Masao Horie

Pax-6 is required for thalamocortical pathway formation in fetal rats.

Pax‐6, a transcription regulatory factor, has been demonstrated to play important roles in eye, nose, and brain development by analyzing mice, rats, and humans with a Pax‐6 gene mutation. We examined the role of Pax‐6 with special attention to the formation of efferent and afferent pathways of the cerebral cortex by using the rat Small eye (rSey2), which has a mutation in the Pax‐6 gene. In rSey2/rSey2 fetuses, cortical efferent axons develop with normal trajectory, at least within the cortical anlage, when examined with immunohistochemistry of the neuronal cell adhesion molecule TAG‐1 and 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate (DiI) labeling from the cortical surface. A remarkable disorder was found in the trajectory of dorsal thalamic axons by immunostaining of the neurofilament and the neural cell adhesion molecule L1 and DiI labeling from the dorsal thalamus. In normal rat fetuses, dorsal thalamic axons curved laterally in the ventral thalamus without invading a Pax‐6‐immunoreactive cell cluster in the ventral part of the ventral thalamus. These axons then coursed up to the cortical anlage, passing just dorsal to another Pax‐6‐immunoreactive cell cluster in the amygdaloid region. In contrast, in rSey2/rSey2 fetuses, dorsal thalamic axons extended downward to converge in the ventrolateral corner of the ventral thalamus and fanned out in the amygdaloid region without reaching the cortical anlage. These results suggest that Pax‐6‐expressing cell clusters along the thalamocortical pathway (ventral part of the ventral thalamus and amygdala) are responsible for the determination of the axonal pathfinding of the thalamocortical pathway. J. Comp. Neurol. 408:147–160, 1999.

Phosphacan and neurocan are repulsive substrata for adhesion and neurite extension of adult rat dorsal root ganglion neurons in vitro.

Phosphacan (PC) and neurocan (NC) are major chondroitin sulfate proteoglycans (CS-PGs) in nervous tissue and are involved in the modulation of cell adhesion and neurite outgrowth during neural development and regeneration. In the present study, we examined the effects of PC and NC on the attachment and neurite extension of adult rat dorsal root ganglion (DRG) neurons in vitro. Treatment with PC and NC on poly-L-lysine (PL) significantly impaired both neuronal attachment and neurite extension in a concentration-dependent manner (10 microg/ml > 1 microg/ml >> 0.1 microg/ml), and they were partially suppressed by chondroitinase ABC (ChABC) digestion. The CS-PGs applied to culture medium (1 microg/ml) also displayed inhibitory effects on neurite extension, which were not altered by ChABC treatment. These results show that PC and NC are repulsive substrata for adhesion and neurite regeneration of adult DRG neurons in vitro and suggest that both chondroitin sulfate moieties and core proteins are responsible for the inhibitory actions of the CS-PGs. We also conducted immunohistochemical analyses with the monoclonal antibodies to core proteins of PC (mAb 6B4) and NC (mAb 1G2), which revealed that only a few neurons in the DRG section were stained with these antibodies. In contrast, most DRG neurons at different stages (12 h, 1 day, 2 days, and 4 days) in culture were immunoreactive to mAb 6B4 and mAb 1G2. Taking these findings together, it is plausible that both CS-PGs expressed in the cultured neurons may play a role in the modulation of attachment, survival, and neurite regeneration.

Aberrant trajectory of ascending dopaminergic pathway in mice lacking Nkx2.1.

In the embryonic brain, the transcription factor Nkx2.1 is localized in the medial ganglionic eminence and the ventromedial part of the hypothalamus. In the present study, we examined the development of mesencephalic dopamine (DA) neuron system in mice lacking Nkx2.1. In normal mice, tyrosine hydroxylase-immunoreactive axons from mesencephalic DA cells extended bilaterally in the lateral hypothalamus at embryonic day 12.5 (E12.5) and project to the ipsilateral striatum by E14.5. In the mutant brain, mesencephalic DA cell groups appeared to develop normally, but the majority of their ascending axons were observed to cross the ventral midline of the caudal hypothalamus and project to the contralateral striatum. DiI, a fluorescent dye, placed in the ventrolateral mesencephalon of E14.5 mutant mice, further revealed that majority of DiI-labeled axons projected to the contralateral striatum, while a minor ipsilateral projection was also observed. In the ventromedial hypothalamus of mutants, the neuroepithelium of third ventricle was missing, and immunoreactivity of semaphorin 3A, a soluble type of axon repellent, which was normally localized in the neuroepithelium, was remarkably reduced. Together with the recent evidence that the expression of slit2, another axon-repellent diffusible factor, is also eliminated in the hypothalamic neuroepithelium of Nkx2.1-deficient mice, the abnormal crossing of ascending DA axons observed may be attributed to the elimination of these chemorepulsive signals in the medial part of the mutant hypothalamus.

Synthesis, localization and externalization of galectin-1 in mature dorsal root ganglion neurons and Schwann cells

We recently confirmed that oxidized galectin‐1 is a novel factor enhancing axonal growth in peripheral nerves after axotomy, but the process of extracellular release and oxidization of endogenous galectin‐1 in the injured nervous tissue remains unknown. In the present study, we examined the distribution of galectin‐1 in adult rat dorsal root ganglia (DRG) in vivo and in vitro. By RT‐PCR analysis and in situ hybridization histochemistry, galectin‐1 mRNA was detected in both DRG neurons and non‐neuronal cells. Immunohistochemical analyses revealed that galectin‐1 was distributed diffusely throughout the cytoplasm in smaller diameter neurons and Schwann cells in DRG sections. In contrast, the immunoreactivity for galectin‐1 was detected in almost all DRG neurons from an early stage in culture (3 h after seeding) and was restricted to the surface and/or extracellular region of neurons and Schwann cells at later stages in culture. In a manner similar to the primary cultured cells, we also observed the surface and extracellular expression of this molecule in immortalized adult mouse Schwann cells (IMS32). Western blot analysis has revealed that both reduced and oxidized forms of galectin‐1 were detected in culture media of DRG neurons and IMS32. These findings suggest that galectin‐1 is externalized from DRG neurons and Schwann cells upon axonal injury. Some of the molecules in the extracellular milieu may be converted to the oxidized form, which lacks lectin activity but could act on neural tissue as a cytokine.

Expression and immunohistochemical localization of heparan sulphate proteoglycan N‐syndecan in the migratory pathway from the rat olfactory placode

N‐syndecan, a membrane‐bound heparan sulphate proteoglycan, is abundantly present in the developing nervous system and thought to play important roles in the neurite outgrowth. In the present study, we examined the distribution of N‐syndecan in the migratory route from the rat olfactory placode using immunohistochemistry and in situ hybridization. At embryonic day 15, both heparan sulphate and N‐syndecan immunoreactivities were localized in and around the migrating cell clusters, which contained luteinizing hormone‐releasing hormone (LHRH) and calbindin D‐28k. Immunoreactivity for other glycosaminoglycan chains, such as chondroitin and keratan sulphate, and core proteins of the chondroitin sulphate proteoglycan, neurocan and phosphacan, were barely detected in the migratory pathway from the olfactory placode. By in situ hybridization histochemistry, N‐syndecan mRNA was localized in virtually all of migrating neurons as well as in cells of the olfactory epithelium and the vomeronasal organ. N‐syndecan immunoreactivity surrounded cells migrating along the vomeronasal nerves that were immunoreactive for neural cell adhesion molecules, NCAM, L1 and TAG‐1. Considering that NCAM is implicated in the migratory process of LHRH neurons and specifically binds to heparan sulphate, it is likely that a heterophilic interaction between NCAM and N‐syndecan participates in the neuronal migration from the rat olfactory placode.

Delineation of a frequency-organized region isolated from the mouse primary auditory cortex

The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice.

Auditory cortical areas activated by slow frequency-modulated sounds in mice

Species-specific vocalizations in mice have frequency-modulated (FM) components slower than the lower limit of FM direction selectivity in the core region of the mouse auditory cortex. To identify cortical areas selective to slow frequency modulation, we investigated tonal responses in the mouse auditory cortex using transcranial flavoprotein fluorescence imaging. For differentiating responses to frequency modulation from those to stimuli at constant frequencies, we focused on transient fluorescence changes after direction reversal of temporally repeated and superimposed FM sweeps. We found that the ultrasonic field (UF) in the belt cortical region selectively responded to the direction reversal. The dorsoposterior field (DP) also responded weakly to the reversal. Regarding the responses in UF, no apparent tonotopic map was found, and the right UF responses were significantly larger in amplitude than the left UF responses. The half-max latency in responses to FM sweeps was shorter in UF compared with that in the primary auditory cortex (A1) or anterior auditory field (AAF). Tracer injection experiments in the functionally identified UF and DP confirmed that these two areas receive afferent inputs from the dorsal part of the medial geniculate nucleus (MG). Calcium imaging of UF neurons stained with fura-2 were performed using a two-photon microscope, and the presence of UF neurons that were selective to both direction and direction reversal of slow frequency modulation was demonstrated. These results strongly suggest a role for UF, and possibly DP, as cortical areas specialized for processing slow frequency modulation in mice.

Dual compartments of the ventral division of the medial geniculate body projecting to the core region of the auditory cortex in C57BL/6 mice

We investigated precise projection patterns from the ventral division of the medial geniculate body (MGv) projecting to the core region of the auditory cortex in C57BL/6 mice. The core region in mice comprises two different tonotopically organized areas, the anterior auditory field (AAF) and the primary auditory cortex (AI). In the present study, AAF and AI were functionally identified using flavoprotein fluorescence imaging. Biotinylated dextran amine (BDA) was injected iontophoretically into the tonotopic bands to 5kHz and 20kHz in AAF, and those to 5kHz, 10kHz, and 20kHz in AI for staining MGv neurons projecting to the injected sites. MGv neurons projecting to AAF were found in the medial part of MGv, while MGv neurons projecting to AI were found in the lateral part. In the medial part of MGv, areas projecting to 5-20kHz bands in AAF were aligned along the medio lateral axis. In the lateral part of MGv, areas projecting to 5-20kHz bands in AI were aligned along the dorso ventral axis. These results indicate that AAF and AI receive auditory information via two different MGv compartments with independent tonotopic axes, respectively.

Quantitative map of multiple auditory cortical regions with a stereotaxic fine-scale atlas of the mouse brain

Optical imaging studies have recently revealed the presence of multiple auditory cortical regions in the mouse brain. We have previously demonstrated, using flavoprotein fluorescence imaging, at least six regions in the mouse auditory cortex, including the anterior auditory field (AAF), primary auditory cortex (AI), the secondary auditory field (AII), dorsoanterior field (DA), dorsomedial field (DM), and dorsoposterior field (DP). While multiple regions in the visual cortex and somatosensory cortex have been annotated and consolidated in recent brain atlases, the multiple auditory cortical regions have not yet been presented from a coronal view. In the current study, we obtained regional coordinates of the six auditory cortical regions of the C57BL/6 mouse brain and illustrated these regions on template coronal brain slices. These results should reinforce the existing mouse brain atlases and support future studies in the auditory cortex.

Disruption of actin-binding domain-containing Dystonin protein causes dystonia musculorum in mice

The Dystonin gene (Dst) is responsible for dystonia musculorum (dt), an inherited mouse model of hereditary neuropathy accompanied by progressive motor symptoms such as dystonia and cerebellar ataxia. Dst‐a isoforms, which contain actin‐binding domains, are predominantly expressed in the nervous system. Although sensory neuron degeneration in the peripheral nervous system during the early postnatal stage is a well‐recognised phenotype in dt, the histological characteristics and neuronal circuits in the central nervous system responsible for motor symptoms remain unclear. To analyse the causative neuronal networks and roles of Dst isoforms, we generated novel multipurpose Dst gene trap mice, in which actin‐binding domain‐containing isoforms are disrupted. Homozygous mice showed typical dt phenotypes with sensory degeneration and progressive motor symptoms. The gene trap allele (DstGt) encodes a mutant Dystonin‐LacZ fusion protein, which is detectable by X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactoside) staining. We observed wide expression of the actin‐binding domain‐containing Dystonin isoforms in the central nervous system (CNS) and peripheral nervous system. This raised the possibility that not only secondary neuronal defects in the CNS subsequent to peripheral sensory degeneration but also cell‐autonomous defects in the CNS contribute to the motor symptoms. Expression analysis of immediate early genes revealed decreased neuronal activity in the cerebellar‐thalamo‐striatal pathway in the homozygous brain, implying the involvement of this pathway in the dt phenotype. These novel DstGt mice showed that a loss‐of‐function mutation in the actin‐binding domain‐containing Dystonin isoforms led to typical dt phenotypes. Furthermore, this novel multipurpose DstGt allele offers a unique tool for analysing the causative neuronal networks involved in the dt phenotype.