Neuroanatomical study on the tecto-suprageniculate-dorsal auditory cortex pathway in the rat.

Previous anatomical and physiological studies suggest that the superior colliculus sends integrated sensory information to the multimodal cortical areas via the thalamic suprageniculate nucleus (SG). However, the detailed distribution of rat tecto-SG axon terminals and SG neurons projecting to the multimodal cortex, as well as synaptic connections between these tectal axons and SG neurons, remains unclear. In this study, the organization of the tecto-thalamo-cortical pathway was investigated via combined injections of anterograde and retrograde tracers followed by light and electron microscopic observations. Injections of a retrograde tracer, cholera toxin B subunit (CTB), into the temporal cortex, area 2, dorsal part (Te2D), and injections of an anterograde tracer, biotinylated dextran amine (BDA), into the deep layers of the superior colliculus produced the following results: (1) Retrogradely CTB-labeled neurons were found throughout SG, predominantly in its rostral part. CTB-labeled neurons were also found in other cortical areas such as the visual cortex, the auditory cortex, the parietal association cortex, and the perirhinal cortex. (2) Anterogradely BDA-labeled axons and their terminals were also observed throughout SG. Dual visualization of BDA and CTB showed that retrogradely labeled SG neurons and anterogradely labeled tectal axon terminal boutons overlapped considerably in the rostral part of SG, and their direct synaptic contacts were also confirmed via electron microscopy. These findings suggest that multimodal information from the superior colliculus can be processed directly in SG neurons projecting to Te2D.

Static, transient and permanent organization of GABA receptor expression in calbindin-positive interneurons in response to amygdala kindled seizures.

We tested the hypothesis that experimentally produced epilepsy (by kindling) may induce changes in GABAA receptor expression in some but not all interneuron populations. Using laser capture microdissection and quantitative polymerase chain reaction (QPCR) analysis, GABAA receptor alpha subunit expression in calbindin- (CBir) and parvalbumin- (Parvir) immunoreactive interneurons was compared between normal brains and brains in which amygdala kindled seizure responses were permanently established. Two weeks after the last seizure response, Cbir neurons in the hilus and/or perirhinal cortex up-regulated the expression of alpha2, alpha3 and alpha5 subunit mRNAs up to 900%. In contrast, no changes were found in Parvir neurons. In Cbir neurons contralateral to the amygdala kindling site alpha1 subunit mRNA expression was increased. In both Cbir and Parvir neurons, the coordinated subunit expression patterns ipsilateral (fully kindled) and contralateral (partially kindled) to the kindling site suggested that permanent and transient co-expressional relationships occur respectively. In the perirhinal cortex alpha2 protein was up-regulated in the processes but not in the cell somas of calbindin-positive neurons, whereas alpha3 subunit protein expression was up-regulated on the cell bodies of Cbir neurons in the hilus. These data indicate that different interneuron populations may selectively reorganize their GABAA subunit expression in response to seizures.

Postnatal development of the retinal projection to the nucleus of the optic tract and accessory optic nuclei in the hooded rat.

Retinal projections to the nucleus of the optic tract (NOT) and accessory optic nuclei (AON) were studied in the postnatal hooded rat after monocular injection of cholera toxin B subunit (CTB) into the vitreous chamber of the eye. At all postnatal ages, retinal axons were labeled sensitively; they revealed dense projections to the contralateral, and sparse but distinct projections to the ipsilateral, NOT and AON. The CTB labeling enabled the first delineation of the complete morphology of developing retinal axons in the ipsilateral NOT and AON. From postnatal day (P) 1 to P3, axons with complex growth cones were seen, and unbranched collaterals with simple growth cones increased and extended gradually. At P6, complex growth cones disappeared while branched collaterals with simple growth cones as well as small-sized varicosities increased. By P12 (two days before eye-opening) the adult-like pattern of terminal arbors appeared. The branched collaterals with tiny, small-sized varicosities present probably represented developing synaptic boutons. At P16 (after eye opening), the pattern of terminal arbors was well developed, almost to the same extent as in the adult. By contrast, a broadly distributed, transient retinal projection around NOT and AON was gradually eliminated; it started to disappear during the first few postnatal days, and was fully retracted by the time of eye-opening time to a pattern normal for the adult.

Metabotropic glutamate receptor 2/3 immunoreactivity in the developing rat cerebellar cortex.

In adult rat cerebellar cortex, the metabotropic glutamate receptors (mGluRs) 2 and 3 (mGluR2/3) are present in somata, dendrites, and terminals of Golgi cells as well as in presumed glial processes (Ohishi et al. [1994], Neuron 13:55-66). In the present study, spatiotemporal changes in immunostaining for mGluR2/3 were examined in postnatal rat cerebellar cortex. mGluR2/3-immunoreactive Golgi cell somata appeared first in the internal granular layer at postnatal day 3 (P3) and were restricted to lobules IX and X; however, by P5, they were present in all lobules. Immunoreactive Golgi cell axons were adult-like, appearing as tortuous fibers with clusters of varicosities. They were observed first in the internal granular layer at P7 and increased in number and complexity with time. It was confirmed that mGluR2/3-immunoreactive Golgi cell axon terminals belong to the synaptic glomerulus by P10. Immunoreactive Golgi cell dendrites extending into the molecular layer became prominent after P15. By that time, the immunostaining pattern was characteristic of Golgi cells, as seen typically in adults. Many intensely immunoreactive radial processes existed at birth (P0). These traversed the molecular and external granular layers, reaching the pial surface in every cerebellar lobule. Because they showed coimmunoreactivity for glial fibrillary acidic protein, they were confirmed to be Bergmann glial fibers. After P9, they began to lose immunoreactivity at the portion corresponding to the molecular layer, while an immunostained granular pattern appeared in that layer. Immunoreactive radial processes, however, remained in the external granular layer, and finally, at P21, they disappeared together along with the external granular layer. Granular staining in the molecular layer reached background levels at this time. These spatiotemporal changes in mGluR2/3 distribution suggested that there may be distinct roles for mGluR2/3 in Golgi cells and Bergmann glial cells during the early postnatal period. mGluR2/3 in Golgi cells might be associated closely with systemic maturation, whereas mGluR2/3 in Bergmann glia might be needed for neuron-glia interactions related to granule cell development.

A light and electron microscopic analysis of the convergent retinal and visual cortical projections to the nucleus of the optic tract (NOT) in the pigmented rat.

It is generally known that the nucleus of the optic tract (NOT) subserves visuomotor relations between the retina and preoculomotor structures as the only subcortical pathway mediating optokinetic responses (OKR) in mammals. We have examined the projections from the retina and visual cortical areas (areas 17, 18a and 18b) to NOT using tracers (wheat germ agglutinin-conjugated horseradish peroxidase, WGA-HRP and cholera toxin B subunit, CTB) in order to clarify how these two different functional inputs to OKR are organized. CTB injection into the vitreous body resulted in anterograde label almost exclusively in the contralateral NOT. Ultrastructually, the size of the retinal axon terminals was small (up to 0.7 micron in diameter), contained round synaptic vesicles and pale mitochondria, and made asymmetrical synaptic contacts with both GABA-positive and GABA-negative NOT neurons. Visual cortical area 17 and the transitional area between area 17 and 18a, or between area 17 and 18b projected their axons to the ipsilateral NOT. Ultrastructually, the size of the cortical axon terminals was small (up to 0.5 micron in diameter), contained round synaptic vesicles, and made asymmetrical synaptic contacts only with GABAnegative NOT neurons. With light and electron microscopical observation, there was a considerable overlap in the cortico-NOT and retino-NOT projection pattern: GABA-negative (presumably NOT projection) neurons simultaneously receive input from both cortical and retinal terminals. From these results, it seems reasonable to postulate that inputs from visual cortical areas in the pigmented rat cooperate with those from the retina in controlling OKR.

The organization of the thalamostriatal projection from the lateral posterior thalamic nuclear complex (LP) in the pigmented rat.

We investigated the thalamostriatal projection of the rat using biotinylated dextran amine (BDA) and wheat-germ agglutinin horseradish peroxidase (WGA-HRP). To obtain the patch/matrix compartments of the striatum (ST), we used mu-opioid receptor (MOR) immunoreaction labeling. Thus, an MOR-positive 'patch' was indicated by a darkly stained spot, while the MOR-negative 'matrix' was displayed as a non-immunoreactive region. A small injection of BDA was made in a subregion of the lateral posterior thalamic nucleus (LP). The LP-ST fibers originated in all subregions of LP and terminated in the dorsocaudal portion of ST, where the corticostriatal fibers from the visual cortex terminate (Serizawa et al. 1994). These LP-ST fibers and terminals were concentrated in the MOR-negative matrix compartment. Electron microscopic observations showed that the LP-ST terminals made asymmetrical synaptic contacts mainly (70%, n = 30) with the dendritic spines of the presumptive ST-output neurons, and fewer (30%) contacted dendritic shafts. The present results provide anatomical support for the contention that ST-output spiny neurons of the matrix that project to the pars reticulata of the substantia nigra or globus pallidus, may be influenced directly by the LP-ST projection.

Normal ontogenic observations on the expression of Eph receptor tyrosine kinase, Cek8, in chick embryos.

The spatio-temporal pattern of expression for the Eph receptor tyrosine kinase, Cek8, was observed in normal chick embryos from H-H stage 6 to 23 by immunohistochemical techniques. Expression of Cek8 was already present in embryos at H-H stage 6, where it was located in the neural plate of the brain region, paraxial mesoderm, and the primitive streak. Regions expressing Cek8 subsequently increased during development to include the neural folds of the brain, rhombomeres 3 and 5, the caudal part of the neural plate, neural crest cells related to the formation of glossopharyngeal nerve ganglia, invaginated cells throughout the primitive groove and the epithelium of the rudiment of the gall bladder. Cek8 was also expressed in the mesenchymal cells of the pharyngeal arches, allantoic stalk and limb buds as well as in the areas surrounding the eye vesicles and nasal pits. Furthermore, cells in the tail bud progressing to the secondary neurulation expressed Cek8. Thus, the spatiotemporal patterns of Cek8 expression appears to have intimate relationships with tissue rebuilding, the maturation of differentiated cells, and the spatial organization of tissues. Consequently, it appears that Cek8 plays an integral role in the developmental events leading to the formation of a wide--though not inclusive--variety of tissues and organ systems.

Cholinergic innervation of the lateralis medialis-suprageniculate nuclear complex (LM-Sg) of the cat's thalamus: a double labeling immunohistochemical study.

The purpose of this study was to investigate morphological characteristics of the synaptic relations of choline acetyltransferase (ChAT)-positive terminals that are made with a variety of post-synaptic profiles in the lateralis medialis-suprageniculate nuclear complex (LM-Sg) using ChAT, gamma-aminobutyric acid (GABA) and glutamate immunohistochemistry in combination with electron microscopical observations. The ChAT immunopositive profiles make asymmetrical synaptic contacts with glutamate immunopositive dendrites that are presumably derived from projection neurons, and/or GABA immunopositive interneurons. The present results indicate that ascending cholinergic mechanisms may be important for modifying information in both the extrinsic and intrinsic circuitries of LM-Sg.

Morphological analysis of the early development of the chick neural tube separated from the floor plate and notochord.

When the neural tube of avian embryos is separated from the notochord and floor plate, motoneurons in the spinal cord fail to develop. In order to investigate the factors involved in this phenomenon, cell proliferation activity and cell death were observed following paramedian incision of the neural tube at the level of the segmental plate using colchicine, BrdU, and TUNEL methods. If the notochord and/or floor plate produces a substance(s) that promotes cell division in the basal plate neuroepithelium or that supports the survival of the motoneuron's neuroblasts, mitotic figures should not be present in the neuroepithelium nor should substantial cell death be observed in the ventral aspect of the notochord- and floor plate-deprived neural tube. Surprisingly, however, neither result was observed in the present experiments, with the exception of a considerable amount of homogeneously distributed cell death. Neuroepithelial cells continued to proliferate and gave rise to neuroblasts. Nevertheless, motoneurons failed to develop, and the neural tube was enveloped by only the basement membrane of the alar plate (S. Hirano and H. Tanaka, 1994, Dev. Growth Differ: 36, 481-488). These morphological results revealed that the cause of the development of the anterior horn lacking a neural tube in the notochord- and neural tube-eliminated embryos is not the elimination of the source of the surviving factor(s) of the motoneuron's neuroblasts, but rather the elimination of the signals to induce the motoneurons, derived from the notochord and/or floor plate. The larger amount of cell death in the neural tube on the experimental side suggests that a nonspecific survival factor(s), necessary for the survival of a variety of types of neuroblasts, is also produced by the notochord and/or floor plate.

Extrinsic and intrinsic connections of the cat's lateral suprasylvian visual area.

The lateral suprasylvian visual area (LS) is known to have numerous interconnections with visual cortical areas as well as with subcortical structures implicated in visually-guided behaviors. In contrast, little data is available regarding connections within the LS itself. In order to obtain information about intra-areal connections and to re-investigate LS connectivity with various cortical and subcortical areas, the traces (biocytin or WGA-HRP) was injected into various loci along the medial and lateral banks of the LS. The anterograde tracer, biocytin injections into both medial and lateral bank produced label contained within the respective bank that extended rostrally and caudally from the infection site. In addition, following medical bank injections, considerable label was distributed throughout the fundus and, to a lesser extent, in the lateral bank. In contrast, no label could be detected in the medial bank after lateral bank injections, and, although label was observed in the fundus, it was restricted to the most lateral aspects. Moderate labeling could be observed in the medial bank following the tracer injection into the most rostral aspect of the lateral bank. It is likely that input derived from various visual cortical areas which project to the medial bank of the LS has access to this intra-areal circuitry. This may provide a route by which visual cortical information can be relayed to other cortical and subcortical structures involved in visually-guided behaviors such as the anterior ectosylvian visual cortex, striatum, and the deep layers of the superior colliculus, despite the fact that these structures themselves do not receive substantial direct projections from the visual cortical areas that are associated with the medial bank. Examination of the laminar location of the cells-of-origin of striate and extrastriate projections to LS using retrograde trace, WGA-HRP, revealed that the supragranular laminae of areas 17, 18 and 19 were the source of LS afferents whereas afferents from the other cortical areas (e.g., 20a, 20b, 21a, 21b, 7 and anterior ectosylvian visual area) were from both supra- and infragranular laminae. In addition, all LS subregions received intra-areal afferent projections from all LS cortical laminae. Thus, although rather clear hierarchical relationship between LS and visual cortical areas appears to exist, the interconnections among LS subregions provide no clear evidence of simple hierarchical relationships between regions LS or may have feed-forward and feed-back pathways.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex.

The distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was immunohistochemically examined in the rat cerebellar cortex at both light and electron microscope levels. An antibody was raised against a fusion protein containing a C-terminal portion of mGluR2. On immunoblot, the antibody reacted with both mGluR2 and mGluR3 in rat brain. mGluR2/3 immunoreactivity was expressed in cell bodies, dendrites, and axon terminals of Golgi cells, as well as in presumed glial processes. Golgi axon terminals with mGluR2/3 immunoreactivity were often encountered in the vicinity of glutamatergic mossy fiber terminals. The results suggest that transmitter glutamate may exert control influences upon Golgi cells not only through dendritic mGluR2/3, but also through axonal mGluR2/3.

Immunohistochemical localization of substance P receptor in the superior colliculus. A light and electron microscope study in the rat.

The superficial layers of the superior colliculus (SC) have been known to contain many axons showing substance P-like immunoreactivity (SP-LI). We, therefore, immunohistochemically examined the distribution of SP receptor (SPR) in the superficial layers of the SC in the rat by using a specific antibody against SPR. The majority of SC neurons with SPR-LI were distributed in the zonal and the superficial gray layers, the rest of them were in the optic layer. Electron microscopy revealed that SPR-immunoreaction products in SC neurons were distributed not only in postsynaptic sites, but also in non-synaptic regions of perikaryal and dendritic profiles.

Role of a metabotropic glutamate receptor in synaptic modulation in the accessory olfactory bulb.

Various functions of glutamate transmission are mediated by both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) consists of at least six different subtypes that are classified into three subgroups, mGluR1/mGluR5, mGluR2/mGluR3, and mGluR4/mGluR6 (refs 1-5), but their physiological roles are largely unknown. Here we report the identification of a very potent agonist for mGluR2/mGluR3, DCG-IV, and the specific localization of mGluR2 in granule cell dendrites that form dendrodendritic synapses with mitral cells in the accessory olfactory bulb. Using the DCG-IV agonist for mGluR2 in combination with slice patch-recording, we demonstrate that the granule cell mGluR2 presynaptically suppresses inhibitory GABA (gamma-aminobutyrate) transmission to the mitral cell. Our results indicate that mGluR2 in granule cells plays an important role in the persistent excitation of olfactory sensory transmission in the accessory olfactory bulb by relieving mitral cells from the GABA inhibition.

Immunocytochemical localization of rat substance P receptor in the striatum.

A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.

Direct projections from the globus pallidus to the midbrain and pons in the cat.

Employing the anterograde and retrograde axonal tracing techniques with Phaseolus vulgaris leucoagglutinin and cholera toxin B subunit, we demonstrated direct projections from the globus pallidus (GP) to the midbrain and pons in the cat. Cells of origin of these projections were localized in the caudal 2/3 of the GP, and their major target sites included the peripeduncular region, nucleus of the brachium of the inferior colliculus, para-lateral lemniscal zone, nucleus sagulum, external and pericentral nuclei of the inferior colliculus, and cuneiform nucleus. A combination of retrograde axonal tracing and immunohistochemistry for choline acetyltransferase revealed that GP neurons giving rise to such descending projections were primarily non-cholinergic.

Substance P-, vasoactive intestinal polypeptide-, and cholecystokinin-like immunoreactive fiber projections from the superior colliculus to the dorsal lateral geniculate nucleus in the rat.

Substance P (SP)-, vasoactive intestinal polypeptide (VIP)-, and cholecystokinin (CCK)-like immunoreactive (LI) neurons were found in the superior colliculus (SC) of the rat, and examined to ascertain whether they sent projection fibers to the dorsal lateral geniculate nucleus (LGNd). Immunocytochemical staining with antisera against SP, VIP, and CCK showed that many immunoreactive neuronal cell bodies were located in the superficial layers of the SC, especially in the stratum griseum superficiale. The pattern of distribution of these immunoreactive neuronal cell bodies in the SC was similar to that of neuronal cell bodies which were retrogradely labeled with WGA-HRP (wheat germ agglutinin-horseradish peroxidase conjugate) injected ipsilaterally into the LGNd. On the other hand, SP-, VIP- and CCK-LI axons were seen most densely in the lateral part of the LGNd, especially in the small-celled LGNd zone adjacent to the optic tract, where anterograde labeling was also observed after injection of WGA-HRP ipsilaterally into the superficial layers of the SC. When a lesion was produced by kainic acid injection into the superficial layers of the SC, axons showing SP-, VIP-, or CCK-LI in the LGNd ipsilateral to the lesion were markedly depleted. The results indicate that SC-LGNd projection neurons contain SP, VIP, and/or CCK in the rat.

Pallidocortical projections to the tempolar polar gyrus in the cat.

Marked projections from the globus pallidus (GP) to the temporal polar gyrus (TPG) of the cat were found by means of the PHA-L (Phaseolus vulgaris leucoagglutinin) and WGA-HRP (horseradish peroxidase conjugated to wheat germ agglutinin) methods. Pallidocortical fibers to the TPG originate mainly from the middle levels of the GP, and terminate all layers of the TPG cortex, especially in layers I, II and III. The GP neurons projecting to the TPG are large-multipolar or small-bipolar neurons. Almost all of these GP neurons show choline acetyltransferase-like immunoreactivity.

Direct projections of non-pyramidal neurons of Ammon's horn to the amygdala and the entorhinal cortex.

When WGA-HRP (wheat germ agglutinin-horseradish peroxidase conjugate) was injected into the amygdala (lateral and basolateral amygdaloid nuclei) or entorhinal cortex of the cat, a number of nonpyramidal neurons in Ammon's horn were retrogradely labeled. The results indicate that some non-pyramidal neurons in Ammon's horn send projection fibers to the amygdala and entorhinal cortex.