Kuehn, M.H., Kim, C.Y., Ostojic, J., Bellin, M., Alward, W.L.M., Stone, E.M., Sakaguchi, D.S., Grozdanic, S.D., Kwon, Y.H. (2006) Local Synthesis and Deposition of Complement Components in the Glaucomatous Retina. (In press: Experimental Eye Research)
Abstract:
Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma.

Ourenik, J., Ourednik, V., Sakaguchi, D.S. and Nilsen-Hamilton, M. (eds.) (2005) Stem Cell Biology: Development and Plasticity. Vol. 1049 of the Annals of the New York Academy of Sciences.

D.S. Sakaguchi, S.J. Van Hoffelen, E. Theusch, E. Parker, J. Orasky, M.M. Harper, A. Benediktsson and M.J. Young. (2004) Transplantation of neural progenitor cells into the developing retina of the Brazilian opossum: An in vivo system for studying neural progenitor cell plasticity. Developmental Neuroscience. 26 (5-6):336-345.
Abstract
In developing cell transplant strategies to repair the diseased or injured retina is essential to consider host-graft interactions and how they may influence the outcome of the transplants. In the present study we evaluated the influence of the host microenvironment upon neural progenitor cells (NPCs) transplanted into the developing and mature retina of the Brazilian opossum, Monodelphis domestica. Monodelphis pups are born in an extremely immature state and the neonatal pups provide a fetal-like environment in which to study the interactions between host tissues and transplanted NPCs. Three different populations of GFP-expressing NPCs were transplanted by intraocular injection in hosts ranging in age from five days postnatal to adult. Extensive survival, differentiation and morphological integration of NPCs was observed within the developing retina. These results suggest that the age of the host environment can strongly influence NPC differentiation and integration.

Klassen, H., Sakaguchi, D.S. and Young, M.J. (2004) Stem Cells and Retinal Repair. Progress in Retinal Eye Research. 23: 149-181.
Abstract
Retinal stem cells (RSCs) are multipotent central nervous system (CNS) precursors that give rise to the retina during the course of development. RSCs are present in the embryonic eyecup of all vertebrate species and remain active in lower vertebrates throughout life. Mammals, however, exhibit little RSC activity in adulthood and thus little capacity for retinal growth or regeneration. Because CNS precursors can now be isolated from immature and mature mammals and expanded ex vivo, it is possible to study these cells in culture as well as following transplantation to the diseased retina. Such experiments have revealed a wealth of unanticipated findings, both in terms of the instructive cues present in the mature mammalian retina as well as the ability of grafted CNS precursors to respond to them. This review examines current knowledge regarding RSCs, together with other CNS precursors, from the perspective of investigators who wish to isolate, propagate, genetically modify, and transplant these cells as a regenerative strategy with application to retinal disease.

Li, M., Babenko, N. and Sakaguchi, D.S. (2004) Inhibition of tyrosine kinase activity disrupts early retinal development. Developmental Biology. 266/1: 209-221.
Abstract
In the present study, we have investigated the role of tyrosine kinase activity during early retinal development in Xenopus laevis. The protein tyrosine kinase (PTK) inhibitors lavendustin A and genistein were used to determine the possible role of tyrosine kinase activity during retinal development in vivo and in vitro. Application of the inhibitors to early embryonic retina disrupted the pattern of lamination in the developing retina. The plexiform layers were severely disorganized or were no longer apparent, and photoreceptor morphogenesis was disrupted. Immunocytochemical analysis verified the presence of focal adhesions in dissociated retinal neuroepithelial cells isolated from St 25 embryos. Application of the PTK inhibitors blocked focal adhesion assembly in these primary cultured cells. To further investigate the regulation of focal adhesions by PTK activity, we examined the effect of lavendustin A on cultured XR1 glial cells. Lavendustin A produced a dose-dependent decrease in the proportion of XR1 cells displaying focal adhesions. Taken together, these results suggest that tyrosine kinase activity is essential for regulating neuroepithelial cell adhesion, migration and morphogenesis during retinal development. Furthermore, the disruption of retinal development may, in part, be due to the inhibition of integrin-mediated signaling.

Jennifer B. Recknor, Justin C. Recknor, Donald S. Sakaguchi, and Surya K. Mallapragada (2004) Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials. 25: 2753-2767.
Abstract
In an effort to develop a permissive environment for neural stem cell differentiation, directional growth of astrocytes has been achieved on polymer substrates in vitro. Manipulating a combination of physical and chemical cues, astrocyte adhesion and alignment in vitro were examined. To provide physical guidance, micropatterned polymer substrates of polystyrene (PS) were fabricated. Laminin was selectively adsorbed onto the grooves of the patterned surface. Rat type-1 astrocytes were seeded onto the micropatterned PS substrates, and the effects of substrate topography and the adsorption of laminin to the PS substrates on the behavior and morphology of the astrocytes were explored. The astrocytes were found to align parallel to the micropatterned grooves
at initial seeding densities of approximately 7500, 13,000, and 20,000 cells/cm2 due to the effects of the physical and chemical guidance mechanisms. Adsorbing laminin in the microgrooves of the micropatterned PS substrates improved cell adhesion and spreading of cytoskeletal ·laments signi·cantly. At these initial seeding densities, over 85% astrocyte alignment in the direction of the grooves was achieved on the micropatterned PS substrates with laminin adsorbed in the grooves. This combination of guidance cues has the potential to provide a permissive substrate for in vivo regeneration within the central nervous system.

Grozdanic SD, Betts DM, Sakaguchi DS, Kwon YH, Kardon RH, Sonea IM. (2003) Temporary elevation of the intraocular pressure by cauterization of vortex and episcleral veins in rats causes functional deficits in the retina and optic nerve. Exp. Eye Res. 2003 Jul;77(1):27-33.
PURPOSE: To evaluate visual function in rats with chronic elevation of intraocular pressure (IOP). METHODS: Chronic ocular hypertension was induced in the left eye of 14 adult Brown Norway rats by cauterizing 3 vortex veins and 2 major episcleral veins; the right eye served as a non-operated control. A control group (n=5) was sham operated on the left eye. Prior to surgery, the IOP was measured with a Tonopen, the pupil light reflex (PLR) evaluated with a custom-made computerized pupillometer and electroretinograms (ERGs) were recorded simultaneously from both eyes post surgically: IOP was measured on weeks 1, 3, 5 and 8 post-operatively, pupil light reflexes on weeks 1, 4 and 8 post-operatively, and ERGs on weeks 4 and 8 post-operatively. Sixty five days postoperatively, rats were euthanized and optic nerves and eye globes were prepared for histological analysis. RESULTS: Seven days after surgery 5/14 rats developed significant elevation of the IOP in operated eyes (control eyes: 25.1+/-0.5mmHg; operated eyes: 34.1+/-0.6mmHg; mean+/-SEM; p=0.0004; Paired t-test). Elevation of the IOP was sustained at 3 (p=0.002) and 5 (p=0.007) weeks postoperatively. However, IOP values did not significantly differ between control and operated eyes 8 weeks postoperatively (p=0.192, Paired t-test). Sham operated animals showed no elevation of the IOP 7 days postoperatively. When the ratio between consensual and direct PLR (PLR(ratio)=consensual/direct PLR; pupil of unoperated eye recorded) was examined in rats which developed elevation of the IOP, preoperative values were 92.2+/-4% (mean+/-SEM), 1 week postoperatively 65+/-4% (significantly different from preoperative values, p<0.05 Repeated Measures ANOVA with Dunnett's Multiple Comparison test, n=5), 4 weeks postoperatively 60.6+/-3.2% (p<0.01, n=5). By 8 weeks postoperatively, pupil responses had essentially recovered 75.4+/-6.9% (p>0.05, n=5). Rats whose IOP values did not rise after surgery and sham operated rats did not develop pupil deficits 4 weeks postoperatively. Rats with elevated IOP displayed a significant decrease in ERG amplitudes in operated eyes at 4 weeks (a-wave(operated)/a-wave(control) (a-wave ratio)=42+/-14% (mean+/-SEM); b-wave(operated)/b-wave(control) (b-wave ratio)=43+/-16%) but not at 8 weeks postoperatively (a-wave ratio=88+/-8.4%; b-wave ratio=82.9+/-9%). Sham operated and rats whose IOP values remained non-elevated after surgery did not develop ERG deficits 4 weeks after surgery. Histological analysis did not reveal any damage in the eyes of animals with elevated intraocular ocular pressure with the exception of one rat, which still had ERG and pupil deficits at the end of experiment. CONCLUSIONS: Development of ERG and PLR deficits are proportional to the elevation of the IOP in the rat model of chronic ocular hypertension. Functional monitoring of the ERG and PLR are useful objective techniques for the detection of retina and optic nerve deficits.

Sakaguchi, D.S., Van Hoffelen, S.J., and Young, M.J. (2003) Differentiation and morphological integration of neural progenitor cells transplanted into the developing mammalian eye. Annals of the New York Academy of Sciences. 995: 127-139.

Transplantation of neural stem/progenitor cells has been proposed as a novel approach for the replacement and repair of damaged CNS tissues. We have evaluated the influence of the host cellular microenvironment upon the survival, differentiation and integration of neural progenitor cells transplanted into the CNS. Using this approach we have investigated the fate of neural progenitor cells in vivo following transplantation into the developing mammalian eye. Murine brain progenitor cells (mBPCs) isolated from neonatal mice expressing the green fluorescent protein (GFP) transgene, were transplanted into the eyes of Brazilian opossums (Monodelphis domestica). Monodelphis pups are born in an extremely immature, fetal-like, state. The eyes of neonatal pups provide a fetal-like environment in which to study cellular interactions between host tissues and transplanted neural progenitor cells. MBPCs were transplanted by intraocular injection into the eye in hosts ranging in age from 5 days postnatal to adult. The transplanted cells were easily identified because of their GFP fluorescence. Extensive survival, differentiation and morphological integration of mBPCs within the host tissue was observed. We found that the younger aged retinas provided a more supportive environment for the morphological integration of the transplanted mBPCs. Cells with morphologies characteristic of specific retinal cell types were observed. Moreover, some transplanted mBPCs were labeled with antibodies characteristic of specific neural/retinal phenotypes. These results suggest that the host environment strongly influences progenitor cell differentiation and that transplantation of neural progenitor cells may be a useful approach aimed at treating degeneration and pathology of the CNS.

Grozdanic S., Sakaguchi D.S., Kwon Y.H., Kardon R.H. and Sonea I. M. (2003) Functional characterization of retina and optic nerve after acute ocular ischemia in rats. Investigative Ophthal. and Visual Sci. 44(6): 2597-2605.
Abstract
Purpose: To functionally characterize the status of the rat retina and optic nerve after acute elevation of the intraocular pressure (IOP) and determine the dynamics of the pathological changes in the ischemic retina and optic nerve.
Methods: Retinal ischemia was induced in rats by acutely increasing the IOP (110 mmHg/60 minutes). Direct and consensual pupil light reflexes (PLR) were recorded from the unoperated eye, and electroretinograms (flash and flicker ERG) were recorded from the operated and control eyes preoperatively and postoperatively. Amplitudes and latency times were calculated for each recording session.
Results: Preoperative values for the PLRratio (ratio=consensual/direct PLR) were 76.7+2.6 (mean+SEM; %). 24h postoperatively the PLRratio was 15.2+12.8, 10 days postoperatively 11.6+9.8, 20 days postoperatively 26.5+8.0 and 28 days postoperatively PLRratio was 33.27+9.3. However, at day 35 the PLR was significantly recovered when compared to the 24h postoperative values (PLRratio=41.1+7.3%, p<0.01, Repeated measures ANOVA). 42 days after surgery the PLR started to decrease once again in the operated eyes (PLRratio=28.7+5.9). Electroretinographic amplitudes (full field flash ERG) followed a similar pattern. Cone responses (flicker ERG) were measured 42 days postoperatively and revealed defects in operated eyes (control eyes: 46.6+2.9mV, operated eyes: 3.4+1.7mV). Histological analysis revealed ischemic damage to all retinal layers with the primary defects localized to central retina.
Conclusions: Acute ocular ischemia causes significant decrease in retinal function as measured by pupillary light reflex and electroretinogram although over time the rat retina and optic nerve show partial regain of function.

Purpose. To investigate the influence of a developing host environment on the survival, differentiation and morphological integration of murine brain progenitor cells (mBPCs) transplanted to the developing mammalian retina.
Methods. Neural stem cells were isolated from newborn brains of eGFP (enhanced green fluorescent protein), transgenic mice. Developing and mature Brazilian opossums, Monodelphis domestica, served as the hosts for these transplant studies. Animals received intravitreal transplants of mBPCs, with host ages ranging from 5 days postnatal (5 PN) to adult. Animals were allowed to survive for up to 4 weeks post transplant at which time the eyes were prepared for immunohistochemical analysis.
Results. Transplanted mBPCs survived and differentiated in vivo and extensive morphological integration was observed within the host retinas. GFP expressing cells often displayed morphologies characteristic of retinal neurons. GFP somata were situated in nuclear layers, and their processes ramified throughout the inner (IPL) and outer (OPL) plexiform layers. Furthermore, in some cases, GFP expressing neurites were confined to specific sublamina within the IPL. The greatest morphological integration and differentiation was observed in the youngest host eyes. Transplanted mBSCs incorporated within the inner retina, often co-expressed the neuronal markers MAP2 and calretinin. Transplanted cells co-expressed GFP and recoverin only in the ONL.
Conclusions. mBPCs survived and morphologically integrated after xenotransplantation. mBPCs incorporated into specific layers of the retina and expressed neuronal markers. The age of the host played a key role in determining cell fate in vivo.

Grozdanic, S., Sakaguchi, D.S., Kwon, Y.H., Kardon, R.H. and Sonea, I. M. (2002) Characterization of the pupil light reflex, electroretinogram and tonometric parameters in healthy rat eyes. Current Eye Research. 25 (2):69-78..
Abstract
Purpose: To characterize the pupil light reflex (PLR), electroretinographic (ERG) and tonometric parameters in healthy rat eyes.
Methods: Brown Norway rats were used for experiments. The PLR was evaluated with a computerized pupillometer (n=27), ERGs were recorded simultaneously from both eyes (n=27) and IOP was measured with a Tonopen (n=15).
Results: The analysis of the PLR parameters confirmed the consensual PLR was significantly smaller in amplitude (p=0.03) and increased latency time (p=0.001) compared to the direct PLR. Electroretinography (1600+200 cd/m2) revealed a-wave amplitude of 207.2+13 mV and the b-wave 554.3+24.5 mV. The flicker ERG recording revealed amplitudes of 40.6+2.4 mV. Tonometry measurements revealed that isoflurane, but not halothane, anesthesia suppressed the IOP (non-anesthetized: 25.3+1.0 mmHg; 1% halothane+30% NO: 26.2+1.1 (p>0.05); 1% isoflurane+30% NO: 20.1+1.6 (p<0.05)).
Conclusions: Consensual PLR in rats has a relative deficit comparing to the direct PLR. Isoflurane anesthesia has suppressive effect on the IOP in healthy rat eyes.

Li., M. and Sakaguchi, D.S. (2002) Expression patterns of focal adhesion proteins in the developing retina. Developmental Dynamics. 225(4): 544-553.

Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study we have investigated the relationships between b1 integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of b1 integrin ECM receptors and cytoplasmic focal adhesion associated proteins, talin, vinculin and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that b1 integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin and phosphotyrosine proteins colocalized with b1 integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer and in the region of the inner and outer limiting membrane. Strong immunoreactivities for b1 integrin, paxillin and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion associated proteins are involved in integrin-mediated adhesion and signaling, and are likely to be essential in regulating retinal morphogenesis.

The SNARE complex is the core machinery required for vesicle fusion events. Numerous structural, functional and genetic studies have led to a better understanding of mechanisms that regulate vesicle fusion events during neural development. Studies using the mammalian retina as a model system have increased our understanding of the dynamic patterns of expression of SNARE proteins. In particular, the SNARE complex protein SNAP-25 is expressed in a dynamic fashion during the development of cholinergic amacrine cells in a number of mammalian species. SNAP-25 is also likely to play a crucial role during the development of vertebrate photoreceptors. The integration of comparative studies examining SNARE proteins, such as SNAP-25, provides a powerful approach for the study of CNS development.

West Greenlee, M.H., Roosevelt, C.R., and Sakaguchi, D.S. Differential Localization of SNARE Complex Proteins SNAP-25, Syntaxin, and VAMP During Development of the Mammalian Retina. J. Comp. Neurol. 430, No. 3: 306-320.pdf.

SNARE complex proteins have critical functions during regulated vesicular release of neurotransmitter. In addition, they play critical roles during neurite outgrowth and synaptogenesis. While it is clear that the function of any one SNARE complex protein during release of neurotransmitter is dependent upon its association with other members of the complex, it is less certain if their function during development and differentiation is dependent upon interaction with one another. Previously, we have observed transient high levels of SNARE complex protein SNAP-25 in developing cholinergic amacrine cells (West Greenlee et al., 1998). In addition, we detected, high levels of SNAP-25 in developing and mature photoreceptors. To better understand the functional significance of these high levels of SNAP-25 expression, we used immunocytochemistry to examine the developmental expression of the three members of the SNARE complex, SNAP-25, Syntaxin and Vesicle associated membrane protein (VAMP/also Synaptobrevin). Our results demonstrate that the high levels of SNAP-25 in cholinergic amacrine cells and photoreceptors are not accompanied by the same relatively high levels of other SNARE complex proteins. These results suggest that high levels of SNAP-25 in specific cell types may function independently of association with Syntaxin and VAMP. In this analysis we characterized the changing patterns of immunoreactivity for the three SNARE complex proteins during the development and differentiation of the mammalian retina. We have compared the pattern of expression of the core SNARE complex proteins in the Brazilian opossum, Monodelphis domestica and in the rat and found common patterns of expression between these diverse mammalian species. We observed temporal differences in the onset of immunoreactivity between these three proteins, and differences in their localization within synaptic layers in the developing and mature mammalian retina. This study is the first to characterize the changing expression patterns of the three SNARE complex proteins in the developing CNS. The differential distribution of SNAP-25, Syntaxin, and VAMP may indicate additional roles for these proteins during vesicle trafficking events, which are independent of their association with one another.

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another
(B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.

Folsom, T.D., and Sakaguchi, D.S. (1999) Disruption of actin-myosin interactions results in the inhibition of focal adhesion assembly in Xenopus XR1 glial cells. Glia 26: 245-259.pdf.

In the present study we have investigated the role of actin-myosin interactions in regulating focal adhesion assembly in XR1 glial cells. Actin-myosin interactions, stress fiber formation, and focal adhesion assembly are thought to allow cells to exert tension in the surrounding extracellular matrix, a process essential during morphogenesis and wound healing. Immunocytochemical analysis has revealed that myosin heavy chain A (MHC-A), the predominant isoform in XR1 cells, was distributed in a filamentous pattern in the central region, but was more diffuse towards the cell periphery. Myosin heavy chain-A - like immunoreactivity (IR) partially colocalized with phalloidin stained F-actin microfilaments in XR1 cells, but not with microtubules. Furthermore, MHC-A-IR colocalized with immunoreactivity for beta1 integrin receptors and vinculin at focal adhesions located more centrally along the ventral surface of the cells. The partial colocalization of MHC-A with the F-actin cytoskeleton, as well as at focal adhesions, provides evidence that actin-myosin interactions may be involved in regulating focal adhesion assembly and stabilization. To examine this possibility we have used drugs which have been shown to inhibit cell contractility: the kinase inhibitors H7 and HA100, and 2,3-butanedione 2-monoxime (BDM) which inhibits muscle and nonmuscle ATPase activity. Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the percentage of cells that displayed focal adhesions. In addition, these cells also displayed disrupted actin cytoskeletons and a similar disruption in myosin filaments. Taken together, these results provide evidence for an important role of actin-myosin generated forces during focal adhesion assembly in glial .

Swanson, J.J., M.C. Kuehl-Kovarik, J.K. Elmquist, D.S. Sakaguchi, and C.D. Jacobson. (1999) Development of the facial and hypoglossal motor nuclei in the neonatal Brazilian opossum brain. Devel. Brain Res. 112(2):159-172 .

The development of the facial and hypoglossal motor nuclei were examined in the neonatal Brazilian opossum (Monodelphis domestica), a marsupial in which postnatal central nervous system development has been well characterized. In this study, we utilized postnatal injection of the retrograde tracer cholera toxin subunit B (CtB) to characterize the formation of the facial and hypoglossal motor nuclei in the developing neonatal opossum brainstem. Injections of CtB were made into the cheek/lip region or tongue of opossum pups to retrogradely label the facial or hypoglossal motor nuclei, respectively. Following a two hour survival time, facial motoneurons in newborn opossum pups (1PN) exhibited CtB labeling, with their cell bodies localized near the developing cranial abducens nucleus. At 3 and 5 PN, following a forty-eight hour survival time, CtB labeled facial motoneurons were observed in and migrating to the region of the adult facial motor nucleus in the rostral medulla. Between 7 and 10PN, almost all facial motoneurons had migrated to their destination within the facial motor nucleus. Hypoglossal motoneurons also exhibited CtB labeling from 1 PN, however, their cell bodies were localized within the hypoglossal motor nucleus at the earliest age examined. Double label studies, to examine guidance of facial motoneurons during migration, demonstrated that CtB labeled facial motoneurons are in close proximity to vimentin-like immunostained radial glial fibers during migration. These results suggest: 1) the migration of facial motoneurons to the facial motor nucleus is a postnatal event, 2) that efferent projections from facial and hypoglossal motoneurons project into the peripheral region of their target muscles from the day of birth, and 3) facial motoneurons migrate to their destination in the brainstem thereafter, in close association with radial glial fibers.

West Greenlee, M.H., Finley, S., Wilson, M.C., Jacobson, C.D., and Sakaguchi, D.S. (1998) Transient, high levels of expression of SNAP-25 in cholinergic amacrine cells during postnatal development of the mammalian retina. J. Comp. Neurol. 394: 374-385.pdf.

Abstract: In the present study we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetlytransferase (anti-ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birthdating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2 and 15 days postnatal (PN). In addition, we have examined the differentiation of the cholinergic amacrine cells using an antibody directed against a presynaptic terminal-associated protein, SNAP-25. Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-immunoreactive amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Sakaguchi, D.S., Janick, L.M., and Reh, T. (1997) Basic fibroblast growth factor (FGF-2) induced transdifferentiation of retinal pigment epithelium: Generation of retinal neurons and glia. Developmental Dynamics. 209(4): 387-398.pdf.

Abstract: In the present study we report that basic fibroblast growth factor (bFGF, FGF-2) promotes the transdifferentiation of Xenopus laevis larval retinal pigment epithelium (RPE) into neural retina. Using specific antibodies we have examined the cellular composition of the regenerated retinal tissue. Our results show that, in addition to retinal neurons and photoreceptors, glial cells were also regenerated from the transdifferentiated RPE. These results were specific to FGF-2, since other factors that were tested, including acidic FGF (aFGF, FGF-1), epidermal growth factor (EGF), laminin, ECL and Matrigel, exhibited no activity in inducing retinal regeneration. These results are the first in amphibians demonstrating the functional role of FGF-2 in inducing RPE transdifferentiation. Transplantation studies were carried out to investigate retinal regeneration from the RPE in an in vivo environment. Sheets of RPE implanted into the lens-less eyes of larval hosts transformed into neurons and glial cells only when under the influence of host retinal factors. In contrast, no retinal transdifferentiation occurred if the RPE was implanted into the enucleated orbit. Taken together, these results show that the amphibian RPE is capable of transdifferentiation into neuronal and glial cell-phenotypes and implicate FGF-2 as an important factor in inducing retinal regeneration in vitro.

Abstract: In the present communication, we have characterized focal adhesions in cultured glial cells derived from the Xenopus retina. Using antibodies directed against focal adhesion proteins we found that beta1 integrin immunoreactivity colocalized with talin, vinculin, and phosphotyrosine immunoreactivities in glial cells from primary cultures of Xenopus retina, as well as in the XR1 glial cell line, an immortal cell line derived from Xenopus retinal neuroepithelium.Beta1 integrin immunoreactivity also colocalized with the termini of rhodamine phalloidin-labeled filamentous-actin at focal adhesions. The regulation of focal adhesion assembly was examined in XR1 glial cells using inhibitors against actin polymerization (cytochalasins) or tyrosine kinase activity (genistein). Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the proportion of cells displaying focal adhesions. Treatment with cytochalasin B also resulted in a dose-dependent decrease in cell area. Mature focal adhesions in XR1 cells with a flattened, spread morphology also were disrupted by the presence of these inhibitors. These results provide strong evidence that an intact actin cytoskeleton and tyrosine kinase activity regulate focal adhesion assembly, and also play important roles in the maintenance of the integrity of focal adhesions in glial cells.

Stone, E. and Sakaguchi, D.S. (1996) Perturbation of the developing Xenopus retinotectal projection following injections of antibodies against beta1 integrin receptors and N-cadherin. Devel. Biol. 180: 297-310.

Abstract: We have examined the function of b1 integrin receptors and N-cadherin in the development of the Xenopus retinotectal projection. In vivo perturbation experiments were performed by injecting antibodies directed against beta1 integrin receptors and N-cadherin into the embryonic optic pathway. The antibodies were present during the initial development of the retinal projection, when the axons of the ganglion cells are migrating through the optic tract and terminating within the optic tectum. When injected individually, the antibodies were insufficient to cause obvious pathfinding errors. However, when injected together, the antibodies caused specific abnormalities in the development of the retinotectal projection. Pathfinding errors most commonly observed included ectopically projecting axons within the optic tract region, meandering and splaying of axons in the optic tectum, and the induction of prominent ipsilateral projections. IgGs and Fab' fragments of the antibodies produced pathfinding errors; these defects were not observed in animals injected with control antibodies. These in vivo results show that beta1 integrin receptors and N-cadherin have important roles during the development of the visual projection and provide evidence that a balance between cell­cell and cell­matrix adhesion may be critical for the normal development of the vertebrate visual system.

Sakaguchi, D.S. and Radke, K. (1996) Beta1 integrins regulate axon outgrowth and glial cell spreading on a glial-derived extracellular matrix during development and regeneration. Devel. Brain Res. 97: 235-250.

Abstract: In the present study we have investigated functional roles for beta1 integrin receptors in regulating axon outgrowth and glial cell adhesion and spreading in the Xenopus retina. The XR1 glial cell line, isolated from Xenopus retinal neuroepithelium, deposits a proteinaceous extracellular matrix (ECM) with potent neurite outgrowth promoting activity. To investigate a potential role of the integrins as cellular receptors for these glial cell-derived ECM components, embryonic and regenerating retinal explants were cultured in the presence of polyclonal antibodies directed against the beta1 integrin receptor complex. The IgGs and Fabs of the anti-beta1 integrin antibody strongly inhibited ganglion cell axon outgrowth on the glial cell-derived ECM, although axons grew freely across the surfaces of glial cells surrounding the explants. The antibodies also inhibited outgrowth on purified laminin containing substrates in a dose-dependent fashion. In addition, the anti-beta1 antibodies were effective at inhibiting the spreading of the glial cell carpets that migrated out from the embryonic explants and also inhibited attachment and spreading of Xenopus XR1 glial cells on ECM substrates. These results show that the beta1 integrins play important functional roles in axon outgrowth during development and regeneration, and also serve in regulating retinal glial cell attachment and spreading in vitro, and thus, are likely to play similar roles in vivo.

West, M.H., Swanson, J.J., Simon, J.J., Elmquist, J.K., Jacobson, C.D. and Sakaguchi, D.S. (1996) Postnatal development and the differential expression of presynaptic terminal-associated proteins in the developing retina of the Brazilian opossum, Monodelphis domestica. Devel. Brain Res. 96: 159-172.

Abstract: In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins Synaptotagmin, Rab3A, Synaptophysin and Synaptosomal-Associated Protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively un-differentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and Synaptotagmin-, Rab3A-, Synaptophysin- and SNAP-25-like immunoreactivities (-IR), exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibit developmentally regulated changes in their cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.

Kuehl-Kovarik, M.C., Sakaguchi, D.S., Iqbal, J., Sonea, I., and Jacobson, C.D. (1995) Monodelphis domestica, the gray short-tailed opossum: A novel model for mammalian development. Lab Animal. 24: 24-29.

Abstract: The Brazilian gray short-tailed opossum, Monodelphis domestica, is a small marsupial from South America. About the size of a hamster, the females weigh approximately 100 grams at puberty, while the males can grow to twice that size. Like other marsupials, Brazilian opossum pups are born in an extremely immature state after a short gestational period, but Brazilian opossums are pouchless. The pups are exposed on the mother's ventrum, allowing easy access for manipulation (Fig. 1a). Additionally, Brazilian opossums are relatively docile when handled in the laboratory. Thus, the Brazilian opossum is an excellent experimental model for studies of mammalian development.

Elmquist, J.K., Swanson, J.J., Sakaguchi, D.S., Ross, L.R., and Jacobson, C.D. (1994) Developmental distribution of GFAP and vimentin in the Brazilian opossum brain. J. Comp. Neurol. 344: 283-296.

Abstract: Cells of glial origin are involved in the morphogenesis of the mammalian central nervous system (CNS). Characterization of glial-associated proteins during neurogenesis and differentiation may aid in understanding the complexity of CNS development. We have utilized immunoblotting and immunohistochemistry to characterize the developmental profiles of glial fibrillary acidic protein (GFAP) and vimentin (VIM) in the brain of the Brazilian opossum, Monodelphis domestica. Typical of marsupials, CNS morphogenesis and neurogenesis in the opossum extend well into the postnatal period. Opossum GFAP and VIM were found as single bands at molecular weights consistent with those reported for other species, thus indicating conservation of the VIM and GFAP proteins through mammalian evolution. Differential developmental trends were observed for both proteins with relative VIM levels decreasing and GFAP levels increasing with age. Vimentin-like immunoreactivity (VIM-IR) was present at day 1 of postnatal life throughout the brain. The density of VIM-IR was maximal at 10 and 15 days postnatal (especially in radial glial elements) and decreased slightly by 25 days postnatal. In the adult brain, VIM-IR was markedly reduced compared to that of younger ages. In contrast, GFAP-like immunoreactivity (GFAP-IR) in the brain of Monodelphis increased dramatically with age. No GFAP was observed in the 1 and 5 day postnatal brains. By 25 days postnatal, the pattern of GFAP-IR in the brainstem resembled that of the adult. In the forebrain, more GFAP-IR was present than at younger ages. The adult distribution of GFAP-IR was very similar to that reported for other mammalian species. These results indicate that GFAP and VIM are reciprocally related during periods of morphogenesis and differentiation of the opossum brain.

Henderson, E., Parpura, V., Sakaguchi, D., and Haydon, P. (1993) Atomic force microscopy of living cells. Polymer Preprints.

Sakaguchi, D.S. and Henderson, E. (1993) Isolation and characterization of glial cell lines from Xenopus retinal epithelium and retinal pigment epithelium. In "NeuroProtocols: Immortalizing Neural Cells" (Calof, ed.) 3: 249-259.

Abstract: We have isolated several immortal cell lines from Xenopus neuroepithelium and retinal pigment epithelium. These cell lines were initially isolated from primary cultures by serial passaging of proliferating cells, followed by subcloning with limiting dilution techniques. Several morphologically distinct cell lines have been isolated using these procedures. On the basis of immunocytochemical characterization using specific antibodies, we have established that three of these cell lines, the XR1, XRpe1 and XRpe2 cell lines are glial-like in nature. These cell lines were extensively labeled by antibodies against glial fibrillary acidic protein and vimentin, markers used to identify glial cells. Monolayers of these cell lines served as useful substrates for axon outgrowth from developing retinal ganglion cells. In addition, cell-free substrates were prepared by treatments of cell line monolayers with Triton X-100. This analysis revealed that the XR1, XRpe1 and XRpe2 cell lines produce an extracellular matrix (ECM) with potent outgrowth promoting activity. In contrast, other established retinal and non-retinal Xenopus cell lines were relatively ineffective and did not support axon outgrowth. We therefore propose that neurite outgrowth promoting activity produced by these cell lines are associated with their ECM and may be glial cell specific. In addition, to further characterize these cell lines, we have recently imaged live cells using the atomic force microscope (AFM). The use of AFM on living, cultured cells provides a new high resolution method for examining dynamic cytoskeletal and morphological events.

Parpura, V., Haydon, P.G., Sakaguchi, D.S., and Henderson, E. (1993) Atomic force microscopy and manipulation of living glial cells. J. Vacuum Sci. Tech. 11(4): 773-775.

Abstract: The atomic force microscope (AFM) is capable of imaging surfaces at very high resolution. The AFM has been used to image living glial cells in culture. Typical images reveal the three-dimensional shape of the cell and often internal cellular structures are visible. In this report, it is shown that by increasing the imaging force, cells can be removed from the surface on which they are grown. Although the forces involved in this process are complex, it is possible to compare relative adhesion of different types of living cells to a particular substrate.

Henderson, E. and Sakaguchi, D.S. (1993) Imaging F-actin in fixed glial cells with a combined optical fluorescence/atomic force microscope. NeuroImage 1(2): 145-150.

Abstract: A prototype combined optical fluorescence/atomic force microscope (OFAFM) designed for use in neurobiology and related disciplines has been constructed and used to study filamentous actin (F-actin) and other cellular structures in fixed Xenopus retinal glial cells (XR1 glial cell line). F-actin was readily observed by both fluorescence and AFM. AMF images of nuclei and other cellular structures were also obtained. The OFAFM consists of an AFM with an interferometer detection mechanism mounted on an inverted optical microscope. Integration of optical and scanned probe imaging methods provides a unique and useful approach to studying glial (and other) cell structure and function.

Henderson, E., Haydon, P.H., and Sakaguchi, D.S. (1992) Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science. 257:1944-1946.

Abstract: Observation of filamentous actin (F-actin) in living cells is currently limited to the resolution of the light microscope. Higher resolution procedures require sample fixation and preclude dynamic studies. The atomic force microscope (AFM) can image and manipulate samples at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting physical interactions between the tip and sample. This study demonstrates that F-actin can be readily resolved in living cells with the AFM and that the dynamic properties of F-actin are easily observed.

Sarkar, D. K., and Sakaguchi, D. S. (1990). Characterization of the neurosecretory activity of hypothalamic beta-endorphin-containing neurons in primary culture. Endocrin. 126: 349-356.

Abstract: To determine the neurosecretory activity of hypothalamic beta-endorphin (bEP)-containing neurons, rat fetal hypothalamic cells were mechanically dispersed and maintained in primary cultures for periods up to 24 days; their electrophysiological properties and regulation by depolarization, calcium and sodium channel-active agents were studied. Under culture conditions, the majority of the cells were immunopositive to neurofilament antibody, and a significant number (7-10%) were reactive to b-EP antibody. Cultured cells were often electrically excitable and possessed voltage-activated ionic conductances. In culture, there was a progressive increase in immunoreactive bEP (IR-bEP) in both cells and media, reaching maximum values at 12-16 days. The majority of IR-bEP in both cells and media corresponded to [125 I]bEP on gel chromatography and was similar to the form previously found in the hypothalamus. These findings suggest viability of the bEP neurons and continuing synthesis of IR-bEP during the culture period. To evaluate the influence of membrane depolarization on IR-bEP release, the cells were challenged with 56 mM potassium. This treatment induced a significant increase in medium IR-bEP. The depolarization-induced IR-BEP release was dependent upon calcium, since a calcium channel blocker, verapamil (0.1 mM), prevented the release; also a calcium ionophore, A23187 (1mM), stimulated IR-BEP release in the cultures. Activation of the sodium channel by veratridine (100mM) also increased the medium content of IR-bEP, and this effect was blocked by tetrodotoxin (1mM). These results suggest that the bEP neurons in primary culture respond to the well defined physiological challenges and that the culture system can be useful in determining the regulation of hypothalamic bEP activity.

Krieg, P. A., Sakaguchi, D.S., and Kintner, C. R. (1989). Primary structure and developmental expression of a large cytoplasmic domain form of Xenopus laevis neural cell adhesion molecule (NCAM). Nucleic Acids Res. 17: 10321-10335.

Abstract: The neural cell adhesion molecule, (NCAM), is involved in cell-cell interactions during development of the vertebrate nervous system. NCAM exists in multiple protein forms and these are selectively expressed in different cells and at different times during development. Here we report the complete amino acid sequence of the large cytoplasmic form of Xenopus laevis NCAM, derived from a full-length cDNA clone. Using specific probes the expression of different NCAM transcripts during Xenopus embryogenesis has been examined. We find that transcripts encoding the large cytoplasmic domain form of NCAM exist in maternal RNA and that these are the only significant NCAM transcripts present until late gastrula when transcripts encoding the small cytoplasmic domain form of NCAM are first detected. No RNA encoding the small surface domain form of NCAM is detected during early development. These results indicate that the expression of NCAM sequences during early development of Xenopus differs from that described in other species.

Sakaguchi, D.S., Moeller, J. F., Coffman, C. R., Gallenson, N., and Harris, W. A. (1989). Growth cone interactions with a glial cell line from embryonic Xenopus retina. Dev. Biol. 134: 158-174.

Abstract: We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory, as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.

Sakaguchi, D.S. (1989). The development of retinal ganglion cells deprived of their targets. Dev. Biol. 134: 103-111.

Abstract: The influence of central targets on the morphological differentiation of retinal ganglion cells was investigated in Xenopus laevis. Since the ganglion cells mature into distinct morphological subtypes after their axons have reached their central targets, it is possible that the target tissues may influence or specify this aspect of neuronal cell development. To test this idea, Xenopus eyebuds were target-deprived by transplantation to the flank region of host embryos where they developed ectopically. The grafted eyes grew at normal rates, but could not make any projections into the central nervous system. To examine the morphological differentiation of the retinal ganglion cells their structures were revealed using an in vitro retinal preparation and intracellular injections of the dye Lucifer yellow. The elaboration and maturation of ganglion cell dendrites were found to be indistinguishable between control and transplanted eyes throughout development. Thus, the development of retinal ganglion cells into distinct morphological classes can occur even when their axons do not interact with the appropriate central targets.

Sakaguchi, D.S., Coffman, C. R., Gallenson, N., and Harris, W. A. (1988). A glial cell line promotes the outgrowth of neurites from embryonic Xenopus retina. Acta Biologica Hungarica 39: 201-209.

Abstract: A glial cell line (XR1 cell line) derived from Xenopus retinal neuroepithelium was examined for neurite outgrowth promoting activity. A monolayer of the XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and freely elaborate neurites. The XR1 neurite outgrowth promoting activity is not secreted into the medium, but is laid down directly on the substrate where it remains active after lysing the cells by hypoosmotic shock. A polyclonal antiserum raised against membranes of the XR1 conditioned factors produced by the XR1 cells are associated with the extracellular matrix and possibly glial specific.

Sakaguchi, D.S., and Murphey, R. K. (1985). Map formation in the developing Xenopus retinotectal system: An examination of ganglion cell terminal arborizations. J. Neurosci. 5: 3228-3245.

Abstract: Single axonal arbors of retinal ganglion cells have been stained by injecting cobalt extracellularly into the retinae of Xenopus embryos and tadpoles. The axonal endings of the earliest retinal axons to arrive in the midbrain were usually simple in appearance, often ended in growth cones, and terminated in tectal regions appropriate to their location in the eye. Thus, a topographic projection exists very early in the development (stages 37 to 39) of the projection, before the elaboration of complex axonal arbors. Retinal axons began acquiring more mature features, exemplified by the elaboration of terminal arbors, by stage 39. The arbors of most ganglion cells were elongated in the rostral-to-caudal dimension during early larval life (stages 40 to 45) and covered a large portion of tectal neuropil. During mid-larval stages (stages 46 to 50), arbors covered a relatively smaller proportion of the tectal neuropil. A quantitative analysis of this change suggests that the apparent decrease in size of the arbors, with respect to the tectum, is due to rapid growth of tectal neuropil and not due to retraction of an initially diffuse arbor. Thus, the refinement in targeting of axonal arbors during development is a phenomenon distinct from that seen during regeneration.

Sakaguchi, D.S., Murphey, R. K., Hunt, R. K., and Tompkins, R. (1984). The development to retinal ganglion cells in a tetraploid strain of Xenopus laevis : A morphological study utilizing intracellular dye injection. J. Comp. Neurol. 224: 231-251.

Abstract: The morphological development of retinal ganglion cells was examined in a tetraploid strain of Xenopus frogs. The enlarged cells of the tetraploid strain facilitate the application of intracellular techniques. Using an in vitro retinal preparation and Nomarski optics, intracellular recording and dye injection were carried out under visual control on ganglion cells in central retina from 2 days of development (stage 24) to metamorphosis (stage 64). We identified three phases in the morphological differentiation of ganglion cells. During the first phase (stages 24-30), all cells were neuroepithelial-like in form and possessed robust resting potentials in the range of -35 to -60 mV, and dye-coupling was occasionally observed between neighboring cells. During the second phase of ganglion cell development (stages 31-45) the neurons had begun to elaborate axons and dendrites. These cells possessing neurites had resting potentials between -15 and -30mV, and no dye-coupling was observed between neighbors. During the third and final phase of maturation, from stage 46 onward, three distinct morphological types of ganglion cells could be identified. Type I cells had the smallest somata and the smallest-diameter dendritic arborizations. The profusely branched dendrites of these cells ramify extensively throughout the inner plexiform layer. Type II cells had large somata, intermediate-diameter dendritic fields, and a highly elaborate dendritic branching pattern. These calls were seen to arborize within two sublamina in the inner plexiform layer. Type III cells had large somata, the largest-diameter dendritic fields, and a dendritic arbor with long primary branches but little higher-order branching. These large dendritic fields were confined to a single sublamina of the inner plexiform layer, abutting the inner nuclear layer. While most phase 3 cells showed radial axon trajectories from the soma to the optic disc, a minority of cells (1-5%) with erratic and nonradial axon trajectories were also observed. Our data provide a morphological description of ganglion cell maturation in the central retina of Xenopus. We show that very early in development (as early as stage 46) three distinct morphological types of retinal ganglion cells are present, which correspond to the three classes of ganglion cells previously described in adult Xenopus (Chung et al., '75).

Murphey, R. K., Johnson, S. E., and Sakaguchi, D.S. (1983). Anatomy and physiology of supernumerary cercal afferents in crickets: Implications for pattern formation. J. Neurosci. 3: 312-325.

Abstract: The afferent projection of supernumerary sensory neurons was examined in the cricket cercal sensory system. When a right cercus was exchanged for a left cercus the growth of supernumerary cerci was induced. The supernumerary cerci which formed tended to appear medial and/or lateral in a simple left->right exchange and were always right handed. Distal to the region where transplant and supernumerary meet, receptors on the supernumerary structure appear in a recognizable pattern typical of control cerci, and individual receptors homologous in position to control receptors could be identified. The supernumerary neurons associated with these identified receptors projected to the same area of neuropil as their homologs and thus uniquely identified neurons by all of the usual criteria were produced by the regeneration process. When the response properties of one postsynaptic neuron were examined it was shown that supernumerary afferents innervated it in a normal manner. The supernumerary neurons provide tests of various hypotheses for the assembly of ordered afferent projections. First, their axon trajectories are often atypical, but the terminal arbors grow into their proper areas in spite of this. Thus, axon trajectory can be uncoupled from target area and is therefore unlikely to be a decisive factor in growth of the arbor. Second, supernumerary neurons are born later in development than their control homologs but their arborizations are completely normal, adding to the existing evidence against a timing mechanism in the assembly of this afferent projection. Third, supernumerary neurons share target regions with their homologs, indicating a lack of competitive interactions. The most economical hypothesis to explain the precise mapping between the cercus and CNS holds that one step in the differentiation of insect sensory neurons is position dependent and that this step determines the choice of a target region within the CNS.

Murphey, R. K., Bacon, J. P., Sakaguchi, D.S., and Johnson, S. E. (1983). Transplantation of cricket sensory neurons to ectopic locations: Arborizations and synaptic connections. J. Neurosci. 3: 659-672.

Abstract: The cerci (abdominal sensory appendages) of crickets were transplanted to a leg stump after amputating the leg. Single identifiable cercal afferents were stained and found to regenerate into the host thoracic ganglia. A given neuron always arborizes in the same area of neuropil of the foreign ganglion and is distinctive in this property from other identified neurons. Taken as a whole, the results show that the afferents from the ectopic cercus are spatially ordered, the destination of a particular afferent within the ganglion being correlated with the location of its sensory cell body on the cercal surface. This is the case for the pro-, meso-, and metathoracic ganglion and the topography of these ectopic projections bears some resemblance to the normal projections found in the terminal ganglion. Thus, the insect segmental ganglion seems to possess a set of markers which are interpretable by all afferent neurons, and this organization is repeated in each ganglion. The ectopic afferents make functional synaptic connections with intersegmental interneurons, one of which is described anatomically here. However, the ectopic afferents do not, as had previously been reported, make functional connections with the medial giant or lateral giant interneuron (the normal targets of cercal sensory neurons in the terminal ganglion).

Sakaguchi, D.S., and Murphey, R. K. (1983). The equilibrium detecting system of the cricket: Physiology and morphology of an identified interneuron. J. Comp. Physiol. 150: 141-152.

Summary: 1. The clavate receptor-to-interneuron system of the cricket, Acheta domesticus, was investigated physiologically and morphologically. 2. Intracellular recordings made during controlled displacements of the cricket's cerci allowed the identification of a pair of position sensitive interneurons (PSIs). Changes in the position of the cerci resulted in modulation of the membrane potential and altered the action potential frequency recorded from the PSIs. 3. Subsequent dye injection demonstrated that the PSIs were a bilaterally symmetric pair of interneurons that received their primary afferent input from club-shaped receptors, called clavate hairs. 4. Receptive fields for the interneurons were determined by recording the extracellular neural activity from the ventral nerve cord while simultaneously displacing the animal in various orientations. The PSI receptive fields were essentially mirror images, each predominantly occupying one quadrant when plotted in polar coordinates. The receptive fields for the left and right PSIs occupy the left posterior and right posterior quadrants respectively. 5. Selective deletion of clavate hairs revealed that receptors located in the proximal, medial region of the clavate array provided the strongest input to the ipsilateral interneuron. 6. Clavate sensory neuron terminal arborizations were stained in order to examine their relationship with the dendrites of the PSIs.

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