Classification of nAChRß2-immunoreactive retinal ganglion cells and their tectal projections in chicks.

The relationship between the type of retinal ganglion cell (RGC) and the retinoreceptive layer of the tectum is investigated by the immunostaining of RGCs with nicotinic acetylcholine receptorß2 (nAChRß2) antibody and intracellular staining by DiI and also by anterograde degeneration and biotinylated dextran amine labeling of retinotectal fibers in chicks. The results strongly suggest that many of the RGCs that express immunoreactivity to nAChRß2 send axons to tectal layer 7 and are mainly classified into the simple-type of Groups II and III, which contain the cells providing middle-sized to large dendritic fields with simple dendritic arborization. These nAChRß2-immunoreactive RGCs receive visual information via the multiple sublayers of the inner plexiform layer.

Morphological properties of chick retinal ganglion cells in relation to their central projections.

Morphological properties of chick retinal ganglion cells (RGCs) were studied in relation to their central projections in 23 chicks. A total of 217 RGCs were retrogradely labeled by applying a carbocyanine dye (DiI) to the thalamus and optic tectum. The labeled RGCs were classified into six groups on the basis of their somal areas, dendritic fields, and branching patterns. The dendrites of these RGCs extended horizontally in the inner plexiform layer (IPL) forming eight dendritic strata. The RGCs in each group showed certain specificities in their central projections. Group Ic predominantly projected to the tectum. Groups IIs and IIIs showed a high thalamic dominance. Groups Is and IIc were nonspecific with regard to their tectal and thalamic projections. Group IVc showed tectal-specific projections. Occurrence rates of the dendritic strata increased progressively toward the inner part of the IPL, i.e., DSs (dendritic strata) 1-4 were scantily distributed, DSs 5 and 6 were moderately distributed, and DSs 7 and 8 were the most frequently distributed. A total of 42 dendritic stratification patterns were identified, and of these, 18 patterns were common to the tectal RGCs (tec-RGCs) and thalamic RGCs (tha-RGCs). The common patterns were detected very frequently in the tec- and tha-RGCs (approximately 85%), and the dendritic strata were largely distributed in the inner part of the IPL (DSs 5-8). In contrast, the remaining 24 noncommon stratification patterns showed low occurrence rates (approximately 15%); however, these dendritic strata were widely distributed in both the outer (DSs 1-4) and inner (DSs 5-8) IPL.

Organization of intrinsic connections of the retrosplenial cortex in the rat.

The retrosplenial cortex consists of areas 29a-d, each of which has different connections with other cortical and subcortical regions. Although these areas also make complex interconnections that constitute part of a neural circuit subserving various functions, such as spatial memory and navigation, the details of such interconnections have not been studied comprehensively. In the study reported here, we investigated the organization of associational and commissural connections of areas 29a-d within the retrosplenial cortex in the rat, using the retrograde tracer cholera toxin B subunit and anterograde tracer biotinylated dextran amine. The results demonstrated that each of these areas has a distinct set of interconnections within the retrosplenial cortex. Each area interconnects strongly along the transverse axis of the retrosplenial cortex: area 29a, area 29b, caudal area 29c, and caudal area 29d connect with each other, and rostral area 29c and rostral area 29d connect with each other. In the longitudinal direction, rostral-to-caudal projections from rostral areas 29c and 29d to areas 29a and 29b and caudal areas 29c and 29d are strong, whereas reciprocal caudal-to-rostral projections are relatively weak. Although most of the intrinsic connections are homotopical, contralateral connections are weaker and less extensive than ipsilateral connections. These findings suggest that each retrosplenial area may not only process specific information somewhat independently but that it may also integrate and transmit such information through intrinsic connections to other areas in order to achieve retrosplenial cortical functions, such as spatial memory and learning.

Organization of anterior cingulate and frontal cortical projections to the retrosplenial cortex in the rat.

The retrosplenial cortex (areas 29a-d), which plays an important role in spatial memory and navigation, is known to provide massive projections to frontal association and motor cortices, which are also essential for spatial behavior. The reciprocal projections originating from these frontal cortices to areas 29a-d, however, have been analyzed to only a limited extent. Here, we report an analysis of the anatomical organization of projections from anterior cingulate area 24 and motor and prefrontal cortices to areas 29a-d in the rat, using the axonal transport of cholera toxin B subunit and biotinylated dextran amine. Area 29a receives projections from rostral area 24a, area 24b, the ventral orbital area, and the caudal secondary motor area. Rostral area 29b receives projections from caudal area 24a, whereas caudal area 29b receives projections from rostral area 24a. Area 29b also receives projections from area 24b and the ventral orbital area. Areas 29c and 29d receive projections from areas 24a and 24b and the secondary motor area in a topographic manner such that the rostrocaudal axis of areas 29c and 29d corresponds to the caudorostral axis of areas 24a and 24b and the secondary motor area. Rostral areas 29c and 29d also receive projections from the caudal primary motor area, and area 29d receives projections from the ventral, lateral, and medial orbital areas. These differential frontal cortical projections to each area of the retrosplenial cortex suggest that each area may contribute to different aspects of retrosplenial cortical function such as spatial memory and behavior.

Role of sympathetic nerves on early embryonic development and immune modulation of uterus in pregnant mice.

To determine the role of sympathetic nerves in the early embryonic development and the immune modulation of maternal uterus during pregnancy, a model of chemical sympathectomy in mice was established by intraperitoneal injection of 6-hydroxydopamine (6-OHDA). The embryonic development and the distribution of maternal uterine immunocytes were investigated during early pregnancy (E1-E9) with methods of histology, immunohistochemistry and ELISA. Our data showed that in the 6-OHDA-treated group, the number of implanted embryos was only 64.4% of that in the control group at E7, and the development of uterine glands and vessels was poor in pregnant mice. In addition, in uterine tissues of 6-OHDA-treated mice, the number of CD8+ T cells increased ten-fold and the concentration of IL-2 increased 3.6-fold at E5. However, no obvious changes to the number of CD4+ T cells and IL-4 were observed. Thus, the CD4+/CD8+ T cells ratio significantly decreased, while the IL-2/IL-4 ratio significantly increased. These findings indicated that the activation of sympathetic nerves might be favorable to fetal survival and development during early pregnancy through influencing on immune function and decidua formation of uterus.

Dimensional differences among the groups of retinal ganglion cells according to the retinal zones in chicks.

The populations of retinal ganglion cell (RGC) groups (Groups I, II, III, IV) were similar each other between the central and intermediate zones, but the population in the peripheral zone were clearly different from those in the central and intermediate zones due to increase of Group III and IV cells and decrease of Group I cells. The dimensions of somal area and dendritic field of Group I cells increased very gradually toward the peripheral zone, but those of other three Groups grew steeply in the peripheral zone. The correlation index between somal area and dendritic field of RGCs showed high coefficient in the central (r=0.73) and intermediate (r=0.77) zones, but lowered clearly in the peripheral zone (r=0.64) due to increase of Group III cells, which showed nonlinear relation between somal area and dendritic field.

Organization of anterior cingulate and frontal cortical projections to the anterior and laterodorsal thalamic nuclei in the rat.

The anterior and laterodorsal thalamic nuclei provide massive projections to the anterior cingulate and frontal cortices in the rat. However, the organization of reciprocal corticothalamic projections has not yet been studied comprehensively. In the present study, we clarified the organization of anterior cingulate and frontal cortical projections to the anterior and laterodorsal thalamic nuclei, using retrograde and anterograde axonal transport methods. The anteromedial nucleus (AM) receives mainly ipsilateral projections from the prelimbic and medial orbital cortices and bilateral projections from the anterior cingulate and secondary motor cortices. The projections from the anterior cingulate cortex are organized such that the rostrocaudal axis of the AM corresponds to the rostrocaudal axis of the cortex, whereas those from the secondary motor cortex are organized such that the rostrocaudal axis of the AM corresponds to the caudorostral axis of the cortex. The ventromedial part of the anteroventral nucleus receives ipsilateral projections from the anterior cingulate cortex and bilateral projections from the secondary motor cortex, in a topographic manner similar to the projections to the AM. The ventromedial part of the laterodorsal nucleus (LD) receives ipsilateral projections from the anterior cingulate and secondary motor cortices. The projections are roughly organized such that more dorsal and ventral regions within the ventromedial LD receive projections preferentially from the anterior cingulate cortex. The difference in anterior cingulate and frontal cortical projections to the anterior and laterodorsal nuclei may suggest that each thalamic nucleus plays a different functional role in spatial memory processing.

Morphological characteristics of tectal neurons of layer I projecting to the nucleus geniculatus lateralis ventralis in chick.

Some visual information is sent to the nucleus geniculatus lateralis ventralis (GLv) via the cells in layer I (I cells) of the tectum in birds and is used for color vision, papillary reflex, and kineoptic functions. To reveal the morphological features of 'I cells' projecting to the GLv, they were retrogradely labeled with DiI (1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate) in chicks. Two different types of neurons, 'spear dendritic I cells' and 'forked dendritic I cells' were identified. The former had small spindle-like soma and an apical dendrite extending to the tectal surface, and the latter had somewhat larger triangular or polygonal soma and plural ascending dendrites. Most of the labeled dendritic endings bifurcated horizontally in layer F, and showed the ending patterns similar to the terminals of optic nerve fibers.

Organization of retrosplenial cortical projections to the anterior cingulate, motor, and prefrontal cortices in the rat.

The retrosplenial cortex (areas 29a-29d) has been implicated in spatial memory, which is essential for performing spatial behavior. Despite this link with behavior, neural connections between areas 29a-29d and frontal association and motor cortices--areas also essential for spatial behavior--have been analyzed only to a limited extent. Here, we report an analysis of the anatomical organization of projections from areas 29a-29d to area 24 and motor and prefrontal cortices in the rat, using the axonal transport of biotinylated dextran amine (BDA) and cholera toxin B subunit (CTb). Area 29a projects to rostral area 24a, whereas area 29b projects to caudodorsal area 24a and ventral area 24b. Caudal area 29c projects to mid-rostrocaudal area 24b, whereas rostral area 29c projects to caudal areas 24a and 24b and caudal parts of primary and secondary motor areas. Caudal area 29d projects to mid-rostrocaudal areas 24a and 24b, whereas rostral area 29d projects to the caudalmost parts of areas 24a and 24b and the secondary motor area and to the mid-rostrocaudal part of the primary motor area. Area 29d also projects weakly to the prefrontal cortex. These differential corticocortical projections may constitute important pathways that transmit spatial information to particular frontal cortical regions, enabling an animal to accomplish spatial behavior.

Morphologic analysis and classification of ganglion cells of the chick retina by intracellular injection of Lucifer Yellow and retrograde labeling with DiI.

Retinal ganglion cells (RGCs) of chicks were labeled by using the techniques of intracellular filling with Lucifer Yellow and retrograde axonal labeling with carbocyanine dye (DiI). Labeled RGCs were morphologically analyzed and classified into four major groups: Group I cells (57.1%) with a small somal area (77.5 microm(2) on average) and narrow dendritic field (17,160 microm(2) on average), Group II cells (28%) with a middle-sized somal area (186 microm(2)) and middle-sized dendritic field (48,800 microm(2)), Group III cells (9.9%) with a middle-sized somal area (203 microm(2)) and wide dendritic field (114,000 microm(2)), and Group IV cells (5%) with a large somal area (399 microm(2)) and wide dendritic field (117,000 microm(2)). Of the four groups, Groups I and II were further subdivided into two types, simple and complex, on the basis of dendritic arborization: Groups Is, Ic, and Groups IIs, IIc. However, Group III and IV showed either a simple or complex type, Group IIIs and Group IVc, respectively. The density of branching points of dendrites was approximately 10 times higher in the complex types (18,350, 6,190, and 3,520 points/mm(2) in Group Ic, IIc, and IVc, respectively) than in the simple types (1,890, 640, and 480 points/mm(2) in Group Is, IIs, and IIIs). The branching density of Group I cells was extremely high in the central zone. The chick inner plexiform layer was divided into eight sublayers by dendritic strata of RGCs and 26 stratification patterns were discriminated. The central and peripheral retinal zones were characterized by branching density of dendrites and composition of RGC groups, respectively.

Morphological features of chick retinal ganglion cells.

On average, in chicks, the total number of retinal ganglion cells is 4.9 x 10(6) and the cell density is 10400 cells/mm2. Two high-density areas, namely the central area (CA) and the dorsal area (DA), are located in the central and dorsal retinas, respectively, in post-hatching day 8 (P8) chicks (19000 cells/mm2 in the CA; 12800 cells/mm2 in the DA). Thirty percent of total cells in the ganglion cell layer are resistant to axotomy of the optic nerve. The distribution of the axotomy resistant cells shows two high-density areas in the central and dorsal retinas, corresponding to the CA (5800 cells/mm2) and DA (3200 cells/mm2). The number of presumptive ganglion cells in P8 chicks is estimated to be 4 x 10(6) (8600 cells/mm2 on average) and the density is 13500 and 10200 cells/mm2 in the CA and DA, respectively, and 4300 cell/mm2 in the temporal periphery (TP). The somal area of presumptive ganglion cells is small in the CA and DA (mean (+/- SD) 35.7 +/- 9.1 and 40.0 +/- 11.3 microm2, respectively) and their size increases towards the periphery (63.4 +/- 29.7 microm2 in the TP), accompanied by a decrease in cell density. Chick ganglion cells are classified according to dendritic field, somal size and branching density of the dendrites as follows: group Ic, Is, IIc, IIs, Ills, IVc. The density of branching points of dendrites is approximately 10-fold higher in the complex type (c) than in the simple type (s) in each group. The chick inner plexiform layer is divided into eight sublayers according to the dendritic strata of retinal ganglion cells and 26 stratification patterns are discriminated.

Changes in somal growth and dendritic patterns of the retinal ganglion cells in the chicks and chick embryos.

Changes in the somal growth and dendritic patterns of retinal ganglion cells (RGCs) were studied in early chick embryos and post-hatching chicks by means of the retrograde axonal transport labeling with DiI. Branching patterns of the dendrites were relatively uniform on E8 (embryonic day 8) and became more complicated on E11. Variety of the branching pattern became plainly abundant after E14. On the other hand, somata of RGCs continued to grow until E14, corresponding the appearance of the central-peripheral gradient of the somal size. After E14, RGCs elaborated on the formation of the dendritic patterns as found in chick retina, and simultaneously the growth of somal sizes almost ceased.

Changes in the distribution of labeled retinal ganglion cells after an implant of DiI into the optic nerve in the chick embryos.

Changes in the distribution of retinal ganglion cells (RGCs) were studied using the retrograde labeling of DiI in chicks and chick embryos. The small retinal area filled with labeled RGCs was observed in the retinal fundus on E8. The labeled retinal area expanded radially toward the peripheral retina as the retina grew, and finally occupied a whole retina by P1. The temporal retina was labeled more rapidly than in the nasal retina. The observed-increasing rate of the labeled area was corrected with the growing rate of the retina. Consequently, the corrected-increasing rate of the labeled area was estimated to be about 390% between E8 and E11, and 20-50% after E11. This means that spreading speed of the maturated RGCs lowered until 1/10-1/20 after E11.