Kousuke Taki

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.

In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins

A new recombinant virus which labeled the infected neurons in a Golgi stainlike fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (pal-GFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.

Preprodynorphin-, preproenkephalin-, and preprotachykinin-expressing neurons in the rat neostriatum: An analysis by immunocytochemistry and retrograde tracing

Specific antibodies were produced against C‐terminal portions of rat preprodynorphin (PPD), preproenkephalin (PPE), and preprotachykinin A (PPT). PPD, PPE, and PPT C‐terminal immunoreactivity was observed in many cell bodies of medium‐sized neurons in the rat neostriatum (caudate‐putamen). Intense PPE immunoreactivity was found in neuropil of the globus pallidus, whereas intense to moderate PPD and PPT immunoreactivity was distributed in neuropil of the substantia nigra and the entopeduncular nucleus. A double‐immunofluorescence analysis revealed that PPE‐immunoreactive neostriatal neurons rarely showed immunoreactivity for PPD (<1%) or PPT (<2%). In contrast, more than 95% of PPD‐immunoreactive neostriatal neurons showed PPT immunoreactivity, and vice versa. No PPD‐, PPE‐, or PPT‐immunoreactive neostriatal neurons showed immunoreactivity for the markers of neostriatal intrinsic neurons, such as calretinin, choline acetyltransferase, parvalbumin, or somatostatin. When tetramethylrhodamine‐dextran amine (TMR‐DA) was injected into the substantia nigra, almost all neurons that were labeled retrogradely with TMR‐DA showed immunoreactivity for PPD (98%) or PPT (99%), but very few of them exhibited PPE immunoreactivity (1%). After injection of TMR‐DA into the globus pallidus, 86%, 17%, and 10% of the retrogradely labeled neurons showed immunoreactivity for PPE, PPD, and PPT, respectively. These results support the notion that the neostriatal projection neurons are divided into at least two groups: The projection neurons of one group contain enkephalins and send projection fibers almost exclusively to the globus pallidus, and the others contain tachykinins and dynorphins/Leu‐enkephalin and send projection fibers mainly to the substantia nigra. J. Comp. Neurol. 386:229–244, 1997.

Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1‐ and VGluT2‐immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1‐immunolabeled terminals were smaller than VGluT2‐labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra‐ and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons. J. Comp. Neurol. 465: 234–249, 2003.

Immunohistochemical detection of L-DOPA-derived dopamine within serotonergic fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats.

On the basis of our previous studies in the normal rat [Arai, R., Karasawa, N., Geffard, M., Nagatsu, I., 1995. L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study. Neurosci. Lett. 195, 195-198; Arai, R., Karasawa, N., Nagatsu, I., 1996a. Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study. Brain Res. 706, 177-179; Arai, R., Karasawa, N., Nagatsu, I., 1996b. Dopamine produced from L-DOPA is degraded by endogenous monoamine oxidase in neurons of the dorsal raphe nucleus of the rat: an immunohistochemical study. Brain Res. 722, 181-184] we have assumed that exogenously administered L-dihydroxyphenylalanine (L-DOPA) is converted into dopamine (DA) in serotonergic (5-HT) fibers within the striatum (ST) and the substantia nigra pars reticulata (SNR). In the present study, an attempt was made to confirm the assumptions in Parkinsonian rats, which were produced by unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC). The rats exhibiting more than 150 total controversial circles were regarded as satisfactory models of Parkinson disease (PD). Using a dual immunofluorescence histochemistry, we examined DA-immunoreactivity in the 5-HT fibers within the ST and the SNR of the PD model rats after L-DOPA was injected intraperitoneally. In experimental cases with the L-DOPA administration, DA-immunoreactivity was detected in 5-HT fibers in both the ST and the SNR on the 6-OHDA injection side; no DA-immunoreactivity was found in 5-HT fibers in the ST or the SNR in control cases without the L-DOPA administration. The results support the assumption that exogenously administered L-DOPA may be converted into DA within the 5-HT fibers in the ST and SNR of the PD model rats.

A group of cortical interneurons expressing μ-opioid receptor-like immunoreactivity : A double immunofluorescence study in the rat cerebral cortex

mu-Opioid receptor-expressing neurons in the rat cerebral neocortex were characterized by an immunolabeling method with an antibody to a carboxyl terminal portion of the receptor. They were small, bipolar, vertically elongated, non-pyramidal neurons, and scattered mainly in layers II-IV. We examined chemical characteristics of mu-opioid receptor-expressing neocortical neurons by the double immunofluorescence method. Almost all neuronal cell bodies expressing mu-opioid receptor-like immunoreactivity showed immunoreactivity for GABA, suggesting that they were cortical inhibitory interneurons. mu-Opioid receptor-immunoreactive neurons were further studied by the double staining method with markers for the subgroups of cortical GABAergic neurons. Immunoreactivities for vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin were found in 92, 79, 67, 35 and 35% of mu-opioid receptor-immunoreactive cortical neurons, respectively. In contrast, less than 10% of mu-opioid receptor-immunoreactive neurons showed immunoreactivity for parvalbumin, calbindin, somatostatin, neuropeptide Y or nitric oxide synthase. Moreover, mu-opioid receptor-immunoreactive neurons very frequently exhibited preproenkephalin immunoreactivity, but not preprodynorphin immunoreactivity. The present results indicate that mu-opioid receptor-expressing neurons belong to a distinct subgroup of neocortical GABAergic neurons, because vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin have often been reported to coexist with one another in single neocortical neurons. Methionine-enkephalin, which is a major product of the preproenkephalin gene, is known to be one of the most potent endogenous ligands for mu-opioid receptor. Thus, the expression of mu-opioid receptor in preproenkephalin-producing neurons suggested that mu-opioid receptor serves as an autoreceptor for the subpopulation of GABAergic interneurons at a single-neuron or population level.

High Range Resolution Ultrasonographic Vascular Imaging Using Frequency Domain Interferometry With the Capon Method

For high range resolution ultrasonographic vascular imaging, we apply frequency domain interferometry with the Capon method to a single frame of in-phase and quadrature (IQ) data acquired using a commercial ultrasonographic device with a 7.5 MHz linear array probe. In order to tailor the adaptive beamforming algorithm for ultrasonography we employ four techniques: frequency averaging, whitening, radio-frequency data oversampling, and the moving average. The proposed method had a range resolution of 0.05 mm in an ideal condition, and experimentally detected the boundary couple 0.17 mm apart, where the boundary couple was indistinguishable from a single boundary utilizing a B-mode image. Further, this algorithm could depict a swine femoral artery with a range beam width of 0.054 mm and an estimation error for the vessel wall thickness of 0.009 mm, whereas using a conventional method the range beam width and estimation error were 0.182 and 0.021 mm, respectively. The proposed method requires 7.7 s on a mobile PC with a single CPU for a 1 × 3 cm region of interest. These findings indicate the potential of the proposed method for the improvement of range resolution in ultrasonography without deterioration in temporal resolution, resulting in enhanced detection of vessel stenosis.

High range resolution medical acoustic vascular imaging with frequency domain interferometry

For high range resolution acoustic vascular imaging we apply frequency domain interferometry and Capon method to a few frames of in-phase and quadrature (IQ) data acquired by a commercial ultrasonographic device. To suit the adaptive beamforming algorithm to medical acoustic imaging we employ three techniques; frequency averaging, whitening, and pseudo-double RF data conversion. The proposed method detected two couples of boundaries 0.26 and 0.19 mm apart using a single frame and two frames of IQ data, respectively, where each couple of boundaries is indistinguishable from a single boundary utilizing B-mode images. Further this algorithm could depict a swine femoral artery with higher range resolution than conventional B-mode imaging. These results indicate the potential of the proposed method for the range resolution improvement in ultrasonography, originating the progress in detection of vessel stenosis.

Adaptive-beamformer with accurate intensity-estimation technique for high-range-resolution vascular ultrasound imaging

For the early detection and for the treatment assessment of cardiovascular disease, improvement in range resolution of vascular ultrasound imaging is highly desirable. The employment of frequency domain interferometry with the Capon method largely improves the range resolution of ultrasound imaging; however, this methodology often underestimates the echo intensity. In this paper, we propose a compensation method to improve the estimation accuracy in echo intensity acquired by the high-range-resolution ultrasound imaging method based on frequency domain interferometry. The method requires 0.18 s/frame using a single CPU PC. The proposed method has the spatial resolution of 0.05 mm with the average estimation intensity error of 0.15 dB on the log scale, where we employ an ultrasound pulse of 7.5 MHz center frequency. We have applied the proposed method to a swine femoral artery in vitro and a human carotid artery in vivo, and verified its efficiency to improve the accuracy in echo intensity estimation.

High range resolution ultrasound imaging of a human carotid artery using frequency domain interferometry

In this study, we propose a high range resolution ultrasound imaging method using frequency domain interferometry with adaptive beamforming technique. We employ multiple reference signals to compensate the variation of the vessel wall slope. The proposed method succeeded to acquire a high range resolution carotid artery image of a normal living human subject, where the method was applied to a single frame IQ data obtained by a commercial ultrasonographic device. This result verified the capability of the proposed method to improve the range resolution in ultrasonography. We believe the proposed method will largely progress medical diagnostics in vascular ultrasound.