Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5'-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5'-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain.

In the adult nervous system, multipotential stem cells of the subventricular zone of the lateral ventricles generate neuron precursors (type-A cells) that migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. The migrating neuroblasts are surrounded by a sheath of astrocytes (type-B cells). Using immunostaining, in situ hybridization and enzyme histochemistry, we demonstrate that the ecto-ATPase nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is expressed in the subventricular zone and the rostral migratory stream of the adult rat brain. This enzyme hydrolyses extracellular nucleoside triphosphates to the respective nucleoside diphosphates and is thought to directly modulate ATP receptor-mediated cell communication. Double labelling for the astrocyte intermediate filament protein GFAP and the glial glutamate transporter GLAST identifies the NTPDase2-positive cells as type-B cells. During development the enzyme protein is first detected at E18, long before expression of the astrocyte marker GFAP. It gradually becomes expressed along the ventricular and subventricular zone of the brain, followed by complete retraction to the adult expression pattern at P21. NTPDase2 is transiently expressed in the outer molecular layer of the dentate gyrus and within the cerebellar white matter and is associated with select microvessels, tanycytes of the third ventricle, and subpial astrocytes of the adult brain. Our results suggest that NTPDase2 can serve as a novel marker for specifying subsets of cells during in vivo and in vitro studies of neural development and raise the possibility that ATP-mediated signalling pathways play a role in neural development and differentiation.

An astrocyte-like cell population corresponding to residual radial glia represents the neuronal progenitors of the adult mammalian hippocampus. We show that radial glia-like cells of the dentate gyrus express surface-located ATP-hydrolyzing activity and are immunopositive for NTPDase2. This enzyme hydrolyzes extracellular nucleoside triphosphates such as ATP and UTP to their nucleoside diphosphates and is thus involved in the control of signaling via P2 receptors. NTPDase2 is expressed from embryonic day 17 onward. In the hippocampus, the embryonic pattern of NTPDase2 expression mirrors that of the dentate migration of neuroblasts. Double-immunolabeling revealed that NTPDase2 is associated with subpopulations of glial fibrillary acidic protein-, nestin- and doublecortin-positive radial cells. It is absent from mature granule cells or S100-positive astrocytes. NTPDase2-positive cells proliferate. Furthermore, after mitosis, progenitor cells preferentially reveal an NTPDase2-positive phenotype. Patch-clamp analysis demonstrates functional nucleotide receptors in progenitor cells expressing nestin promotor-driven green fluorescent protein. Our results identify the ecto-nucleotidase NTPDase2 and functional P2X receptors at hippocampal progenitor cells. We infer that signaling pathways via extracellular nucleotides may play a role in the control of hippocampal neurogenesis.

Extracellular nucleotides are ubiquitous extracellular mediators that interact with and activate nucleotide type 2 (P2) receptors. These receptors initiate a wide variety of signalling pathways that appear important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain. Ectonucleotidases are extracellular nucleotide-metabolizing enzymes that modulate P2 receptor-mediated signalling by the regulated hydrolysis of these agonists. A considerable number of ectoenzyme species with partially overlapping substrate and tissue distributions have been described. Major candidates for expression in the brain are members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase or CD39) family. The production of cd39-/- mice and specific reagents have enabled us to analyse the specific cellular distribution of NTPDase1 (CD39), the prototype member of the enzyme family, in the mouse brain. Using monospecific antibodies and enzyme histochemical staining, we have identified NTPDase1 as a major ectonucleotidase associated with both microglia and the endothelial and smooth muscle cells of the vasculature. NTPDase1 is not expressed by neurons and astrocytes. Additional unidentified ectonucleotidase functional activity is observed at lower levels throughout the brain parenchyma. NTPDase1 may regulate P2 receptor-mediated functions of microglia as well as influence nucleotide signalling between neurons or astrocytes that are associated with multiple microglial ramifications. The expression of NTPDase1 by cerebrovascular endothelial and smooth muscle cells also suggests involvement in the regulation of blood flow and thrombogenesis.

Cellular signaling via extracellular nucleotides appears to play a major role in the functioning of the peripheral nervous system. Information regarding the functional characterization of nucleotide P2 receptors or their expression pattern has been accumulating rapidly; however, very little is known regarding the distribution of ecto-nucleotidases in the periphery. The extracellular level of nucleotides is controlled by ecto-nucleotidases, whereby the three membrane-bound members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family are of special functional importance. Using enzyme histochemistry and immunostaining, we demonstrate that NTPDase2 is associated with nonmyelinating Schwann cells of the rat sciatic nerve, whereas NTPDase1 is restricted to blood vessel walls. NTPDase2 immunoreactivity was detected from embryonic day E18 onward, suggesting that immature Schwann cells express the enzyme. With the onset of myelination, NTPDase2 immunoreactivity remained associated solely with nonmyelinating Schwann cells. NTPDase2 was absent from perisynaptic Schwann cells but was associated with fibroblasts covering the endplate at some distance. In addition, NTPDase2 immunoreactivity was associated with the satellite glial cells in dorsal root ganglia and sympathetic ganglia, and with the enteric glia surrounding the cell bodies of ganglionic neurons of the myenteric and the submucous plexus. In contrast to NTPDase1, NTPDase2 preferentially hydrolyzes nucleoside triphosphates over nucleoside diphosphates and thus can act either in inactivating or in producing P2 receptor ligands. Our results suggest that NTPDase2 plays an important role in the control of nucleotide-mediated activation of peripheral neurons or glia and in the dialogue between these two cell types.