric synapses between hippocampal neurons

Within the framework of the quantum hypothesis of synaptic transmission, the amount of a neurotransmitter released in a unitary event of calcium-dependent exocytosis corresponds to the content of a synaptic vesicle (SV). The number of these organelles in the presynaptic terminal is an important index characterizing the functional state of the given synapse. The technique of estimation of the dimension of the total SV pool, which is based on mathematical modeling and is realized in a computer experiment, is described. This technique allows one to interpret quantitative estimations obtained in the course of the analysis of images of random ultrathin sections of presynaptic terminals in the terms of 3D space. The capabilities of this technique are illustrated using an example of estimation of the size of the total SV pool in asymmetric synapses between neurons of the radial layer of the murine hippocampal CA1 area. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 219–223, May–June, 2006.     

APOE genotype affects the pre‐synaptic compartment of glutamatergic nerve terminals

Apolipoprotein E (APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock‐in mice, we have previously shown that APOE‐ε4 Targeted Replacement (TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post‐synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate–glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE‐ε3 TR mice, APOE‐ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE‐ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre‐synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4–5 months and 1 year. Using high‐frequency ¹³C/¹H nuclear magnetic resonance spectroscopy, we found that APOE‐ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE‐ε4, and also act as surrogate markers for Alzheimer's disease risk.     

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

Types of degenerating geniculocortical axon terminals and their contribution to layer IV of area 17 in the squirrel monkey (Saimiri)

Summary Radiofrequency lesions were made in the lateral geniculate nuclei of six squirrel monkeys. The resulting degenerating terminals and their postsynaptic structures in layer IV of area 17 were quantitatively categorized on photomontages covering large areas of neuropil. Two to five days after the lesion, numerous axon terminals were affected by a variety of degenerative changes, i.e., enlargement and distortion of synaptic vesicles, neurofilamentous hyperplasia, electron-lucent and electron-dense reactions. Based on the aggregation of electron-dense material beneath the postsynaptic membrane, the degenerating terminals were considered to be of the asymmetric type. Among the degenerating boutons were the largest axon endings that occur in layer IV. Three days postoperatively, degenerating boutons contributed an average of 16.2% to the total synapse population; five days postoperatively, the average had increased to 19.3 %. The percentage of degenerating boutons on individual montages, however, amounted to as much as 29%. This amount probably reflects more closely the actual contribution of the geniculocortical fiber system to layer IV of striate cortex. The postsynaptic structure most frequently contacted by degenerating axon endings was the dendritic spine, followed by dendrites of small diameter. To account for the diversity of degenerative changes in the same fiber system, we offer the tentative suggestion that heterogeneously degenerating axon terminals arise from a heterogeneous population of neurons in the lateral geniculate nucleus, i.e., from magnocellular versus parvocellular laminae.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

AP‐1/σ1B‐adaptin mediates endosomal synaptic vesicle recycling,learning and memory

Synaptic vesicle recycling involves AP‐2/clathrin‐mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue‐specific AP‐1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP‐1–σ1A complex mediates protein sorting between the trans‐Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B‐deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B‐deficient mice have reduced motor coordination and severely impaired long‐term spatial memory. These data reveal a molecular mechanism for a severe human X‐chromosome‐linked mental retardation.     

On the distribution of axonal terminals containing spheroidal and flattened synaptic vesicles in the hippocampus and dentate gyrus of the rat and cat

Summary A quantitative analysis has been made of the distribution of presynaptic profiles containing round (or spheroidal) and flattened (or ellipsoidal) synaptic vesicles in the apical and basal dendritic zones and in the layer of pyramidal cell somata of fields CA₁ and CA₃ of the hippocampus, and in the molecular and granular layers of the dentate gyrus of the rat and cat.In the apical and basal dendritic zones of fields CA₁ and CA₃ the overwhelming majority of the synapses are of the asymmetrical variety, the axon terminals ending principally upon dendritic spines, and to a lesser extent upon the shafts and secondary or tertiary branches of the dendrites. Between 1 and 8% of the axon terminals in these zones contained flattened vesicles: all of these formed symmetrical contacts upon medium-sized or large dendritic shafts. In the molecular layer of the dentate gyrus a slightly higher percentage of flattened vesicle containing profiles was observed (10%); again these formed symmetrical contacts upon dendritic shafts. In the stratum pyramidale of the hippocampal fields and the stratum granulosum of the dentate gyrus of the rat, flattened vesicle containing synapses are two or three times more numerous than those with spheroidal vesicles. In the cat hippocampus the axosomatic synapses are about equally distributed between those containing round, and those with flattened vesicles.The finding that at the focus of post-synaptic inhibition, at the level of the pyramidal cell somata, the majority of the axon terminals contains flattened synaptic vesicles, whereas in the region of termination of the extrinsic, commissural and long association pathways (all of which are excitatory) virtually all the synapses contain round vesicles, strongly supports the view that endings containing flattened vesicles mediate post-synaptic inhibition in the hippocampal formation.Supported in part by Grant EY-00599 from the National Eye Institute.We should like to thank Mr. Paul Myers and Mr. Milburn W. Rhoades for their technical assistance, and Mrs. Doris Stevenson for secretarial help.     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals

There have been numerous studies on the activity and localization of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) in brain tissue. However, there is still a controversy as to the specific roles and relative importance of these enzymes in glutamate and glutamine metabolism in astrocytes and neurons or synaptic terminals. There are many reports documenting GDH activity in synaptic terminals, yet the misconception that it is a glial enzyme persists. Furthermore, there is evidence that this tightly regulated enzyme may have an increased role in synaptic metabolism in adverse conditions such as low glucose and hyperammonemia that could compromise synaptic function. In the present study, we report high activity of both AAT and GDH in mitochondrial subfractions from cortical synaptic terminals. The relative amount of GDH/AAT activity was higher in SM2 mitochondria, compared to SM1 mitochondria. Such a differential distribution of enzymes can contribute significantly to the compartmentation of metabolism. There is evidence that the metabolic capabilities of the SM1 and SM2 subfractions of synaptic mitochondria are compatible with the compartments A and B of neuronal metabolism proposed by Waagepetersen et al. (1998b. Dev. Neurosci. 20, 310-320).     

DCLK1 phosphorylates the microtubule‐associated protein MAP7D1 to promote axon elongation in cortical neurons

Doublecortin‐like kinase 1 (DCLK1) is a member of the neuronal microtubule‐associated doublecortin (DCX) family and functions in multiple stages of neural development including radial migration and axon growth of cortical neurons. DCLK1 is suggested to play the roles in part through its protein kinase activity, yet the kinase substrates of DCLK1 remain largely unknown. Here we have identified MAP7D1 (microtubule‐associated protein 7 domain containing 1) as a novel substrate of DCLK1 by using proteomic analysis. MAP7D1 is expressed in developing cortical neurons, and knockdown of MAP7D1 in layer 2/3 cortical neurons results in a significant impairment of callosal axon elongation, but not of radial migration, in corticogenesis. We have further defined the serine 315 (Ser 315) of MAP7D1 as a DCLK1‐induced phosphorylation site and shown that overexpression of a phosphomimetic MAP7D1 mutant in which Ser 315 is substituted with glutamic acid (MAP7D1 S315E), but not wild‐type MAP7D1, fully rescues the axon elongation defects in Dclk1 knockdown neurons. These data demonstrate that DCLK1 phosphorylates MAP7D1 on Ser 315 to facilitate axon elongation of cortical neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels

The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were subjected to nanoscale-liquid chromatography electrospray ionization-tandem mass spectrometry. Combining this method with 2-dimensional benzyldimethyl- n -hexadecylammonium chloride/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre-synapse. Four maxi-channels, three isoforms of voltage-dependent anion channels and the tweety homolog 1 were co-isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis.     

Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles

Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1–3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.