Dendritic Localization and Exocytosis of NAAG in the Rat Hippocampus.

While a lot is known about classical, anterograde neurotransmission, less is known about the mechanisms and molecules involved in retrograde neurotransmission. Our hypothesis is that N-acetylaspartylglutamate (NAAG), the most abundant dipeptide in the brain, may act as a retrograde transmitter in the brain. NAAG was predominantly localized in dendritic compartments of glutamatergic synapses in the intact hippocampus, where it was present in close proximity to synaptic-like vesicles. In acute hippocampal slices, NAAG was depleted from postsynaptic dendritic elements during neuronal stimulation induced by depolarizing concentrations of potassium or by exposure to glutamate receptor (GluR) agonists. The depletion was completely blocked by botulinum toxin B and strictly dependent on extracellular calcium, indicating exocytotic release. In contrast, there were low levels of NAAG and no effect by depolarization or GluR agonists in presynaptic glutamatergic terminals or GABAergic pre- and postsynaptic elements. Together these data suggest a possible role for NAAG as a retrograde signaling molecule at glutamatergic synapses via exocytotic release.

Vesicular glutamate transporter-3 in the rodent brain: vesicular colocalization with vesicular γ-aminobutyric acid transporter.

Vesicular glutamate transporters (VGLUT1-3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ-aminobutyric acid (GABA)-ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT-positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light-microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals.

Pre- and postsynaptic localization of NMDA receptor subunits at hippocampal mossy fibre synapses.

The N-methyl-D-aspartate (NMDA) type of glutamate receptors is involved in synaptic plasticity in hippocampal mossy fibre-CA3 pyramidal neuron synapses. The ultrastructural localization of NMDA receptor subunits at this synapse type is not known. By postembedding electron microscopic immunogold cytochemistry we show that the NMDA receptor subunits GluN1, GluN2A, GluN2B, GluN2C and GluN2D are located in postsynaptic membranes of mossy fibre as well as CA3 recurrent associational commissural synapses. In the mossy fibres the GluN1, GluN2B and GluN2D labelling patterns suggested that these subunits were located also presynaptically in nerve terminal membranes and in mossy fibre axons. GluN3B was predominantly present in mossy fibre synapses as compared to recurrent associational commissural synapses, showing a presynaptic labelling pattern. In conclusion, while the postsynaptic localization of GluN1, GluN2A, GluN2B, and GluN2D is in good agreement with the recent finding of NMDA receptor-dependent long term potentiation (LTP) at CA3 mossy fibre synapses, we propose that presynaptic GluN1, GluN2B, GluN2D and GluN3B subunits could be involved in plastic phenomena such as certain types of LTP and recurrent mossy fibre growth.

Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Protein aggregation in Parkinson's disease.

Parkinson's disease (PD) is a primary neurodegenerative movement disorder. In most cases it occurs as a sporadic type of disease, but there are also rare familial forms. Pathologically Parkinson's disease is characterized by loss of dopaminergic neurons in the compact part of substantia nigra. As a part of the neurodegenerative process protein aggregates will accumulate as Lewy bodies in dopaminergic neurons (1). In addition, non-dopaminergic neurons are known to be affected in Parkinsons's disease, for example, in several brain stem nuclei and the olfactoric bulb (2-4). The pathogenic process underlying the death of dopaminergic neurons is far from fully understood. Along with mitochondrial dysfunction, excitotoxicity, neuroinflammation and oxidative stress (5-8), recent evidence indicates that accumulation of protein filaments in Lewy bodies actively takes part in the degeneration of neurons. This will be further discussed below.

Complementary expression of SN1 and SAT2 in the islets of Langerhans suggests concerted action of glutamine transport in the regulation of insulin secretion.

Insulin and glucagon secretion from the islets of Langerhans is highly regulated. Although an increased plasma glucose level is the major stimulus for insulin exocytosis, roles for glutamine and glutamate have been suggested. Interestingly, the islet cells display elements associated with synaptic transmission. In the central nervous system (CNS), glutamine transport by SN1 and SAT2 sustain the generation of neurotransmitter glutamate. We hypothesized that the same transporters are essential for glutamine transport into the islet cells and for subsequent formation of glutamate acting as an intracellular signaling molecule. We demonstrate that islet cells express several transporters which can mediate glutamine transport. In particular, we show pronounced expression of SN1 and SAT2 in B-cells and A-cells, respectively. The cell-specific expression of these transporters together with their functional characteristics suggest an important role for glutamine in the regulation of insulin secretion.

Morphological evidence for vesicular glutamate release from astrocytes.

There is now growing evidence that astrocytes, like neurons, can release transmitters. One transmitter that in a vast number of studies has been shown to be released from astrocytes is glutamate. Although asytrocytic glutamate may be released by several mechanisms, the evidence in favor of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Here we review the morphological data supporting that astrocytes possess the machinery for exocytosis of glutamate. We describe the presence of small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters in astrocytes, as well as NMDA receptors situated in vicinity of the astrocytic vesicles.

A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2).

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

Co-localization of excitatory and inhibitory transmitters in the brain.

OBJECTIVES: To review the understanding about co-localisation of amino acid transmitters in the brain. RESULTS: The idea that neurons release the same transmitter at all their synapses is associated with Henry Dale and formulated as Dale's principle by John Eccles. This idea has been modified during the last years based on several studies showing that transmitters can co-exist at the same synapse. First, a large body of evidence was presented showing that a classical transmitter can be co-localized with different types of neuropeptides. Then, several studies showed that a synapse could co-release two classical transmitters. CONCLUSION: This review presents and discusses data from our laboratory showing co-release of glutamate/gamma-aminobutyric acid (GABA), aspartate/glutamate and aspartate/GABA from different types of hippocampal synapses.

Comparison of major and trace element concentrations in Danish greenhouse tomatoes (Lycopersicon esculentum Cv. Aromata F1) cultivated in different substrates.

The concentration of major and trace elements was determined for tomato (Lycopersicon esculentumcv. Aromata F1) fruits grown in three different substrate systems. The systems were soil and rockwool irrigated with a normal nutrient solution and rockwool irrigated with a nutrient solution with elevated electrical conductivity (EC). At three harvest times, tomato fruits were analyzed for Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Sr, and Zn by ICP-AES and for Cd, Cr, Mo, Ni, Pb, Sn, and V by HR-ICPMS. The concentrations of Ca, Cd, Fe, Mn, Mo, Na, Ni, Sr, and Zn were significantly different (p < 0.05) for tomato fruits grown on the different substrates. Between the harvest times different levels (p < 0.05) were shown for Ca, Cd, Fe, Mn Na, Ni, Sr, Zn Cu, K, Mg, P, Sn, and V. The concentration of Cd was >15 times higher and the concentration of Ca was 50-115% higher in soil-grown fruits than in rockwool-grown fruits. Principal component analysis applied on each harvest split the data into two groups. One group includes soil-grown fruits, and the other group includes rockwool-grown fruits with the two different nutrient solutions.

Redistribution of neuroactive amino acids in hippocampus and striatum during hypoglycemia: a quantitative immunogold study.

Postembedding immunocytochemistry was used to localize aspartate, glutamate, gamma-aminobutyric acid (GABA), and glutamine in hippocampus and striatum during normo- and hypoglycemia in rat. In both brain regions, hypoglycemia caused aspartatelike immunoreactivity to increase. In hippocampus, this increase was evident particularly in the terminals of known excitatory afferents-in GABA-ergic neurons and myelinated axons. Aspartate was enriched along with glutamate in nerve terminals forming asymmetric synapses on spines and with GABA in terminals forming symmetric synapses on granule and pyramidal cell bodies. In both types of terminal, aspartate was associated with clusters of synaptic vesicles. Glutamate and glutamine immunolabeling were markedly reduced in all tissue elements in both brain regions, but less in the terminals than in the dendrosomatic compartments of excitatory neurons. In glial cells, glutamine labeling showed only slight attenuation. The level of GABA immunolabeling did not change significantly during hypoglycemia. The results support the view that glutamate and glutamine are used as energy substrates in hypoglycemia. Under these conditions both excitatory and inhibitory terminals are enriched with aspartate, which may be released from these nerve endings and thus contribute to the pattern of neuronal death characteristic of hypoglycemia.

Comparative investigation of concentrations of major and trace elements in organic and conventional Danish agricultural crops. 1. Onions (Allium cepa Hysam) and peas (Pisum sativum ping pong).

210 samples of onions (Allium cepa Hysam) from 11 conventionally and 10 organically cultivated sites and 190 samples of peas (Pisum sativum Ping Pong) from 10 conventionally and 9 organically cultivated sites in Denmark were collected and analyzed for 63 and 55 major and trace elements, respectively, by high-resolution inductively coupled plasma mass spectrometry. Sampling, sample preparation, and analysis of the samples were performed under carefully controlled contamination-free conditions. Comparative statistical tests of the element concentration mean values for each site show significantly (p < 0.05) different levels of Ca, Mg, B, Bi, Dy, Eu, Gd, Lu, Rb, Sb, Se, Sr, Ti, U, and Y between the organically and conventionally grown onions and significantly (p < 0.05) different levels of P, Gd, and Ti between the organically and conventionally grown peas. Principal component analysis (PCA) applied to the 63 elements measured in the individual onion samples from the 21 sites split up the sites into two groups according to the cultivation method when the scores of the first and third principal components were plotted against each other. Correspondingly, for peas, a PCA applied to the 55 elements measured as mean values for each site split up the 19 sites into two groups according to the cultivation method when the scores of the third and fourth principal component were plotted against each other. The methodology may be used as authenticity control for organic cultivation after further method development.

Concentrations of 50 major and trace elements in Danish agricultural crops measured by inductively coupled plasma mass spectrometry. 3. Potato (Solanum tuberosum folva).

The multi-element (Ag, Al, Au, Ba, Bi, Ca, Cd, Co, Cr, Cs, Cu, Dy, Er, Fe, Ga, Gd, Ho, In, Ir, La, Lu, Mn, Mo, Nb, Nd, P, Pb, Pd, Pr, Pt, Rb, Re, Rh, Ru, Sb, Sc, Sm, Sn, Sr, Ta, Tb, Th, Ti, Tl, Tm, U, V, Y, Yb, and Zn) concentrations (microg/kg, fresh weight) in potatoes (Solanum tuberosum, Folva) were investigated in this study. The potatoes were grown in two fertilization practices; one with pig slurry and one with calcium ammonium nitrate at three levels of N fertilization (0, 60, and 120 kg of N/ha). The experiment field was located at the Riso National Laboratory Agronomy Farms in Roskilde, Denmark. High-resolution-inductively coupled plasma mass spectrometry (HR-ICPMS) was used for analyses of the samples. The effect of three levels of N fertilization on elemental concentrations of the crop are evaluated by use of discriminant partial least-squares regression (PLS). The results provide useful biological and nutritional information on potatoes.

Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus.

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.

A high affinity glutamate/aspartate transport system in pancreatic islets of Langerhans modulates glucose-stimulated insulin secretion.

To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3H]glutamate uptake activity was Na(+)-dependent, and it was blocked by L-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a Vmax of 8.48 +/- 1.47 fmol/min/islet (n = 4), which corresponds to 102.2 +/- 17.7 pmol/min/mg islet protein. The apparent Km of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mM), the apparent Km was 34.7 +/- 7.8 microM (n = 3). However, in high glucose (16.7 mM) the apparent Km increased to 112.7 +/- 16.5 microM (n = 3) with little or no change in Vmax. Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported D-aspartate. Anti-D-aspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-beta cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids, L-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mM glucose potentiated insulin secretion 23.3 +/- 2.3% (n = 3) compared with 8.3 mM glucose alone. This effect was abolished in the presence of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 +/- 5.9% (n = 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion.

Selective excitatory amino acid uptake in glutamatergic nerve terminals and in glia in the rat striatum: quantitative electron microscopic immunocytochemistry of exogenous (D)-aspartate and endogenous glutamate and GABA.

To characterize glutamate/aspartate uptake activity in various cellular and subcellular elements in the striatum, rat striatal slices were exposed to 10 and 50 mu M exogenous (D)-aspartate. After fixation with glutaraldehyde/formaldehyde the distribution of (D)-aspartate was analysed by postembedding immunocytochemistry and the ultrastructural distribution was compared with the distributions of endogenous glutamate and GABA. Light microscopically, (D)-aspartate-like immunoreactivity was localized in conspicuous dots along very weakly labelled dendritic profiles and neuron cell bodies. At the electron microscope level gold particles signalling (D)-aspartate occurred at highest density in nerve terminals making asymmetrical contacts with postsynaptic spines (i.e. resembling synapses of cortical afferents). Astrocytic processes also contained gold particles, but at a lower density than nerve endings. In contrast, dendritic spines were only weakly (D)-aspartate-positive. The difference in labelling at 10 and 50 mu M (D)-aspartate was consistent with 'high-affinity' uptake. Neighbouring sections processed with other antibodies showed that the D-aspartate labelling. Occurred in nerve terminals strongly immunoreactive for glutamate, rather than in terminals very weakly glutamate-immunopositive or in nerve endings immunoreactive for GABA. Glutamate labelling of perfusion-fixed striatum confirmed that terminals forming asymmetrical synaptic contacts with spines were enriched with gold particles, suggesting that these terminals use glutamate as a transmitter. This study demonstrates that high-affinity uptake sites for excitatory amino acids in the striatum are most strongly expressed on presumed glutamatergic nerve terminals and on astrocytes.

Quantification of excitatory amino acid uptake at intact glutamatergic synapses by immunocytochemistry of exogenous D-aspartate.

To study the localization and efficiency of glutamate/aspartate membrane transport in the vicinity of intact glutamatergic synapses, the avascular lamprey spinal cord was incubated with D-aspartate, a metabolically inert transporter substrate. The exogenous D-aspartate was localized by immunocytochemistry after aldehyde fixation. Incubation at 50 or 500 microM D-aspartate for 1 hr caused a prominent D-aspartate labeling of glial processes at glutamatergic synapses, while presynaptic axons and postsynaptic dendrites remained unlabeled. The glial processes surrounding glutamatergic sensory axons with a predominantly tonical firing pattern contained significantly higher levels of D-aspartate than did processes surrounding glutamatergic reticulospinal axons, which fire rarely and in brief bursts. Preparations incubated for 10 hr with 500 microM D-aspartate showed D-aspartate immunolabeling in glia as well as in the two types of glutamatergic axon, but no evidence was obtained for uptake into synaptic vesicles. Nor was such evidence obtained after high-frequency electrical stimulation. The observations suggest that excitatory amino acids delivered diffusely to the extracellular space in the intact CNS are transported almost exclusively into glia. The avid uptake in glial processes, combined with their spatial arrangement around glutamatergic synapses, appears to limit the access of exogenous D-aspartate to the nerve terminal glutamate/aspartate transporter. In physiological conditions, the glial processes are likely to impede the exchange of glutamate between the synaptic cleft and the rest of the extracellular space. The transport was more efficient in glial processes located near tonically active synapses than in ones located near synapses releasing transmitter sporadically. D-Aspartate is not a substrate of vesicular glutamate transport sites at these intact synapses.

Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum.

Despite physiological evidence that cholecystokinin (CCK) is an excitatory neurotransmitter in the brain, little is known about its mechanism of action. CCK immunoreactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phosphorylation of a dopamine- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) in striatal neurons. The effect of CCK-8S is prevented by antagonists of CCKB and N-methyl-D-aspartate receptors. Our results support a model in which CCK-8S, originating from CCK or CCK/glutamate corticostriatal neurons, promotes the release of an excitatory neurotransmitter that causes the dephosphorylation and inactivation of DARPP-32, a potent protein phosphatase inhibitor, thereby modulating neuronal excitability.