Kynurenic acid (KYNA), a metabolite of the kynurenine pathway of tryptophan degradation, is a neuroinhibitory agent present in the mammalian brain. Endogenous KYNA preferentially affects the alpha7 nicotinic acetylcholine (alpha7nACh) receptor and, possibly, the glycine co-agonist (glycineB) site of the NMDA receptor. Functionally relevant fluctuations in brain KYNA occur under both physiological and pathological conditions, affecting cholinergic and glutamatergic neurotransmission. Kynurenine aminotransferase II (KAT II), the major biosynthetic enzyme of KYNA in the rat brain, catalyzes the irreversible formation of KYNA from its immediate bioprecursor, kynurenine. We now purified rat kidney KAT II to homogeneity, generated a polyclonal rabbit anti-rat KAT II antibody, and purified the antibody using routine biochemical methods. The antibody selectively recognized KAT II by Western blot analysis and in immunotitration experiments. Used for immunocytochemistry, the antibody revealed discrete, specific staining of KAT II-positive astrocyte-like cells throughout the adult rat brain. The presence of KAT II in astrocytes was confirmed by double fluorescence immunostaining with an antibody against the astrocyte-specific marker glial fibrillary acidic protein (GFAP). No specific labeling was detected in neurons or microglia. However, KAT II-positive astrocytes were intimately associated with select neuron populations, supporting a neuromodulatory role of KYNA. Intense staining was frequently seen around brain capillaries, with astrocytic end feet contacting the capillary wall. This may explain the rapid access of blood-derived kynurenine to KAT II-containing astrocytes. The new anti-KAT II antibody should be useful in the further elucidation of the presumed role of KYNA in brain physiology and pathology.

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain gamma-aminobutyric acid (GABA)-ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti-GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA-immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA-negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid-contacting (CSF-c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15-30-mm-long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF-c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF-c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF-c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early-appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non-CSF-c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999] J. Comp. Neurol. 414:391-422) is discussed in view of these results and information obtained with ancillary markers.

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

The renin–angiotensin system (RAS) was initially considered as a circulating humoral system controlling blood pressure, being kidney the key control organ. In addition to the ‘classical' humoral RAS, a second level in RAS, local or tissular RAS, has been identified in a variety of tissues, in which local RAS play a key role in degenerative and aging-related diseases. The local brain RAS plays a major role in brain function and neurodegeneration. It is normally assumed that the effects are mediated by the cell-surface-specific G-protein-coupled angiotensin type 1 and 2 receptors (AT1 and AT2). A combination of in vivo (rats, wild-type mice and knockout mice) and in vitro (primary mesencephalic cultures, dopaminergic neuron cell line cultures) experimental approaches (confocal microscopy, electron microscopy, laser capture microdissection, transfection of fluorescent-tagged receptors, treatments with fluorescent angiotensin, western blot, polymerase chain reaction, HPLC, mitochondrial respirometry and other functional assays) were used in the present study. We report the discovery of AT1 and AT2 receptors in brain mitochondria, particularly mitochondria of dopaminergic neurons. Activation of AT1 receptors in mitochondria regulates superoxide production, via Nox4, and increases respiration. Mitochondrial AT2 receptors are much more abundant and increase after treatment of cells with oxidative stress inducers, and produce, via nitric oxide, a decrease in mitochondrial respiration. Mitochondria from the nigral region of aged rats displayed altered expression of AT1 and AT2 receptors. AT2-mediated regulation of mitochondrial respiration represents an unrecognized primary line of defence against oxidative stress, which may be particularly important in neurons with increased levels of oxidative stress such as dopaminergic neurons. Altered expression of AT1 and AT2 receptors with aging may induce mitochondrial dysfunction, the main risk factor for neurodegeneration.

The ‘classical’ renin–angiotensin system (RAS) is a circulating system that controls blood pressure. Local/paracrine RAS, identified in a variety of tissues, including the brain, is involved in different functions and diseases, and RAS blockers are commonly used in clinical practice. A third type of RAS (intracellular/intracrine RAS) has been observed in some types of cells, including neurons. However, its role is still unknown. The present results indicate that in brain cells the intracellular RAS counteracts the intracellular superoxide/H2O2 and oxidative stress induced by the extracellular/paracrine angiotensin II acting on plasma membrane receptors. Activation of nuclear receptors by intracellular or internalized angiotensin triggers a number of mechanisms that protect the cell, such as an increase in the levels of protective angiotensin type 2 receptors, intracellular angiotensin, PGC-1α and IGF-1/SIRT1. Interestingly, this protective mechanism is altered in isolated nuclei from brains of aged animals. The present results indicate that at least in the brain, AT1 receptor blockers acting only on the extracellular or paracrine RAS may offer better protection of cells.