Yoshinori Koshimizu

Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents

Expression of vesicular glutamate transporters (VGLUTs: VGLUT1, VGLUT2 and VGLUT3) in muscle spindle afferents was examined in rats. VGLUT1 immunoreactivity was detected in the sensory endings on the equatorial and juxta-equatarial regions of intrafusal fibers as well as in many axon terminals within lamina IX of the spinal cord. VGLUT1 might be expressed not only in the central axon terminals but also in the peripheral sensory endings of muscle-spindle afferents.

Production and histological application of affinity-purified antibodies to heat-denatured green fluorescent protein.

Enhanced green fluorescent protein (GFP) irreversibly loses not only fluorescence but also antigenicity recognized with conventional anti-GFP antibodies by heat denaturation. This hinders combinatory applications of the GFP immunodetection technique with heat-requiring procedures, such as in situ hybridization histochemistry, antigen retrieval, and Western blot. Here we produced new rabbit and guinea pig antibodies against heat-denatured GFP. The polyclonal antibodies affinity-purified with the antigen column detected a single band corresponding to the molecular size of GFP in Western blot analysis, with mouse brain expressing GFP from the GAD67 locus. By immunofluorescence labeling, the new antibodies detected GFP molecules in heat (≥70C)-treated sections but not in untreated sections of the mouse brain. When the sections were incubated at ≥37C with in situ hybridization buffer containing 50% formamide, a denaturing reagent, the sections lost immunoreactivity with the conventional anti-GFP antibodies but acquired immunoreactivity with the new antibodies to heat-denatured GFP. Finally, GFP immunofluorescence was successfully visualized with the new antibodies in sections of the GFP-expressing mice labeled by fluorescence in situ hybridization histochemistry against GAD67 mRNA. Thus, the antibodies produced in this study may provide an opportunity to combine GFP immunodetection with procedures requiring heat treatment. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Singular localization of sodium channel β4 subunit in unmyelinated fibres and its role in the striatum

Voltage-gated Na(+) channel β-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. β-subunits including β4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse β4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report β4 dimer expression in the mouse brain, with high levels of β4 dimers in the striatal projection fascicles, suggesting a specific role of β4 in those fibres. Scn4b-deficient mice show a resurgent Na(+) current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of β4 in the striatum.

Paucity of enkephalin production in neostriatal striosomal neurons: analysis with preproenkephalin-green fluorescent protein transgenic mice.

Whether or not the striosome compartment of the neostriatum contained preproenkephalin (PPE)‐expressing neurons remained unresolved. To address this question by developing a sensitive detection method, we generated transgenic mice expressing enhanced green fluorescent protein (GFP) under the specific transcriptional control of the PPE gene. Eight transgenic lines were established, and three of them showed GFP expression which was distributed in agreement with the reported localization of PPE mRNA in the central nervous system. Furthermore, in the matrix compartment of the neostriatum of the three lines, intense GFP immunoreactivity was densely distributed in the neuronal cell bodies and neuropil, and matrix neurons displayed > 94% co‐localization for GFP and PPE immunoreactivities. In sharp contrast, GFP immunoreactivity was very weak in the striosome compartment, which was characterized by intense immunoreactivity for mu‐opioid receptors (MOR). Although neostriatal neurons were divided into GFP‐immunopositive and ‐negative groups in both the striosome and matrix compartments, GFP immunoreactivity of cell bodies was much weaker (∼1/5) in GFP‐positive striosomal neurons than in GFP‐positive matrix neurons. A similar reciprocal organization of PPE and MOR expression was also suggested in the ventral striatum, because GFP immunoreactivity was weaker in intensely MOR‐immunopositive regions than in the surrounding MOR‐negative regions. As PPE‐derived peptides are endogenous ligands for MOR in the neostriatum and few axon collaterals of matrix neurons enter the striosome compartment, the present results raised the question of the target of those peptides produced abundantly by matrix neurons.

Neurochemical properties of enkephalinergic neurons in lumbar spinal dorsal horn revealed by preproenkephalin-green fluorescent protein transgenic mice.

J. Neurochem. (2010) 113, 1555–1564.

Quantitative analysis of axon bouton distribution of subthalamic nucleus neurons in the rat by single neuron visualization with a viral vector

The subthalamic nucleus (STN) of the basal ganglia plays a key role in motor control, and STN efferents are known to mainly target the external segment of the globus pallidus (GPe), entopeduncular nucleus (Ep), and substantia nigra (SN) with some axon collaterals to the other regions. However, it remains to be clarified how each STN neuron projects axon fibers and collaterals to those target nuclei of the STN. Here we visualized the whole axonal arborization of single STN neurons in the rat brain by using a viral vector expressing membrane‐targeted green fluorescent protein, and examined the distribution of axon boutons in those target nuclei. The vast majority (8–9) of 10 reconstructed STN neurons projected to the GPe, SN, caudate‐putamen (CPu), and Ep, which received, on average ± SD, 457 ± 425, 400 ± 347, 126 ± 143, and 106 ± 100 axon boutons per STN neuron, respectively. Furthermore, the density of axon boutons in the GPe was highest among these nuclei. Although these target nuclei were divided into calbindin‐rich and ‐poor portions, STN projection showed no exclusive preference for those portions. Since STN neurons mainly projected not only to the GPe, SN, and Ep but also to the CPu, the subthalamostriatal projection might serve as a positive feedback path for the striato‐GPe‐subthalamic disinhibitory pathway, or work as another route of cortical inputs to the striatum through the corticosubthalamostriatal disynaptic excitatory pathway. J. Comp. Neurol. 521:2125–2146, 2013.

Single subthalamic neurons target the striatum in rat: Complete visualization with a viral vector

P2-h09 Selective removal of the “hyperdirect” corticosubthalamic pathway of the primate basal ganglia by immnotoxin-mediated neuronal ablation in combination with the use of a modified lentiviral vector with enhanced retrograde transfer Ken-ichi Inoue 1 , Daisuke Koketsu 2, Shigeki Kato 3, Kazuto Kobayashi 3, Atsushi Nambu 2, Masahiko Takada 1 1 Systems Neurosci., Primate Res. Inst., Kyoto Univ., Inuyama, Japan 2 Div. System Neurophysiol., NIPS, Okazaki, Japan 3 Dept. Mol. Genetics, Fukushima Med. Univ., Fukushima, Japan