Fumino Fujiyama

Single Nigrostriatal Dopaminergic Neurons Form Widely Spread and Highly Dense Axonal Arborizations in the Neostriatum

The axonal arbors of single nigrostriatal dopaminergic neurons were visualized with a viral vector expressing membrane-targeted green fluorescent protein in rat brain. All eight reconstructed tyrosine hydroxylase-positive dopaminergic neurons possessed widely spread and highly dense axonal arborizations in the neostriatum. All of them emitted very little axon collateral arborization outside of the striatum except for tiny arborization in the external pallidum. The striatal axonal bush of each reconstructed dopaminergic neuron covered 0.45–5.7% (mean ± SD = 2.7 ± 1.5%) of the total volume of the neostriatum. Furthermore, all the dopaminergic neurons innervated both striosome and matrix compartments of the neostriatum, although each neurons arborization tended to favor one of these compartments. Our findings demonstrate that individual dopaminergic neurons of the substantia nigra can broadcast a dopamine signal and exert strong influence over a large number of striatal neurons. This divergent signaling should be a key to the function of the nigrostriatal system in dopamine-based learning and suggests that neurodegeneration of individual nigral neurons can affect multiple neurons in the striatum. Thus, these results would also contribute to understanding the clinicopathology of Parkinsons disease and related syndromes.

Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain

Vesicular glutamate transporter 1 (VGluT1) is one of the best markers for glutamatergic neurons, because it accumulates transmitter glutamate into synaptic vesicles. Differentiation‐associated Na+‐dependent inorganic phosphate cotransporter (DNPI) shows 82% amino acid identity to VGluT1, and is another candidate for vesicular glutamate transporters. Here, we report the immunocytochemical localization of DNPI and compare it with that of VGluT1 in the adult rat brain. Both DNPI and VGluT1 immunoreactivities were found mostly in neuropil, presumably in axon terminals, throughout the brain. In the telencephalic regions, intense DNPI immunoreactivity was observed in the glomeruli of the olfactory bulb, layer IV of the neocortex, granular layer of the dentate gyrus, presubiculum, and postsubiculum. In contrast, VGluT1 immunoreactivity was intense in the olfactory tubercle, layers I‐III of the neocortex, piriform cortex, entorhinal cortex, hippocampus, dentate gyrus, and subiculum. In the thalamic nuclei, DNPI‐immunoreactive terminal‐like profiles were much larger than VGluT1‐immunoreactive ones, suggesting that DNPI immunoreactivity was subcortical in origin. DNPI immunoreactivity was much more intense than VGluT1 immunoreactivity in many brainstem and spinal cord regions, except the pontine nuclei, interpeduncular nucleus, cochlear nuclei, and external cuneate nucleus. In the molecular layer of the cerebellar cortex, climbing‐like fibers showed intense DNPI immunoreactivity, whereas neuropil contained dense VGluT1‐immnoreactive deposits. Both DNPI and VGluT1 immunoreactivities were observed as mossy fiber terminal‐like profiles in the cerebellar granular layer. DNPI and VGluT1 immunoreactivities appeared associated with synaptic vesicles in the axon terminals forming asymmetric synapses in several regions examined electron microscopically. The present results indicate that DNPI and VGluT1 are used by different neural components in most, if not all, brain regions, suggesting the complementary functions of DNPI and VGluT1. J. Comp. Neurol. 444:39–62, 2002.

Complementary distribution of vesicular glutamate transporters in the central nervous system.

Two vesicular glutamate transporters (VGluTs) have been identified at the molecular level very recently and revealed to possess similar pharmacological characteristics for glutamate uptake. Vesicular glutamate transporter 1 (VGluT1), which was originally named brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), is mainly expressed in telencephalic regions, whereas vesicular glutamate transporter 2 (VGluT2), formerly referred to as differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), is produced principally in diencephalic and lower brainstem regions. Since no other proteins show as high molecular similarity to VGluT1 or VGluT2 as the two transporters exhibit, it is likely that the mammalian central nervous system use only two gene products for vesicular glutamate uptake. Immunoelectron-microscopic analysis has revealed that the two VGluTs are located on synaptic vesicles in axon terminals making an asymmetric type of synapses, supporting that they serve as vesicular transporters in excitatory terminals. Furthermore, mRNA and immunoreactivity for VGluTs are distributed largely in a complementary fashion to distinct populations of excitatory neurons; for example, in the cerebral cortex, thalamocortical axon terminals use VGluT2, whereas excitatory axon terminals of corticocortical or intracortical fibers seem to apply VGluT1 for glutamate uptake. This complementary distribution might suggest that the two VGluTs have an as yet unknown difference in functions.

Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex

Brain‐specific Na+‐dependent inorganic phosphate cotransporter (BNPI) was recently reported to serve as a vesicular glutamate transporter (VGluT), and was renamed VGluT1 (Bellocchio et al. [ 2000] Science 289:957–960; Takamori et al. [2000] Nature 407:189–194). Ahead of these reports, cDNA encoding another brain‐specific inorganic phosphate transporter, which showed 82% amino acid identity to VGluT1, was cloned and designated differentiation‐associated Na+‐dependent inorganic phosphate cotransporter (DNPI; Aihara et al. [2000] J Neurochem 74:2622–2625). In the present study, we produced a specific antibody against a C‐terminal portion of DNPI, and studied the immunohistochemical localization of DNPI in the rat cerebral cortex in comparison with that of VGluT1. DNPI immunoreactivity was enriched in neuropil of layers I and IV and to a lesser extent in the upper portion of layer VI of the cerebral neocortex, whereas VGluT1 immunoreactivity was distributed more evenly in neuropil of the neocortex. Electron microscopic observation revealed that both DNPI and VGluT1 immunoreactivities were mainly located on synaptic vesicles in nerve terminals which made asymmetrical contacts in the neocortex. Furthermore, neither DNPI nor VGluT1 immunoreactivity in the neocortex was colocalized with gamma aminobutyric acid (GABA)ergic axon terminal markers, immunoreactivity for glutamic acid decarboxylase or vesicular GABA transporter. Neuronal depletion in the ventrobasal thalamic nuclei produced by the kainic acid injection resulted in a clear reduction of DNPI immunoreactivity in layers I, IV, and VI of the somatosensory cortex. These results indicate that DNPI is located on the membrane of synaptic vesicles in thalamocortical axon terminals, and that it may be a candidate for VGluT of thalamocortical glutamatergic neurons. J. Comp. Neurol. 435:379–387, 2001.

Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions

Sympathetic premotor neurons directly control sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column (IML) of the thoracic spinal cord, and many of these premotor neurons are localized in the medulla oblongata. The rostral ventrolateral medulla contains premotor neurons controlling the cardiovascular conditions, whereas rostral medullary raphe regions are a candidate source of sympathetic premotor neurons for thermoregulatory functions. Here, we show that these medullary raphe regions contain putative glutamatergic neurons and that these neurons directly control thermoregulatory SPNs. Neurons expressing vesicular glutamate transporter 3 (VGLUT3) were distributed in the rat medullary raphe regions, including the raphe magnus and rostral raphe pallidus nuclei, and mostly lacked serotonin immunoreactivity. These VGLUT3-positive neurons expressed Fos in response to cold exposure or to central administration of prostaglandin E2, a pyrogenic mediator. Transneuronal retrograde labeling after inoculation of pseudorabies virus into the interscapular brown adipose tissue (BAT) or the tail indicated that those VGLUT3-expressing medullary raphe neurons innervated these thermoregulatory effector organs multisynaptically through SPNs of specific thoracic segments, and microinjection of glutamate into the IML of the BAT-controlling segments produced BAT thermogenesis. An anterograde tracing study further showed a direct projection of those VGLUT3-expressing medullary raphe neurons to the dendrites of SPNs. Furthermore, intra-IML application of glutamate receptor antagonists blocked BAT thermogenesis triggered by disinhibition of the medullary raphe regions. The present results suggest that VGLUT3-expressing neurons in the medullary raphe regions constitute excitatory neurons that could be categorized as a novel group of sympathetic premotor neurons for thermoregulatory functions, including fever.

Exclusive and common targets of neostriatofugal projections of rat striosome neurons: a single neuron-tracing study using a viral vector

The rat neostriatum has a mosaic organization composed of striosome/patch compartments embedded in a more extensive matrix compartment, which are distinguished from each other by the input–output organization as well as by the expression of many molecular markers. The matrix compartment gives rise to the dual γ‐aminobutyric acid (GABA)ergic striatofugal systems, i.e. direct and indirect pathway neurons, whereas the striosome compartment is considered to involve direct pathway neurons alone. Although the whole axonal arborization of matrix striatofugal neurons has been examined in vivo by intracellular staining, that of striosome neurons has never been studied at the single neuron level. In the present study, the axonal arborizations of single striosome projection neurons in rat neostriatum were visualized in their entirety using a viral vector expressing membrane‐targeted green fluorescent protein, and compared with that of matrix projection neurons. We found that not only matrix but also striosome compartments contained direct and indirect pathway neurons. Furthermore, only striatonigral neurons in the striosome compartment projected directly to the substantia nigra pars compacta (SNc), although they sent a substantial number of axon collaterals to the globus pallidus, entopeduncular nucleus and/or substantia nigra pars reticulata. These results suggest that striosome neurons play a more important role in the formation of reward‐related signals of SNc dopaminergic neurons than do matrix neurons. Together with data from previous studies in the reinforcement learning theory, our results suggest that these direct and indirect striosome–SNc pathways together with nigrostriatal dopaminergic neurons may help striosome neurons to acquire the state‐value function.

Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex

The chemical organization of excitatory axon terminals in the rat cerebellar cortex was examined by immunocytochemistry and in situ hybridization histochemistry of vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2). Chemical depletion of the inferior olivary complex neurons by 3-acetylpyridine treatment almost completely removed VGluT2 immunoreactivity from the molecular layer, leaving VGluT1 immunoreactivity apparently intact. On the other hand, neuronal deprivation of the cerebellar cortex by kainic acid injection induced a large loss of VGluT1 immunoreactivity in the molecular layer. In the cerebellar granular layer, both VGluT1 and VGluT2 immunoreactivities were found in mossy fiber terminals, and the two immunoreactivities were mostly colocalized in single-axon terminals. Signals for mRNA encoding VGluT2 were found in the inferior olivary complex, and those for VGluT1 and VGluT2 mRNAs were observed in most brainstem precerebellar nuclei sending mossy fibers, such as the pontine, pontine tegmental reticular, lateral reticular and external cuneate nuclei. These results indicate that climbing and parallel fibers selectively use VGluT2 and VGluT1, respectively, whereas mossy fibers apply both VGluT1 and VGluT2 together to accumulate glutamate into synaptic vesicles. Since climbing-fiber and parallel-fiber terminals are known to make depressing and facilitating synapses, respectively, VGluT1 and VGluT2 might have distinct properties associated with those synaptic characteristics. Thus, it would be the next interesting issue to determine whether mossy-fiber terminals co-expressing VGluT1 and VGluT2 show synaptic facilitation or depression.

Vesicular Glutamate Transporter 2 Is Required for Central Respiratory Rhythm Generation But Not for Locomotor Central Pattern Generation

Glutamatergic excitatory neurotransmission is dependent on glutamate release from presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6–8, which function as vesicular glutamate transporters (VGLUTs). Here, we show that VGLUT2 (Slc17a6) is required for life ex utero. Vglut2 null mutant mice die immediately after birth because of the absence of respiratory behavior. Investigations at embryonic stages revealed that neural circuits in the location of the pre-Bötzinger (PBC) inspiratory rhythm generator failed to become active. However, neurons with bursting pacemaker properties and anatomical integrity of the PBC area were preserved. Vesicles at asymmetric synapses were fewer and malformed in the Vglut2 null mutant hindbrain, probably causing the complete disruption of AMPA/kainate receptor-mediated synaptic activity in mutant PBC cells. The functional deficit results from an inability of PBC neurons to achieve synchronous activation. In contrast to respiratory rhythm generation, the locomotor central pattern generator of Vglut2 null mutant mice displayed normal rhythmic and coordinated activity, suggesting differences in their operating principles. Hence, the present study identifies VGLUT2-mediated signaling as an obligatory component of the developing respiratory rhythm generator.

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex

γ‐Aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67‐green fluorescent protein (GFP) knock‐in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP‐positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long‐range projections by tracing GFP‐labeled GABAergic neurons with axon branches traveled rostro‐caudally and medio‐laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.

Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain

Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single‐ and double‐immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2‐loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal‐like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate‐putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2‐loaded glutamatergic axons maturate earlier than VGluT1‐laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood. J. Comp. Neurol. 492:263–288, 2005.