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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.

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.

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.

Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co-localization of the receptors and their functional cross-talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine- and ATP-mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co-localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross-talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1–P2Y1 receptor co-localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine-mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.

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.

Na+-dependent “binding” of acidic amino acids in brain plasma membranes was examined by procedures similar to those employed in earlier studies, using the metabolically inert D-[3H]aspartate as a probe. The “binding” showed characteristics similar to those described before in terms of affinity (KD, 400 nM). density of sites (Bmax, 300 pmol/mg protein), sensitivity to D,L-threo-3-hydroxyaspartate. and requirement for Na+. It turned out that the “binding” represents uptake into membrane-bounded saccules (which according to the inulin and H2O spaces constituted 3.4 μ/mg protein and comprised about 50% of the volume of the sedimented membranes), rather than binding to the transport carrier. This conclusion is based on the observations that the “binding” of D-aspartate (1) was released by osmotic shock; (2) was abolished by thorough washing of membranes in H2O prior to assay, Which removed endogenous contents of amino acids, and could be recovered by loading the washed membranes with glutamate; (3) was reduced by prior freezing and thawing; (4) was low on incubation at 0°C; (5) had a bell-shaped time course similar to that reported for uptake; and (6) had a slow rate of reversal compared to the apparent KD. True binding would have considerably lower apparent Bmax than the carrier-mediated uptake. This and its likely rapid rate of dissociation would make binding to the carrier difficult to detect by the methods used up to now.

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

Three closely related proteins transport glutamate into synaptic vesicles for release by exocytosis. Complementary patterns of expression in glutamatergic terminals have been reported for VGLUT1 and VGLUT2. VGLUT3 shows expression by many cells not considered to be glutamatergic. Here we describe the changes in VGLUT expression that occur during development. VGLUT1 expression increases gradually after birth and eventually predominates over the other isoforms in telencephalic regions. Expressed at high levels shortly after birth, VGLUT2 declines with age in multiple regions, in the cerebellum by 14-fold. In contrast, Coexpression of the two isoforms occurs transiently during development as well as permanently in a restricted subset of glutamatergic terminals in the adult. VGLUT3 is transiently expressed at high levels by select neuronal populations, including terminals in the cerebellar nuclei, scattered neurons in the cortex, and progenitor-like cells, implicating exocytotic glutamate release in morphogenesis and development. VGLUT3 also colocalizes extensively during development with the neuronal vesicular monoamine transporter VMAT2, with the vesicular acetylcholine transporter VAChT, and with the vesicular gamma-aminobutyric acid transporter VGAT. Such coexpression occurs particularly at some specific developmental stages and is restricted to certain sets of cells. In skeletal muscle, VGLUT3 localizes to granular organelles in the axon terminal as well as in the muscle sarcoplasm. The results suggest novel mechanisms and roles for regulated transmitter release.

Glutamine is involved in a variety of metabolic processes, including recycling of the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). The system N transporter SN1 mediates efflux as well as influx of glutamine in glial cells [Chaudhry et al. (1999), Cell, 99, 769-780]. We here report qualitative and quantitative data on SN1 protein expression in rat. The total tissue concentrations of SN1 in brain and in kidney are half and one-quarter, respectively, of that in liver, but the average concentration of SN1 could be higher in astrocytes than in hepatocytes. Light and electron microscopic immunocytochemistry shows that glutamatergic, GABAergic and, surprisingly, purely glycinergic boutons are ensheathed by astrocytic SN1 laden processes, indicating a role of glutamine in the production of all three rapid transmitters. A dedication of SN1 to neurotransmitter recycling is further supported by the lack of SN1 immunoreactivity in oligodendrocytes (cells rich in glutamine but without perisynaptic processes). All neuronal structures appear unlabelled implying that a different protein mediates glutamine uptake into nerve endings. In several regions, SN1 immunoreactivity is higher in association with GABAergic than glutamatergic synapses, in agreement with observations that exogenous glutamine increases output of transmitter glutamate but not GABA. Nerve terminals with low transmitter reuptake or high prevailing firing frequency are associated with high SN1 immunoreactivity in adjacent glia. Bergmann glia and certain other astroglia contain very low levels of SN1 immunoreactivity compared to most astroglia, including retinal Muller cells, indicating the possible existence of SN isoforms and alternative mechanisms for transmitter recycling.

Numerous features of its primary structure demonstrate that the orphan transporter Rxt1 belongs to the Na+/Cl--dependent neurotransmitter plasma membrane transporter superfamily, which includes the dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA) transporters. Initial immunocytochemical investigations with affinity-purified antibodies have established that Rxt1 is localized, almost exclusively, in axon terminals of glutamatergic neurons and subsets of GABAergic neurons in the CNS. Further studies were carried out to determine its subcellular distribution. In a first series of experiments, PC-12 cells were transfected with plasmids encoding either the dopamine transporter or Rxt1. Immunofluorescence experiments showed that the dopamine transporter was expressed in these cells, and, as expected, addressed to their plasma membrane. Surprisingly, this was never the case with Rxt1, which was targeted to the same subcellular compartment as synaptophysin, a vesicular protein. In a second set of experiments, subcellular fractionation of rat striatum showed that Rxt1, but not the dopamine transporter, was relatively abundant in the purified synaptic vesicle fraction. Finally, electron microscopic immunocytochemistry with anti-Rxt1 antibodies showed peroxidase as well as pre- and post-embedding immunogold labelling confined to the intracellular compartment in various brain regions. Moreover, quantitative analysis of post-embedding experiments demonstrated that the immunogold particles corresponding to Rxt1 immunoreactivity were mostly localized to small synaptic vesicles. These data indicate that, in contrast with the other members of the Na+/Cl--dependent neurotransmitter transporter superfamily, which are targeted to the plasma membrane, Rxt1 is distributed as a vesicular protein in the CNS.

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.

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.