Takahiro Sonomura

A Morphological Analysis of Thalamocortical Axon Fibers of Rat Posterior Thalamic Nuclei: A Single Neuron Tracing Study with Viral Vectors

The rostral sector of the posterior thalamic nuclei (POm) is, together with the ventral posterior nuclei (VP), involved in somatosensory information processing in rodents. The POm receives inputs from the spinal cord and trigeminal nuclei and projects to the primary somatosensory (S1) cortex and other cortical areas. Although thalamocortical axons of single VP neurons are well known to innervate layer (L) 4 of the S1 cortex with distinct columnar organization, those of POm neurons have not been elucidated yet. In the present study, we investigated complete axonal and dendritic arborizations of single POm neurons in rats by visualizing the processes with Sindbis viruses expressing membrane-targeted fluorescent protein. When we divided the POm into anterior and posterior parts according to calbindin immunoreactivity, dendrites of posterior POm neurons were wider but less numerous than those of anterior neurons. More interestingly, axon fibers of anterior POm neurons were preferentially distributed in L5 of the S1 cortex, whereas those of posterior neurons were principally spread in L1 with wider and sparser arborization than those of anterior neurons. These results suggest that the POm is functionally segregated into anterior and posterior parts and that the 2 parts may play different roles in somatosensory information processing.

Effectable application of vascular endothelial growth factor to critical sized rat calvaria defects.

OBJECTIVE An early vascular response for angiogenesis is essential for the normal progression of bone defect healing. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis. The aim of this study was to evaluate the effects of a poly (L,D-lactic-co-glycolic acid) (PLGA) membrane with VEGF encapsulated into PLGA microspheres on bone regeneration at bone defects in rat calvaria. STUDY DESIGN Microspheres of PLGA incorporating VEGF(165) (VEGF microspheres) were prepared, and critical-size bone defects were created in rat calvaria. The VEGF microspheres, PLGA microspheres, or VEGF microspheres plus PLGA membrane were applied to the defects. Bone regeneration was evaluated using image analysis based on soft radiographic and histologic examination. RESULTS Mature thick bone regeneration was observed in selected sites at bone defects that had been applied with VEGF microspheres/PLGA membrane compared with those that had been applied with the other treatments. CONCLUSION A combination of VEGF microspheres and a PLGA membrane effectively enhances bone regeneration.

Parvalbumin-producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites.

To examine inputs to parvalbumin (PV)‐producing interneurons, we generated transgenic mice expressing somatodendritic membrane‐targeted green fluorescent protein specifically in the interneurons, and completely visualized their dendrites and somata. Using immunolabeling for vesicular glutamate transporter (VGluT)1, VGluT2, and vesicular GABA transporter, we found that VGluT1‐positive terminals made contacts 4‐ and 3.1‐fold more frequently with PV‐producing interneurons than VGluT2‐positive and GABAergic terminals, respectively, in the primary somatosensory cortex. Even in layer 4, where VGluT2‐positive terminals were most densely distributed, VGluT1‐positive inputs to PV‐producing interneurons were 2.4‐fold more frequent than VGluT2‐positive inputs. Furthermore, although GABAergic inputs to PV‐producing interneurons were as numerous as VGluT2‐positive inputs in most cortical layers, GABAergic inputs clearly preferred the proximal dendrites and somata of the interneurons, indicating that the sites of GABAergic inputs were more optimized than those of VGluT2‐positive inputs. Simulation analysis with a PV‐producing interneuron model compatible with the present morphological data revealed a plausible reason for this observation, by showing that GABAergic and glutamatergic postsynaptic potentials evoked by inputs to distal dendrites were attenuated to 60 and 87%, respectively, of those evoked by somatic inputs. As VGluT1‐positive and VGluT2‐positive axon terminals were presumed to be cortical and thalamic glutamatergic inputs, respectively, cortical excitatory inputs to PV‐producing interneurons outnumbered the thalamic excitatory and intrinsic inhibitory inputs more than two‐fold in any cortical layer. Although thalamic inputs are known to evoke about two‐fold larger unitary excitatory postsynaptic potentials than cortical ones, the present results suggest that cortical inputs control PV‐producing interneurons at least as strongly as thalamic inputs.

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.

Local Connections of Excitatory Neurons to Corticothalamic Neurons in the Rat Barrel Cortex

Corticothalamic projection neurons in the cerebral cortex constitute an important component of the thalamocortical reciprocal circuit, an essential input/output organization for cortical information processing. However, the spatial organization of local excitatory connections to corticothalamic neurons is only partially understood. In the present study, we first developed an adenovirus vector expressing somatodendritic membrane-targeted green fluorescent protein. After injection of the adenovirus vector into the ventrobasal thalamic complex, a band of layer (L) 6 corticothalamic neurons in the rat barrel cortex were retrogradely labeled. In addition to their cell bodies, fine dendritic spines of corticothalamic neurons were well visualized without the labeling of their axon collaterals or thalamocortical axons. In cortical slices containing retrogradely labeled L6 corticothalamic neurons, we intracellularly stained single pyramidal/spiny neurons of L2–6. We examined the spatial distribution of contact sites between the local axon collaterals of each pyramidal neuron and the dendrites of corticothalamic neurons. We found that corticothalamic neurons received strong and focused connections from L4 neurons just above them, and that the most numerous nearby and distant sources of local excitatory connections to corticothalamic neurons were corticothalamic neurons themselves and L6 putative corticocortical neurons, respectively. These results suggest that L4 neurons may serve as an important source of local excitatory inputs in shaping the cortical modulation of thalamic activity.

Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

Development of deciduous and permanent dentitions in the upper jaw of the house shrew (Suncus murinus)

The diphyodont tooth replacement in mammals is characterized by a single replacement of a deciduous dentition by a permanent dentition. Despite its significance in mammalian biology and paleontology, little is known about the developmental mechanisms regulating the diphyodont replacement. Because the mouse never replaces its teeth, this study used the house shrew, Suncus murinus, as a model to investigate the control of the diphyodont replacement of a deciduous dentition by successions and additions of permanent teeth. Using morphological and gene expression analyses of serial sections, we have demonstrated the development of the upper dentition of the house shrew. In this species, the deciduous tooth germs are formed but soon become vestigial, whereas the successional and accessional (molar) germs are subsequently formed and developed. There are distinct Shh expression domains in the deciduous, successional, and accessional tooth germs, and those of the latter two germs are identified from the appearance of their primary enamel knots. The developmental sequence of tooth germs in the house shrew indicates that two adjacent primary enamel knots of the successional and accessional germs do not develop simultaneously, but with a constant time lag. We suggest that this mode of tooth succession and accession can be explained by a sequential inhibitory cascade model in which the timing of initiation and the spacing of tooth development are determined by the inhibition from the primary enamel knots of developmentally preceding adjacent tooth germs.

Expression of D1 but not D2 dopamine receptors in striatal neurons producing neurokinin B in rats

Neostriatal projection neurons are known to be largely divided into two groups, striatoentopeduncular/striatonigral and striatopallidal neurons, which mainly express D1 and D2 dopamine receptors, respectively. Recently, a small population of neostriatal neurons have been reported to produce neurokinin B (NKB), and send their axons mainly to the basal forebrain regions. To reveal which type of dopamine receptors were expressed by these NKB‐producing neurons, we examined rat striatal neurons by combining immunofluorescence labeling for preprotachykinin B (PPTB), the precursor of NKB, and fluorescence in situ hybridization labeling for dopamine receptors. Fluorescent signals for D1 receptor mRNA were detected in 85–89% of PPTB‐immunopositive neurons in the neostriatum, accumbens nucleus and lateral stripe of the striatum, whereas almost no signal for D2 receptor was observed in PPTB‐positive striatal neurons. To further reveal intracellular signaling downstream of D1 receptor in PPTB‐producing neurons, we used a double immunofluorescence labeling method to study the localization of some substrates for protein kinase A (PKA), which was known to be activated by D1 receptor. Although only 3–7% of PPTB‐immunopositive striatal neurons displayed immunoreactivity for dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa, a well‐known PKA substrate expressed in the two major groups of neostriatal projection neurons, 60–64% of PPTB‐positive striatal neurons exhibited immunoreactivity for striatal‐enriched tyrosine phosphatase. These results suggest that NKB‐producing neostriatal neurons are similar to striatoentopeduncular/striatonigral neurons in the usage of dopamine receptor subtypes, but different from the two major groups of neostriatal projection neurons in terms of the downstream signaling of dopamine receptors.

Topographical Representation of Motoneurons Innervating the Transverse Mandibular Muscle in the Trigeminal Motor Nucleus, with Special Reference to Rats

Localization of motoneurons of the transverse mandibular muscle (TM) was examined by a retrograde tracing method (horseradish peroxidase (HRP) and wheat germ agglutinin-HRP (WGA-HRP) ) in the rat (rodent), house musk shrew (Suncus murinus, insectivore) and miniature pig (even-toed, ungulate). In the injection of HRP solution into the TM of the rat, labeled neurons with HRP appeared at the lateral and ventral margins of the dorsolateral division in the rostral two-thirds level of the trigeminal motor nucleus. This distribution area was close to or overlapped that of medial pterygoid and lateral pterygoid motoneurons. TM motoneurons of the shrew and pig were located in the ventromedial part of the dorsolateral division at the rostral half level of the nucleus. The distribution patterns of each masticatory muscle motoneuron in the rat were also confirmed for comparison with those of TM motoneurons. Electromyography during feeding in the rat showed that TM activity almost synchronized with the activities of both jaw-closer and jaw-opener muscles.

Excitatory and inhibitory inputs to PV-positive cortical neurons: A quantitative analysis with BAC transgenic mice

We have developed a novel experimental system for introduction of genetically encoded tools by an adenovirus-mediated gene transfer technique. Here we tested the validity of the system by analyzing the expression pattern of introduced fluorescent proteins. We found that fluorescent cells are pyramidal cells in the cerebral cortex (NeuN-positive, GABA-negative, and GFAP-negative) and Purkinje cells in the cerebellum (IP3R1-positive). Interestingly, the expression pattern in the cortex showed a specific pattern depending on the time when the adenovirus-injection was performed: the injection at embryonic day (E) 12.5 led to the preferential expression in cerebral layer 5/6 neurons (∼90% of expressing neurons), whereas the injection at E14.5 led to the preferential expression in layer 2/3 neurons (∼70% of expressing neurons). Our novel experimental system enables us to introduce genetically encoded tools in specific subtypes of neurons and thus would be a promising way to perform optical recording/manipulation of neural activities in vivo.