Search

Aim: Adult neurogenesis occurs in two major niches in the brain: the subgranular zone of the hippocampal formation and the ventricular-subventricular zone. Neurogenesis in both niches is reduced in ageing and neurological disease involving dementia. Exercise can rescue memory by enhancing hippocampal neurogenesis, but whether exercise affects adult neurogenesis in the ventricular-subventricular zone remains unresolved. Previously, we reported that exercise induces angiogenesis through activation of the lactate receptor HCA1. The aim of the present study is to investigate HCA1-dependent effects on neurogenesis in the two main neurogenic niches. Methods: Wild-type and HCA1 knock-out mice received high intensity interval exercise, subcutaneous injections of L-lactate, or saline injections, five days per week for seven weeks. Well-established markers for proliferating cells (Ki-67) and immature neurons (doublecortin), were used to investigate neurogenesis in the subgranular zone and the ventricular-subventricular zone. Results: We demonstrated that neurogenesis in the ventricular-subventricular zone is enhanced by HCA1 activation: Treatment with exercise or lactate resulted in increased neurogenesis in wild-type, but not in HCA1 knock-out mice. In the subgranular zone, neurogenesis was induced by exercise in both genotypes, but unaffected by lactate treatment. Conclusion: Our study demonstrates that neurogenesis in the two main neurogenic niches in the brain is regulated differently: Neurogenesis in both niches was induced by exercise, but only in the ventricular-subventricular zone was neurogenesis induced by lactate through HCA1 activation. This opens for a role of HCA1 in the physiological control of neurogenesis, and potentially in counteracting age-related cognitive decline.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA&mdash, i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)&mdash, which is enriched on parvalbumin+ GABAergic neurons&mdash, and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

The volume, composition, and movement of the cerebrospinal fluid (CSF) are important for brain physiology, pathology, and diagnostics. Nevertheless, few studies have focused on the main structure that produces CSF, the choroid plexus (CP). Due to the presence of monocarboxylate transporters (MCTs) in the CP, changes in blood and brain lactate levels are reflected in the CSF. A lactate receptor, the hydroxycarboxylic acid receptor 1 (HCA1), is present in the brain, but whether it is located in the CP or in other periventricular structures has not been studied. Here, we investigated the distribution of HCA1 in the cerebral ventricular system using monomeric red fluorescent protein (mRFP)-HCA1 reporter mice. The reporter signal was only detected in the dorsal part of the third ventricle, where strong mRFP-HCA1 labeling was present in cells of the CP, the tela choroidea, and the neuroepithelial ventricular lining. Co-labeling experiments identified these cells as fibroblasts (in the CP, the tela choroidea, and the ventricle lining) and ependymal cells (in the tela choroidea and the ventricle lining). Our data suggest that the HCA1-containing fibroblasts and ependymal cells have the ability to respond to alterations in CSF lactate in body&ndash, brain signaling, but also as a sign of neuropathology (e.g., stroke and Alzheimer&rsquo, s disease biomarker).

A ketogenic diet (KD; high-fat, low-carbohydrate) can benefit refractory epilepsy, but underlying mechanisms are unknown. We used mice inducibly expressing a mutated form of the mitochondrial DNA repair enzyme UNG1 (mutUNG1) to cause progressive mitochondrial dysfunction selectively in forebrain neurons. We examined the levels of mRNAs and proteins crucial for mitochondrial biogenesis and dynamics. We show that hippocampal pyramidal neurons in mutUNG1 mice, as well as cultured rat hippocampal neurons and human fibroblasts with H2O2 induced oxidative stress, improve markers of mitochondrial biogenesis, dynamics and function when fed on a KD, and when exposed to the ketone body β-hydroxybutyrate, respectively, by upregulating PGC1α, SIRT3 and UCP2, and (in cultured cells) increasing the oxygen consumption rate (OCR) and the NAD+/NADH ratio. The mitochondrial level of UCP2 was significantly higher in the perikarya and axon terminals of hippocampus CA1 pyramidal neurons in KD treated mutUNG1 mice compared with mutUNG1 mice fed a standard diet. The β-hydroxybutyrate receptor GPR109a (HCAR2), but not the structurally closely related lactate receptor GPR81 (HCAR1), was upregulated in mutUNG1 mice on a KD, suggesting a selective influence of KD on ketone body receptor mechanisms. We conclude that progressive mitochondrial dysfunction in mutUNG1 expressing mice causes oxidative stress, and that exposure of animals to KD, or of cells to ketone body in vitro, elicits compensatory mechanisms acting to augment mitochondrial mass and bioenergetics via the PGC1α-SIRT3-UCP2 axis (The compensatory processes are overwhelmed in the mutUNG1 mice by all the newly formed mitochondria being dysfunctional).

The energy deficit hypothesis of attention-deficit/hyperactivity disorder (ADHD) suggests that low lactate production by brain astrocytes causes the symptoms of the disorder. Astrocytes are the main producers of lactate in the brain; however, skeletal muscles can produce the most lactate in the body. The lactate production by skeletal muscles increases with physical activity, as does the expression of the lactate transporter monocarboxylate transporter 1 (MCT1) at the blood-brain barrier (BBB). We hypothesise that children with ADHD, by being hyperactive, increase lactate production by skeletal muscles and transport it into the brain to compensate for low supply by astrocytes. The aim of this study was to explore whether the level of MCT1 is altered in the brain in an animal model of ADHD. The MCT1 expression was quantified on hippocampal brain sections from the best available rat model of ADHD, i.e., the spontaneously hypertensive rat (SHR) (n=12), and the relevant control, the Wistar Kyoto rat (WKY) (n=12), by the use of quantitative immunofluorescence laser scanning microscopy and postembedding immunogold electron microscopy. The results revealed significantly higher levels of hippocampal MCT1 immunoreactivity in SHR compared to WKY, particularly at the BBB. These results indicate that lactate flux through MCT1 between the body and the brain could be upregulated in children with ADHD. This study adds to previous research suggesting hyperactivity may be beneficial in ADHD; Children with ADHD possibly display a hyperactive behaviour in order to raise skeletal muscle lactate production, MCT1 expression and flux over the BBB to supply the brain with lactate. This work was supported by the Norwegian National Research Network for ADHD and the Research Council of Norway (RCN). Norges forskningsråd 214458 Andre 211871

Detailed knowledge about the neural circuitry connecting the hippocampus and entorhinal cortex is necessary to understand how this system contributes to spatial navigation and episodic memory. The two principal cell types of the dentate gyrus, mossy cells and granule cells, are interconnected in a positive feedback loop, by which mossy cells can influence information passing from the entorhinal cortex via granule cells to hippocampal pyramidal cells. Mossy cells, like CA3 pyramidal cells, are characterized by thorny excrescences on their proximal dendrites, postsynaptic to giant terminals of granule cell axons. In addition to disynaptic input from the entorhinal cortex and perforant path via granule cells, mossy cells may also receive monosynaptic input from the perforant path via special dendrites ascending to the molecular layer. We here report qualitative and quantitative descriptions of Golgi-stained hippocampal mossy cells in mink, based on light microscopic observations and three-dimensional reconstructions. The main focus is on the location, branching pattern, and length of dendrites, particularly those ascending to the granular and molecular layers. In mink, the latter dendrites are more numerous than in rat, but fewer than in primates. They form on average 12% (and up to 29%) of the total dendritic length, and appear to cover the terminal fields of both the lateral and medial perforant paths. In further contrast to rat, the main mossy cell dendrites in mink branch more extensively with distal dendrites encroaching upon the CA3 field. The dendritic arbors extend both along and across the septotemporal axis of the dentate gyrus, not conforming to the lamellar pattern of the hippocampus. The findings suggest that the afferent input to the mossy cells becomes more complex in species closer to primates.

We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells.

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.

Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor., Physical exercise promotes brain angiogenesis through an unknown signalling cascade. Morland et al. identify the elusive muscle-brain communication and show that lactate produced by muscle activity binds to its receptor HCAR1 in brain vessel-surrounding cells, stimulating VEGF production and brain angiogenesis.

Glial cells in their plurality pervade the human brain and impact on brain structure and function. A principal component of the emerging glial doctrine is the hypothesis that astrocytes, the most abundant type of glial cells, trigger major molecular processes leading to brain ageing. Astrocyte biology has been examined using molecular, biochemical and structural methods, as well as 3D brain imaging in live animals and humans. Exosomes are extracelluar membrane vesicles that facilitate communication between glia, and have significant potential for biomarker discovery and drug delivery. Polymorphisms in DNA repair genes may indirectly influence the structure and function of membrane proteins expressed in glial cells and predispose specific cell subgroups to degeneration. Physical exercise may reduce or retard age-related brain deterioration by a mechanism involving neuro-glial processes. It is most likely that additional information about the distribution, structure and function of glial cells will yield novel insight into human brain ageing. Systematic studies of glia and their functions are expected to eventually lead to earlier detection of ageing-related brain dysfunction and to interventions that could delay, reduce or prevent brain dysfunction.

The Kavli Prize in Neuroscience was awarded for the third time in September 2012, by the Norwegian Academy of Science and Letters in Oslo. The accompanying Kavli Prize Symposium on Neuroscience, held in Bergen and Trondheim, was a showcase of excellence in neuroscience research. The common theme of the Symposium presentations was the mechanisms by which animals adapt to their environment. The symposium speakers--Michael Greenberg, Erin Schuman, Chiara Cirelli, Michael Meaney, Catherine Dulac, Hopi Hoekstra, and Stanislas Dehaene--covered topics ranging from the molecular and cellular levels to the systems level and behavior. Thus a single amino acid change in a transcriptional repressor can disrupt gene regulation through neural activity (Greenberg). Deep sequencing analysis of the neuropil transcriptome indicates that a large fraction of the synaptic proteome is synthesized in situ in axons and dendrites, permitting local regulation (Schuman). The nature of the 'reset' function that makes animals dependent of sleep is being revealed (Cirelli). Maternal behavior can cause changes in gene expression that stably modify behavior in the offspring (Meaney). Removal of a single sensory channel protein in the vomero-nasal organ can switch off male-specific and switch on female-specific innate behavior of mice in response to environmental stimulation (Dulac). Innate behaviors can be stably transmitted from parent to offspring through generations even when those behaviors cannot be expressed, as illustrated by the elaborate burrowing behavior in a rodent species, in which independent genetic regions regulate distinct modules of the burrowing pattern (Hoekstra). Finally, at the other extreme of the nature-nurture scale, functional magnetic resonance imaging (fMRI) analysis in children and adults identified a brain area specifically involved in reading (Dehaene). As the area must originally have developed for a purpose other than reading, such as shape recognition, this illustrates the use of a previously formed neural structure to tackle a new challenge.

Granulocyte/macrophage colony forming cells (GM-CFC) require colony stimulating factors (CSF) for growth in vitro. Crude preparations of CSF also contain inhibitors. To investigate whether this inhibition involves free radicals, the following scavenging agents were tested on human GM-CFC grown in agar: cysteine, reduced glutathione, dithiothreitol, sodium selenite, alpha-tocopherol, superoxide dismutase, catalase and ascorbic acid. All except ascorbic acid and catalase had enhancing and stabilising effects on colony formation. The colonies also contained more cells but no changes in cell composition were detected. Cysteine, the most extensively tested substance, was active at concentrations present in body fluids and enhanced colony formation without an exogenous source of CSF. Our results are consistent with an imbalance between the production and removal of free radicals generated in the human GM-CFC assay in vitro. This hampers investigation of regulators of granulopoiesis and results in underestimation of GM-CFC, but can be alleviated by the addition of free radical scavengers.

A new malignant human T-cell line-labelled PFI-285-has been isolated from a boy with malignant lymphoma. Morphologically, the cells had characteristics of malignant lymphoid cells. The cells presented surface antigens as early cortical lymphocytes and proliferated non-adherently as single cells, independent of T-cell growth factor (IL-2), in liquid culture. The cells had undetectable levels of receptors for IL-2, were not clonogenic in soft agar, but did form tumors in nude mice. Their establishment and continuous growth in vitro was dependent on the number of cells inoculated and on the growth medium used. Cytogenetic alteration, HTLV-1 or reverse transcriptase activity were not detected. The production of known T-cell derived lymphokines such as IL-2, B-cell growth factor(s), alpha-interferon or granulocyte/macrophage colony stimulating or inhibiting factor(s) was not detected. The cells had 5-8% natural killer (NK)-cell activity against NK-cell sensitive target cells (K562) and were not sensitive for NK cells. A most unusual characteristic was the pronounced sensitivity of the cells to ascorbic acid. Concentrations down to 50 mumol/l killed the cells within hours.

This special issue of Neuroscience, “From Cochlea to Cortex: Recent Advances in Auditory Neuroscience,” was organized as a tribute to the pioneering contributions to the field by Kirsten Kjelsberg Osen, Professor Emeritus, The University of Oslo. In this introduction, we summarize her professional career, highlighting her numerous scientific achievements, and provide references to all of her published papers. In addition, we briefly summarize the contents of the 35 papers that make up the issue.

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.

In propionic acidemia, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors. Little is known about the cerebral metabolism of propionate, although clinical effects of propionic acidemia are largely neurological. We found that propionate was metabolized oxidatively by glia: [3-(14)C]propionate injected into mouse striatum or cortex, gave a specific activity of glutamine that was 5-6 times that of glutamate, indicating metabolism in cells that express glutamine synthetase, i.e., glia. Further, cultured cerebellar astrocytes metabolized [3-(14)C]propionate; cultured neurons did not. However, both cultured cerebellar neurons and astrocytes took up [3H]propionate, and propionate exposure increased histone acetylation in cultured neurons and astrocytes as well as in hippocampal CA3 pyramidal neurons of wake mice. The inability of neurons to metabolize propionate may be due to lack of mitochondrial propionyl-CoA synthetase activity or transport of propionyl residues into mitochondria, as cultured neurons expressed propionyl-CoA carboxylase, a mitochondrial matrix enzyme, and oxidized isoleucine, which becomes converted into propionyl-CoA intramitochondrially. The glial metabolism of propionate suggests astrocytic vulnerability in propionic acidemia when intramitochondrial propionyl-CoA may accumulate. Propionic acidemia may alter both neuronal and glial gene expression by affecting histone acetylation.

Purpose In the brain and in adipose tissue activation of the lactate receptor GPR81 is known to promote downregulation of cAMP. Lactate is hereby involved in excitability, metabolism and inflammation. Neurodegenerative diseases in retina may in a similar manner be linked to disturbed lactate homeostasis. Methods qPCR was performed on retina and brain extracts to investigate whether the lactate receptor GPR81 is expressed in the retina. Immunocytochemistry was performed on primary cell cultures of Muller cells (MC) and retinal ganglion cells (RGC) from mice to evaluate the presence of lactate receptors in neuronal and glial cells in the retina. Lactate assays were made to show the changes in lactate concentrations during different conditions of stress. Results GPR81 mRNA was twice as high in retina as in hippocampus or cerebral cortex. Immunocytochemistry indicated lactate receptors in both RGC and MC. Lactate assays showed a decreased release of lactate from MC upon energy restriction. Interestingly, the combination of inhibited mitochondrial function and energy restriction significantly increased the amount of released lactate. Conclusions The presence of lactate receptors in the retina as well as the changed levels of lactate in response to stress support the suggestion that lactate could be of great importance in retinal homeostasis and as such in the pathogenesis of inner retinal diseases.

In retina, like in brain, lactate equilibrates across cell membranes via monocarboxylate transporters and in the extracellular space by diffusion, forming a basis for the action of lactate as a transmitter of metabolic signals. In the present paper, we argue that the lactate receptor GPR81, also known as HCAR1, may contribute importantly to the control of retinal cell functions in health and disease. GPR81, a G-protein coupled receptor, is known to downregulate cAMP both in adipose and nervous tissue. The receptor also acts through other down-stream mechanisms to control functions, such as excitability, metabolism and inflammation. Recent publications predict effects of the lactate receptor on neurodegeneration. Neurodegenerative diseases in retina, where the retinal ganglion cells die, notably glaucoma and diabetic retinopathy, may be linked to disturbed lactate homeostasis. Pilot studies reveal high GPR81 mRNA in retina and indicate GPR81 localization in Muller cells and retinal ganglion cells. Moreover, monocarboxylate transporters are expressed in retinal cells. We envision that lactate receptors and transporters could be useful future targets of novel therapeutic strategies to protect neurons and prevent or counteract glaucoma as well as other retinal diseases.

During chronic metabolic acidosis (CMA), the plasma levels of glutamine are increased and so is glutamine metabolism in the kidney tubule cells. Degradation of glutamine results in the formation of ammonium (NH(4)(+)) and bicarbonate (HCO(3)(-)) ions, which are excreted in the pre-urine and transported to the peritubular blood, respectively. This process contributes to counteract acidosis and to restore normal pH, but the molecular mechanism, the localization of the proteins involved and the regulation of glutamine transport into the renal tubular cells, remains unknown. SN1, a Na(+)- and H(+)-dependent glutamine transporter has previously been identified molecularly, and its mRNA has been detected in tubule cells in the medulla of the kidney. Now shown is the selective targeting of the protein to the basolateral membranes of the renal tubule cells of the S3 segment throughout development of the normal rat kidney. During CMA, SN1 expression increases five- to six-fold and appears also in cortical tubule cells in parallel with the increased expression and activity of phosphate-activated glutaminase, a mitochondrial enzyme involved in ammoniagenesis. However, SN1 remains sorted to the basolateral membranes. The unique ability of SN1 to change transport direction according to physiologic changes in transmembrane gradients of [glutamine] and pH and its sorting to the basolateral membranes and the presence of a putative pH responsive element in the 3' untranslated region (UTR) of the gene (supported here by the demonstration in CMA kidney of a protein that binds SN1 mRNA) are conducive to the function of this transporter in pH regulation.

Previous findings, mainly in in vitro systems, have shown that the density of vesicles and the synaptic efficacy at excitatory synapses are reduced in the absence of synapsins, despite the fact that transgenic mice lacking synapsins develop an epileptic phenotype. Here we study glutamate receptors by quantitative immunoblotting and by quantitative electron microscopic postembedding immunocytochemistry in hippocampus of perfusion fixed control wild type and double knock-out mice lacking synapsins I and II. In wild type hippocampus the densities of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits were higher (indicated for glutamate receptor subunit 1, highly significant for glutamate receptor subunits 2/3) in mossy fiber-to-cornu ammonis 3 pyramidal cell synapses than in the Schaffer collateral/commissural-to-cornu ammonis 1 pyramidal cell synapses, the two synapse categories that carry the main excitatory throughput of the hippocampus. The opposite was true for N-methyl-D-aspartate receptors. The difference in localization of glutamate receptor subunit 1 receptor subunits was increased in the double knock-out mice while there was no change in the overall expression of the glutamate receptors in hippocampus as shown by quantitative Western blotting. The increased level of glutamate receptor subunit 1 at the mossy fiber-to-cornu ammonis 3 pyramidal cell synapse may result in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with reduced proportions of glutamate receptor subunit 2, and hence increased Ca2+ influx, which could cause increased excitability despite of impaired synaptic function (cf. [Krestel HE, Shimshek DR, Jensen V, Nevian T, Kim J, Geng Y, Bast T, Depaulis A, Schonig K, Schwenk F, Bujard H, Hvalby O, Sprengel R, Seeburg PH (2004) A genetic switch for epilepsy in adult mice. J Neurosci 24:10568-10578]), possibly underlying the seizure proneness in the synapsin double knock-out mice. In addition, the tendency to increased predominance of N-methyl-d-aspartate receptors at the main type of excitatory synapse onto cornu ammonis 1 pyramidal cells might contribute to the seizure susceptibility of the synapsin deficient mice. The results showed no significant changes in the proportion of 'silent' Schaffer collateral/commissural synapses lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors or in the synaptic membrane size, indicating that plasticity involving these parameters is not preferentially triggered due to lack of synapsins.

It has been established that there is a strong functional link between sensory neural circuits on the two sides of the spinal cord. In one of our recent studies we provided a morphological confirmation of this functional phenomenon, presenting evidence for the presence of a direct commissural connection between the lateral aspects of the dorsal horn on the two sides of the lumbar spinal cord. By using a combination of neural tracing and immunocytochemical detection of neural markers like vesicular glutamate transporters, glutamic acid decarboxylase, glycine transporter, and met-enkephalin (which are characteristic of various subsets of excitatory and inhibitory neurons), we investigated here the distribution, synaptic relations, and neurochemical characteristics of the commissural axon terminals. We found that the cells of origin of commissural fibers in the lateral aspect of the dorsal horn were confined to laminae III–IV and projected to the corresponding area of the contralateral gray matter. Most of the commissural axon terminals established synaptic contacts with dendrites. Axospinous or axosomatic synaptic contacts were found in limited numbers. We demonstrated that interactions among commissural neurons also exist. More than three-fourths of the labeled axon terminals were immunostained for glutamic acid decarboxylase and/or glycine transporter, but none of them showed positive immunoreaction for met-enkephalin and vesicular glutamate transporters. The results indicate that there is a substantial reciprocal commissural synaptic interaction between the lateral aspects of laminae III–IV on the two sides of the lumbar spinal cord and that this pathway may transmit both inhibitory and excitatory signals to their postsynaptic targets. J. Comp. Neurol. 480:364 –377, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: spinal dorsal horn; propriospinal neurons; neural tracing; immunocytochemistry; amino acid neurotransmitters

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset ofexcitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.

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.

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 Ca(2+)-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.

The prognosis of infantile myofibromatosis (IMF) depends on the organs involved: the prognosis is very poor if vital viscera are affected, but excellent if there is no visceral involvement. We report the case of a boy presenting with a pathological fracture at the age of 6 weeks. Progressive osteolytic lesions in the whole skeleton until the age of 8 months led to respiratory failure due to a softened thoracic wall requiring mechanical ventilation for 11 months. No pulmonary, laryngeal or other visceral involvement was found. In spite of the rapidly progressing disease and serious complications only supportive therapy was given. The lesions subsided gradually leaving slight deformities but normal function. At the age of 3.5 years the boy has an excellent quality of life.This case illustrates that even in progressing, complicated multifocal infantile myofibromatosis (without visceral involvement) the lesions can resolve without antitumour treatment if high quality intensive care is supplemented.

Previous studies of the cochlear nuclei in cat, rat, and guinea pig have demonstrated neural structures that are enriched in the inhibitory neurotransmitter amino acids γ-aminobutyric acid (GABA) and glycine. In these mammals, inhibitory terminals are widely distributed throughout the nuclear complex, but somata of inhibitory neurons are concentrated in the dorsal cochlear nucleus, in granule cell regions, and in the cap area. Because these are the subdivisions that undergo the most pronounced phylogenetic changes in primates, we wanted to see whether the inhibitory systems are influenced by changes in cytoarchitecture. Therefore, we applied light microscopic postembedding immunostaining and optical densitometry to the cochlear nuclei of an anthropoid primate, the Senegalese baboon (Papio anubis). Our results demonstrate that, in baboon 1) glycinergic neurons and axons in the ventral cochlear nucleus seem to form a commissural system similar to that of other mammals; 2) the tuberculoventral system appears to be unchanged in morphology but exhibits a higher level of colocalization of GABA with glycine; 3) there is a reduction of the granule/cartwheel cell system, which is reflected in lesser numbers of inhibitory cartwheel, Golgi, and molecular layer stellate cells; 4) the cap area is larger than in rodents and carnivores and contains many neurons that colocalize GABA and glycine; and 5) throughout the nuclear complex, a higher proportion of the inhibitory terminals colocalize GABA and glycine. We conclude that modulation of the ascending auditory pathway in baboon is likely to differ from that in rodents and cat. © 1996 Wiley-Liss, Inc.

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.

Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.

Background/Aims: The inhibitory neurotransmitter γ-aminobutyrate (GABA) has been shown to coexist with insulin in pancreatic β-cells. We have presently investigated whether GABA also colocalizes with gastrin in G cells in rat antral mucosa. Methods: Three alternative approaches were used: (1) gastrin in situ hybridization and GABA immunocytochemistry on consecutive cryostat sections; (2) GABA immunocytochemistry and gastrin immunocytochemistry on adjacent semithin and ultrathin sections; and (3) double-immunogold labeling of GABA and gastrin in the same ultrathin section. Results: Colocalization of GABA and gastrin was observed with each of the three approaches. In the double-immunogold labeled cells, the G-cell granules displayed a high gold-particle density indicating gastrin and a low particle density indicating GABA, whereas the converse was true for the extragranular cytoplasmic matrix. The gold-particle ratios between these compartments were 11 (for gastrin) and 0.36 (for GABA), respectively. GABA labeling was also observed in two other antral endocrine cell types, classified by morphological criteria as somatostatin producing D cells and serotonin producing ECn cells. Conclusions: This is the first direct demonstration of GABA in gastrointestinal G cells. Our findings suggest that GABA may have a paracrine function in the stomach mucosa, analogous to its presumed role in the pancreatic islets.

Functional studies indicate that the dopamine D5 receptor is involved in synaptic transmission in the hippocampus. However, previous anatomical studies have detected D5 receptor labelling primarily on the soma and main dendrites of CA1 pyramidal cells and on dendritic spines in monkey but not in rats. In order to get a better understanding of putative dopamine function in the hippocampus, we quantified the D5 receptor immunoreactivity on the pyramidal cell somas and on spines and dendrites in stratum radiatum and stratum oriens in the hippocampal CA1 region of rats by quantitative immunofluorescence and immunogold electron microscopy. The quantitative immunogold results revealed a higher labelling density on dendritic spines, notably at their synaptic membranes, compared to pyramidal cell somas and dendrites. Hence, dopamine could have effects on spines as well as on somas and dendrites. The labelling density was similar on spines in stratum oriens and stratum radiatum, but the presence of labelling varied between the spines within each stratum, indicating that the effect of dopamine could be diverse between different spines.

Slide-mounted 12-µm cryostat sections of rat brain were incubated with [3H]bilirubin, washed, dried, and apposed to emulsion-coated coverslips for autoradiography. The binding appeared to be nonsaturable. Within the gray matter, binding was concentrated over neuronal cell bodies (particularly evident over hippocampal pyramidal and granular cells, and over cerebellar Purkinje cells), suggesting that the preferential neuronal toxicity of bilirubin may be related to neuronal binding. Regional differences in bilirubin binding were not demonstrated.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na + + K + ]coupled l -glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the l -glutamate transport activity—as assayed upon reconstitution—from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al . (1990) Biochemistry 29 , 6734–6740] closely correlated with the l -glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the l -glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate l -glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.

The cellular localization of γ-aminobutyric acid (GABA) has been visualized immunocytochemically in the nervous system of Oikopleura dioica by using an antiserum to glutaraldehyde fixation complexes of GABA. The results show GABA-like immunoreactivity in neurons of the brain, in cells of the sensory vesicle, in the caudal ganglion, and in the nerve cord. Positive reactions were also found at the neuromuscular terminals in the tail.

The cochlear root nucleus is part of the cochlear nuclear complex in small rodents. Its cells, the large root neurons, have a superficial resemblance to the globular neurons of the ventral cochlear nucleus. It has been a matter of debate, therefore, whether the root neurons and globular neurons represent the same or different types of cell. In the present study the two cell types with adjacent neuropil structures were compared by light microscopic, postembedding immunocytochemistry. Pairs of 0.5 microns sections of resin-embedded, glutaraldehyde-fixed material were treated with purified antisera raised against GABA- and glycine-glutaraldehyde-protein conjugates, respectively. Both types of cell were found to be immunonegative. Striking differences, however, occurred in what was interpreted as afferent nerve terminals. The globular cells appeared to receive numerous afferents with GABA- or glycine-like immunoreactivity on their somata. Immunoreactive terminals on the root neurons, on the contrary, were mostly GABA-positive and located on the dendrites. Although of unknown origin, the immunoreactive afferents were clearly different from the primary fibres as demonstrated both by the immunonegativity of the latter and by the different size and distribution of the terminals labelled anterogradely after horseradish peroxidase injections into the spiral ganglion.

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal.

Polyclonal antisera raised against conjugated GABA were used to study the distribution of GABAergic neurons in the spinal cords of lampreys ( Lampetra fluviatilis and Ichtyomyzon unicuspis ) using immunofluorescence and peroxidase-antiperoxidase techniques. Three morphologically distinct types of GABA-immunoreactive (GABA-ir) cell bodies were observed, multipolar neurons in the lateral grey cell column, apparently bipolar cells in the ventral aspect of the dorsal horn, and small liquor-contacting cells surrounding the central canal. A high density of immunoreactive fibers of spinal origin were present in the lateral and ventral funiculi, whereas the dorsal column had a relatively low density. Dense GABA-ir plexuses were situated in the lateral spinal margin, and in the dorsal part of the dorsal horn. A chronic lesion of the rostral spinal cord did not result in any observable loss of GABA-ir fibers below or above the lesion, suggesting that the 3 types o f segmental GABA-ir neurons are the main sources of the GABAergic innervation of the lamprey spinal cord.

A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.

To elucidate the possible roles of the CNS neurotransmitters glycine and GABA in neuroendocrine paracrine signalling, we investigated their localizations, and those of their transport proteins, by confocal immunofluorescence and quantitative post-embedding immuno-electron microscopy in the pancreatic islets of Langerhans. We show that A-cells contain glycine in synaptic-like microvesicles as well as in secretory granules. A-cells express the macromolecules necessary to: (1) concentrate glycine within both organelle types before release (the vesicular GABA/glycine transporter VGAT=VIAAT); and to (2) take up the transmitter from the extracellular space (the plasma membrane glycine transporter GLYT2). Also B-cells have glycine in their microvesicles and granules, but the microvesicle/cytosol ratio is lower than in A-cells, consistent with the presence of GABA (which competes with glycine for vesicular uptake) in the cytosol at a much higher concentration in B-cells than in A-cells. Both A- and B-cells contain GABA in their microvesicles and secretory granules, and the membranes of the two organelle types contain VGAT in both cell types. A-cells as well as B-cells express a plasma membrane transporter GAT3 that mediates uptake of GABA. The localization of VGAT in the cores of A-cell secretory granules, and in the secretory granule membranes in both cell types, indicates novel aspects of the mechanisms for release of glycine and GABA. The discovery that both A- and B-cells possess the molecular machinery for the evoked release of both glycine and GABA from synaptic-like microvesicles suggests that both of the principal inhibitory transmitters in the brain participate in paracrine signalling in the pancreas.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex.Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 μm) or intracellular Cl-concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

We previously found evidence for the exocytosis of aspartate from excitatory nerve terminals in hippocampus [J. Neurosci. 18, (1998) 6059]. Here we show, by immunogold electron microscopy in hippocampal slices that aspartate is co-localized and co-exocytosed with GABA from synaptic vesicles in nerve endings assumed to be inhibitory on dentate granule cells and CA1 pyramidal cells. By immunogold double labeling cytochemistry in perfusion fixed hippocampus, we further find that GABA-positive terminals forming symmetric synaptic specializations on perikarya of granule and pyramidal cells express NMDA receptors. In addition, NMDA receptors are present at synapses on granule cell bodies that have asymmetric synaptic specializations and contain high levels of GABA. Glutamate levels are low in the described types of GABA-positive nerve terminals, but high in terminals making asymmetric synapses on dendritic spines, whereas aspartate is localized with high levels in all of these types of terminal. We propose that aspartate is exocytotically released not only from glutamatergic terminals, but also from GABAergic terminals to act on NMDA receptors and may have a role in the regulation of synaptic transmission. Under pathological conditions, release of an excitatory transmitter at an inhibitory synapse could contribute to the development of, for example, epilepsy.

After axotomy in the ventral funiculus of the cat spinal cord, about half of the population of lesioned motoneurons die at 1–3 weeks postoperatively, whereas the other half survives and generates new axons through the lesion area. To identify conditions that may promote survival and regeneration of motoneurons subjected to this kind of injury, the authors examined ultrastructurally lesion-induced changes in the number and distribution of nerve terminals on the somata and proximal dendrites of α-motoneurons in the 7th lumbar spinal segment (L7) of the cat spinal cord. Intramedullary axotomy resulted in a profound reduction in the number of nerve terminals impinging on the somata and proximal dendrites, with the maximal effect seen at 3 weeks postlesion. At that time, only 12–25% of the normal number of terminals remained on the cell somata, and 22–33% remained on proximal dendrites. Thereafter, a gradual increase in terminal numbers occurred, reaching normal levels at 34 weeks after the lesion. Already at 2 days postoperatively and, most obviously, at 3 weeks postoperatively, type S nerve terminals were eliminated to a larger degree than type F terminals. Postembedding immunohistochemistry confirmed that the largest reduction at 3 weeks was seen for excitatory glutamate-immunopositive type S nerve terminals (90%), whereas inhibitory glycine-immunoreactive and γ-aminobutyric acid (GABA)-immunoreactive type F terminals were affected less (70% reduction). This led to a distinct shift in the ratio between the numbers of terminals that were immunopositive for glycine and GABA and the numbers of terminals that were labeled for glutamate. For the cell body, this ratio increased from 3.7 in normal material to 14.5 in lesioned motoneurons, whereas the corresponding values for proximal dendrites were 3.8 and 7.5. The preferential elimination of glutamatergic inputs to lesioned motoneurons may reflect an active reorganization of the synaptic input to diminish the excitotoxic influence on these neurons, thereby promoting the survival of motoneurons after intramedullary axotomy. J. Comp. Neurol. 425:10–23, 2000. © 2000 Wiley-Liss, Inc.

Antibodies to an excitatory amino acid transporter (EAAT4) label a glycoprotein of ∼65 kDa strongly in the cerebellum and weakly in the forebrain. Cross-linking of cerebellar proteins with bis(sulfosuccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters GLAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cerebellar molecular layer, being strongly expressed in parasagittal zones. It is located in cerebellar Purkinje cells, and the highest concentrations are seen in ones expressing high levels of zebrin II (aldolase C). The labeling of Purkinje cell spines and thin dendrites is stronger than that of large diameter dendrites and cell bodies. EAAT4 is present at low concentrations in the synaptic membrane, but is highly enriched in the parts of the dendritic and spine membranes facing astrocytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and multivesicular organelles may reflect turnover of transporter in the plasma membrane. The total Purkinje cell spine surface and the EAAT4 concentration were found to be 1.1 m2/cm3and 0.2 mg/cm3, respectively, in the molecular layer, corresponding to 1800 molecules/μm2. The juxtasynaptic location of EAAT4 may explain electrophysiological observations predicting the presence of a neuronal glutamate transporter near the release site at a Purkinje cell spine synapse. EAAT4 may function as a combined transporter and inhibitory glutamate receptor.

The main pathway for transmitter glutamate turnover in excitatory synapses is thought to involve an uptake in glial processes, a conversion into glutamine, which recycles to the presynaptic terminal to serve as the main precursor for new synthesis of glutamate. To investigate whether the mechanisms of glutamine and glutamate turnover are linked with the properties of different glutamate synapses, the distribution of glutamine was studied in two types of glutamate synapse in the lamprey spinal cord using immunogold post-embedding electron microscopy. The synapses examined are formed by primary afferent axons (dorsal column axons), which predominantly exhibit a tonic firing pattern, and by giant reticulospinal axons, which primarily fire in brief bursts. Glial cell processes and postsynaptic dendrites displayed the highest density of glutamine labeling in both types of synapse. The level of glutamine was significantly higher in the glial cell processes surrounding the tonic dorsal column synapses, as compared to those surrounding the reticulospinal synapses. The axoplasmic matrix and presynaptic mitochondria, as well as postsynaptic dendrites, contained similar levels of glutamine labeling in both cases. The glutamate labeling in glial processes was also similar at the two types of synapse, while axoplasmic matrix and presynaptic mitochondria displayed four to six times higher levels in the tonic axons. In conjunction with our previous results, showing a different transport activity in glial processes of the two types of excitatory synapse, the results of the present study suggest that the glial pool of neurotransmitter precursor is linked to the rate of transmitter synthesis and release in adjacent synapses.

A long line of studies emphasizes the contribution of serotonergic fibres descending from the rostral ventromedial medulla in the control of spinal nociceptive information processing. A growing body of evidence, however, suggests that the relative contribution of serotonin to the mediation of spinal neuronal activity from the rostral ventromedial medulla may require re-evaluation. It has recently been substantiated that, in addition to the serotonergic fibres, the spinal dorsal horn receives an abundant non-serotonergic projection from the rostral ventromedial medulla. Furthermore, stimulation in the rostral ventromedial medulla could result in a powerful inhibition of nociceptive spinothalamic tract cells without any detectable serotonin release in the dorsal horn. After labelling raphe-spinal axons and axon terminals in the rat by iontophoretic injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into the central region of the rostral ventromedial medulla (nucleus raphe magnus) and revealing GABA and glycine immunoreactivites of the labelled raphe-spinal terminals and their postsynaptic targets by postembedding immunocytochemical methods, here we demonstrate an extensive GABAergic projection from the rostral ventromedial medulla to the spinal dorsal horn. We show that the majority of the labelled raphe-spinal terminals in laminae I-IIo and IV-V contain GABA and some of the GABA-immunoreactive terminals are also immunoreactive for glycine. We also disclose that GABA-immunoreactive raphe-spinal terminals establish synaptic contacts primarily with GABA- and glycine-negative, presumably excitatory, spinal neurons, including Calbindin-D28k-as well as parvalbuminimmunoreactive cells in both laminae I–IIo and IV–V. The results suggest that volleys in fibres descending from the rostral ventromedial medulla may evoke GABA release from raphe-spinal terminals, and the released GABA, in some cases probably acting together with glycine, might play a crucial, as yet mostly unidentified, role in the inhibition of nociceptive information processing in the dorsal horn of the spinal cord.

The monosynaptic connection between muscle spindle Ia afferents and motoneurones is one of the most extensively studied reflex pathways in the central nervous system. Physiological evidence has pointed to glutamate as the fast transmitter in this synaptic connection. In this study of the spinal cord segment C2, we used dual labelling with cholera toxin subunit B-horseradish peroxidase (B-HRP) and postembedding immunohistochemistry to show that group Ia primary afferent boutons synapsing on retrogradely labelled central cervical nucleus (CCN) neurones and motoneurones are enriched in glutamate-like immunoreactivity (LI). All double-labelled Ia terminals studied could be classified as S-type (CCN neurones and motoneurones) or M-type (motoneurones) boutons.

The present paper addresses the identity of the neurotransmitter(s) of the parallel fibers in the molecular layer of the dorsal cochlear nucleus, A brainstem center in the pathway for sound perception. The distribution of putative neurotransmitter amino acids was studied by using postembedding single- and double-immunolabeling procedures. Perfusion-fixed brains and immersion-fixed slices from in vitro release experiments were evaluated. Quantitative immunogold analyses revealed that the parallel fiber terminals were significantly enriched with glutamate immunoreactivity compared with other terminals, dendrites, and glial processes. Within the parallel fiber terminals, the gold particles signaling the presence of glutamate were concentrated over vesicle clusters relative to the axoplasmic matrix. Furthermore, the parallel fiber terminals, but not the parent granule cell bodies, could be depleted of glutamate immunoreactivity by exposure to depolarizing concentrations of K+ in vitro. This depletion was partly dependent on Ca2+In double-labeled preparations, the glutamine:glutamate ratio was by far higher in glial processes than in other types of profile. Aspartate immunoreactivity was mainly concentrated in neuronal cell bodies and dendrites and was very low in fiber terminals, particularly in those of the parallel fibers. These data indicate that parallel fiber terminals contain a glutamate pool that is associated with synaptic vesicles and that can be subject to release. The glial processes that are found in proximity to the terminals may provide them with the glutamine required for glutamate replenishment. No evidence was found for a neurotransmitter role of aspartate in the parallel fibers. © 1995 Wiley-Liss, Inc.

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.