Results of TRIO-14, a phase II, multicenter, randomized, placebo-controlled trial of carboplatin-paclitaxel versus carboplatin-paclitaxel-ganitumab in newly diagnosed epithelial ovarian cancer.

PURPOSE: Insulin-like growth factor (IGF) signaling is implicated in pathogenesis and chemotherapy resistance of epithelial ovarian cancer (EOC). We explored efficacy and safety of adding ganitumab, a monoclonal antibody targeting IGF-1R, to carboplatin/paclitaxel (CP) chemotherapy in patients with primary EOC. DESIGN: Patients were randomly assigned to receive CP/ganitumab (18 mg/kg q3w) or CP/placebo for 6 cycles followed by 6 cycles of single agent ganitumab/placebo maintenance therapy as front-line therapy. Primary endpoint was progression free survival. Secondary endpoints were time to progression and overall survival. Pretreatment samples were prospectively collected for retrospective biomarker analyses. RESULTS: 170 patients enrolled. 165 patients assessable for toxicity. Median PFS was 15.7 months with CP/ganitumab and 16.7 months with CP/placebo (HR 1.23; 95% CI 0.82-1.83, P = 0.313). All grade neutropenia (84.1% vs 71.4%), thrombocytopenia (75.3% vs 57.1%) and hyperglycemia (15.9% vs 2.6%) were more common in the ganitumab group compared to the placebo group. Ganitumab/placebo related serious adverse events were reported in 26.1% of the patients with ganitumab and in 6.5% with placebo. Non-progression related fatal events were more common with ganitumab (5 versus 2 patients). The ganitumab group experienced more dose delays which resulted in lower relative dose intensity of chemotherapy in the experimental group. In an exploratory model IGFBP2 expression was predictive of ganitumab response (treatment interaction; PFS, P = 0.03; OS, P = 0.01). CONCLUSION: Addition of ganitumab to CP chemotherapy in primary EOC did not improve PFS. Our results do not support further study of ganitumab in unselected EOC patients.

The foveal avascular region of developing human retina.

OBJECTIVE: To study the development of the perifoveal retinal vasculature. METHODS: We studied 7 retinas aged between 26 weeks' gestation and 1 week postnatal (41 weeks' gestation). Sections were imaged using high-resolution digital photography and blood vessel profiles identified at 200% to 300% magnification. Flat mounts were immunolabeled using antibodies to CD31 and factor VIII to identify blood vessels and antibodies to rhodopsin to identify the rod-free zone. RESULTS: The foveal region was identified by the absence of rod photoreceptors in the outer retina and/or presence of a shallow depression in the inner retina. The whole mount at 26 weeks' gestation showed a blood vessel-free region centered on the rod-free zone that was open along the horizontal meridian on the temporal side. At 37 weeks' gestation, the foveal avascular zone formed a complete circle. In sections, the foveal avascular zone was approximately 500 microm in diameter at 35 weeks' gestation and 300 to 350 microm at 40 weeks' gestation; in whole mounts, it was 150 to 170 microm in diameter at 37 and 41 weeks' gestation. CONCLUSIONS: The foveal region is normally avascular during development, as in adult life. We found no evidence of foveal vascularization during development of the human retina. Clinical Relevance Instances of vascularization of the foveal region are not due to failed regression of a transient vasculature.

Development of the primate area of high acuity, 3: temporal relationships between pit formation, retinal elongation and cone packing.

By establishing an avascular, highly elastic, region within the fetal area of high acuity (AHA), the developing primate eye has created a unique substrate on which the mechanical forces of intraocular pressure (IOP) and growth-induced retinal stretch (stretch) can act. We proposed (Springer & Hendrickson, 2004b) that these forces generate both the pit and high cone density found in the adult AHA. In this paper, we use quantitative measures to determine the temporal relationships between nasal and temporal retinal elongation, changes in pit depth, cone packing, and cone morphology over M. nemestrina retinal development. Retinal length increased rapidly to about 105 days postconception (dpc; Phase 1) and then elongation virtually ceased (Phase 2) until just after birth (180 dpc). Retinal elongation due to stretch resumed during Phase 3 until approximately 315 dpc (4-5 months), after which time the retina appeared mature (Phase 4). The pit appeared during the quiescent Phase 2, suggesting that IOP acts, in conjunction with molecular changes in the inner retina, on the highly elastic, avascular, AHA to generate a deep, narrow pit and causes inner retinal cellular displacements. Subsequently (Phase 3), the pit widened, became 50% shallower and central inner retinal lamina thinned slightly due to a small amount of retinal stretch occurring in the AHA. Centripetal movement of cones was minimal until just after birth when the pit reached 88% of its maximal depth. Accelerated cone packing during Phase 3 was temporally correlated with increased stretch.

Development of the primate area of high acuity. 1. Use of finite element analysis models to identify mechanical variables affecting pit formation.

Most primate retinas have an area dedicated for high visual acuity called the fovea centralis. Little is known about specific mechanisms that drive development of this complex central retinal specialization. The primate area of high acuity (AHA) is characterized by the presence of a pit that displaces the inner retinal layers. Virtual engineering models were analyzed with finite element analysis (FEA) to identify mechanical mechanisms potentially critical for pit formation. Our hypothesis is that the pit emerges within the AHA because it contains an avascular zone (AZ). The absence of blood vessels makes the tissue within the AZ more elastic and malleable than the surrounding vascularized retina. Models evaluated the contribution to pit formation of varying elasticity ratios between the AZ and surrounding retina, AZ shape, and width. The separate and interactive effects of two mechanical variables, intraocular pressure (IOP) and ocular growth-induced retinal stretch, on pit formation were also evaluated. Either stretch or IOP alone produced a pit when applied to a FEA model having a highly elastic AZ surrounded by a less elastic region. Pit depth and width increased when the elasticity ratio increased, but a pit could not be generated in models lacking differential elasticity. IOP alone produced a deeper pit than did stretch alone and the deepest pit resulted from the combined effects of IOP and stretch. These models predict that the pit in the AHA is formed because an absence of vasculature makes the inner retinal tissue of the AZ very deformable. Once a differential elasticity gradient is established, pit formation can be driven by either IOP or ocular growth-induced retinal stretch.

Development of the primate area of high acuity. 2. Quantitative morphological changes associated with retinal and pars plana growth.

Mechanisms underlying the development of the primate area of high acuity (AHA) remain poorly understood. Finite-element models have identified retinal stretch and intraocular pressure (IOP) as possible mechanical forces that can form a pit (Springer & Hendrickson, 2004). A series of Macaca nemestrina monkey retinas between 68 days postconception (dpc) and adult were used to quantify growth and morphological changes. Retinal and pars plana length, optic disc diameter, disc-pit distance, and inner and outer retinal laminar thickness were measured over development to identify when and where IOP or stretch might operate. Horizontal optic disc diameter increased 500 mum between 115 dpc and 2 months after birth when it reached adult diameter. Disc growth mainly influences the immediate surrounding retina, presumably displacing retinal tissue centrifugally. Pars plana elongation also began at 115 dpc and continued steadily to 3-4 years postnatal, so its influence would be relatively constant over retinal development. Unexpectedly, horizontal retinal length showed nonlinear growth, divided into distinct phases. Retinal length increased rapidly until 115 dpc and then remained unchanged (quiescent phase) between 115-180 dpc. After birth, the retina grew rapidly for 3 months and then very slowly into adulthood. The onset of pit development overlapped the late fetal quiescent phase, suggesting that the major mechanical factor initiating pit formation is IOP, not retinal growth-induced stretch. Developmental changes in the thickness of retinal layers were different for inner and outer retina at many, but not all, of the ten eccentricities examined.

Astrocytes and blood vessels define the foveal rim during primate retinal development.

PURPOSE: To investigate the relationship between development of the perifoveal blood vessels and formation of the foveal depression. METHODS: Retinal sections and flatmounts from monkeys aged between fetal day (Fd)80 and 2 years of age were double labeled using antisera to CD31 or von Willebrand factor to detect vascular endothelial cells and antiserum to glial fibrillary acidic protein to detect astrocytes. Sections were studied by fluorescence or confocal microscopy. RESULTS: From Fd88 to 115, vessels on the horizontal meridian were found only at the level of the ganglion cell layer (GCL)-inner plexiform layer (IPL) border where they form the ganglion cell layer plexus (GCP). Stellate astrocytes accompany GCP vessels and extend closer to the fovea than vessels. The foveal avascular zone was present within the GCP at Fd101, and at Fd105 a shallow foveal depression encircled by the GCP was present. The GCP foveal margin had the same dimensions as the adult foveal pit. Both blood vessels and astrocytes were excluded from the emerging fovea throughout development. After Fd140, capillary plexuses in the outer retina anastomosed with the GCP on the foveal slope to form a perifoveal plexus, but this plexus did not mature until a month or more after birth. After Fd142, astrocytes rapidly disappeared from the GCP and most of central retina. CONCLUSIONS: An avascular area is outlined by the GCP before the foveal pit begins to form, suggesting that molecular factors in this region exclude both vessels and astrocytes. These factors may also guide neuronal migration to form the pit. Because the perifoveal plexus is formed during late gestation, both capillary growth and foveal development may be affected adversely by prematurity.

Expression of glycine and the glycine transporter Glyt-1 in the developing rat retina.

Previous studies show that glycine transporter-1 (glyt-1) is a consistent membrane marker of adult retinal neurons that are likely to release glycine at their synaptic terminals (Pow, 1998; Vaney et al., 1998; Pow & Hendrickson, 1999). The current study investigated when glyt-1 immunoreactivity appeared in the postnatal rat retina, and whether all glycine-containing neurons also labelled for glyt-1. Ganglion cells, horizontal cells, and photoreceptors showed transient labelling. Many cells in the ganglion cell layer are immunoreactive for both glycine and glyt-1 at postnatal day (Pd) 1 but both are minimal by Pd5. Transient immunoreactivity for both glyt-1 and glycine was observed in presumptive horizontal cells between Pd5 and Pd10. At Pd1 many cells in the outer part of the retina which resembled immature photoreceptors were heavily labelled for glycine, but did not express glyt-1 these disappeared at older ages. These findings suggest diverse mechanisms and transient roles for glycine in the developing rat retina. In the adult rat retina, a subpopulation of amacrine cells are prominently immunoreactive for both glycine and glyt-1. These cells labelled for glycine at Pd1, but did not express significant levels of glyt-1 until Pd5. Processes from these amacrine cells did not reach the inner half of the inner plexiform layer until Pd10-14. Bipolar cells became glycine-IR between Pd10 and Pd14, but consistently lacked any glyt-1 immunoreactivity. This temporal pattern of labelling strongly indicates that bipolar cells label for glycine when gap junctions become functional between glycine/glyt-1 immunoreactive amacrine cells and cone bipolar cells.

Localization of tubby-like protein 1 in developing and adult human retinas.

PURPOSE: To localize tubby-like protein 1 (TULP1) in developing and adult human retinas. METHODS: TULP1 was localized by immunofluorescence microscopy in human retinas, aged 8.4 fetal weeks to adult. TULP1-positive cells were identified by double labeling with antibodies specific for cones, rods, and astrocytes. RESULTS: In adult retinas, anti-TULP1 labels cone and rod inner segments, somata, and synapses; outer segments are TULP1-negative. A few inner nuclear and ganglion cells are weakly TULP1-positive. In fetal retinas, cells at the outer retinal border are TULP1-positive at 8.4 weeks. At 11 weeks, the differentiating central cones are strongly TULP1-reactive and some are positive for blue cone opsin. At 15.4 weeks, all central cones are strongly positive for TULP1 and many are reactive for red/green cone opsin. At 17.4 weeks, central rods are weakly TULP-reactive. In peripheral retina at 15.4 weeks to 1 month after birth, displaced cones in the nerve fiber layer are positive for TULP1, recoverin, and blue cone opsin. Some ganglion cells are weakly reactive for TULP1 at 11 weeks and later, but astrocytes and the optic nerve are TULP1-negative at all ages examined. CONCLUSIONS: The finding of TULP1 labeling of cones before they are reactive for blue or red/green cone opsin suggests an important role for TULP1 in development. TULP1 expression in both developing and mature cones and rods is consistent with a primary photoreceptor defect in retinitis pigmentosa (RP) caused by TULP1 mutations. Weak TULP1-immunolabeling of some inner retinal neurons in developing and adult retinas suggests that optic disc changes in patients with RP who have TULP1 mutations may be primary as well as secondary to photoreceptor degeneration.

Expression of glycine and the glycine transporter glyt-1 in the developing rat retina.

Previous studies show that glycine transporter-1 (glyt-1) is a consistent membrane marker of adult retinal neurons that are likely to release glycine at their synaptic terminals (Pow, 1998; Vaney et al., 1998; Pow & Hendrickson, 1999). The current study investigated when glyt-1 immunoreactivity appeared in the postnatal rat retina, and whether all glycine-containing neurons also labelled for glyt-1. Ganglion cells, horizontal cells, and photoreceptors showed transient labelling. Many cells in the ganglion cell layer are immunoreactive for both glycine and glyt-1 at postnatal day (Pd) 1 but both are minimal by Pd5. Transient immunoreactivity for both glyt-1 and glycine was observed in presumptive horizontal cells between Pd5 and Pd10. At Pd1 many cells in the outer part of the retina which resembled immature photoreceptors were heavily labelled for glycine, but did not express glyt-1; these disappeared at older ages. These findings suggest diverse mechanisms and transient roles for glycine in the developing rat retina. In the adult rat retina, a subpopulation of amacrine cells are prominently immunoreactive for both glycine and glyt-1. These cells labelled for glycine at Pd1, but did not express significant levels of glyt-1 until Pd5. Processes from these amacrine cells did not reach the inner half of the inner plexiform layer until Pd10-14. Bipolar cells became glycine-IR between Pd10 and Pd14, but consistently lacked any glyt-1 immunoreactivity. This temporal pattern of labelling strongly indicates that bipolar cells label for glycine when gap junctions become functional between glycine/glyt-1 immunoreactive amacrine cells and cone bipolar cells.

Distribution of the glycine transporter glyt-1 in mammalian and nonmammalian retinae.

We have examined the distribution of the glycine transporter glyt-1 in retinae of macaques, cats, rabbits, rats, and chickens. In all species, all glycine-containing amacrine cells expressed immunoreactivity for glyt-1, though the intensity of immunoreactivity for glyt-1 did not appear to directly correlate with the intensity of immunoreactivity for glycine in individual cells. A small subpopulation of glycine-immunoreactive displaced amacrine cells or ganglion cells also expressed glyt-1 in retinae from macaques, cats, chickens, and rats but not in retinae from rabbits. In addition, in all species examined, some displaced amacrine cells also contained glycine but did not express glyt-1. In monkeys, cats, and rats, populations of cells which we interpret as being glycine-containing interplexiform cells expressed glyt-1: these cells lacked a content of glutamate, suggesting they are not bipolar cells. The glycine-containing bipolar cells did not express glyt-1, suggesting that these cells probably acquired their content of glycine by other means such as via gap junctional connections with glycine-containing amacrine cells.

SPARC is expressed by ganglion cells and astrocytes in bovine retina.

SPARC (secreted protein, acidic and rich in cysteine)/osteonectin is a matricellular, counteradhesive glycoprotein that disrupts cell-matrix interactions, interacts with growth factors and components of extracellular matrix, and modulates the cell cycle, but appears to subserve only minor structural roles. SPARC is expressed in a variety of tissues during embryogenesis and remodeling and is believed to regulate vascular morphogenesis and cellular differentiation. Although usually limited in normal adult tissues, SPARC is expressed at significant levels in the adult central nervous system. Using a monoclonal antibody against bovine bone osteonectin, we have determined the localization of SPARC in newborn (3-day-old) and adult (4-8-year-old) normal bovine retinas. SPARC was present in the soma of ganglion cells and strong reactivity was found in ganglion cell axons. Muller cells displayed no immunoreactivity, but SPARC was present in retinal astrocytes that were identified by the astrocyte marker glial fibrillary acidic protein (GFAP). Newborn calf retina showed a staining pattern similar to that of adult retina but exhibited significantly reduced levels of SPARC. Minimal levels of SPARC protein were also detected in some capillaries of the inner retina of both newborn and adult animals, whereas large vessels were negative. The presence of SPARC in the retina was confirmed by Western blotting of retinal extracts. These data indicate that SPARC originating from bot h neurons and glia of the inner retina may be an important modulator of retinal angiogenesis. The increased expression of SPARC in adult relative to newborn retinal tissue also indicates that SPARC has an ongoing role in the maintenance of retinal functions.

Development of astrocytes and their relation to blood vessels in fetal monkey retina.

PURPOSE: To determine the development of astrocytes and their vascular relations in Macaca monkey retina. METHODS: Sections and wholemounts of retinas from fetal day (Fd) 65 to adult animals were analyzed immunohistochemically to detect glial fibrillary acidic protein (GFAP) and vimentin. RESULTS: Astrocytes appeared first near the optic disc, then subsequently further peripherally, but avoided the fovea. In the nerve fiber layer, round and ovoid cells extended processes parallel to ganglion cell axons. In the ganglion cell layer, ovoid and stellate cells exhibited anisotropic processes or a honeycomb network. The inner lamina of astrocytes developed ahead of the outer lamina, and both reached their final positions before birth. Astrocytes lay more peripherally than did developing blood vessels, and the growing edge of nerve fiber layer vessels lay between the two astrocytic layers. Spindle cells, which may be vascular precursor cells, often aligned along linear astrocytic processes. Occasional spindle-shaped cells containing GFAP or vimentin were identified as immature glia. Astrocytes and blood vessels coincided regionally during development, but astrocyte processes were typically not in register with the meshwork of growing blood vessels. Astrocyte-vessel associations increased during fetal life and postnatally. CONCLUSIONS: During development, astrocytes display the same bilaminar pattern and morphologies present in adult retina. Astrocytes and blood vessels exhibit a similar regional distribution, but develop in distinct spatial patterns. Vessel investment by astrocytic processes increases during fetal life but is variable at all ages.

Primary culture and characterization of microvascular endothelial cells from Macaca monkey retina.

PURPOSE: To develop methods for the culture of microvascular endothelial cells (EC) from Macaca monkey retina and to investigate their propagation and survival in vitro. METHODS: Endothelial cells from capillary fragments were cultured on fibronectin-coated dishes in QB-58 serum-free medium containing 20 microliters/ml bovine retinal extract, 90 micrograms/ml heparin, 10% fetal bovine serum, and 10% monkey serum. Non-EC were removed manually. Endothelial cell-specific properties were assessed by endocytosis of acetylated low-density lipoprotein (ac-LDL) and by immunocytochemical staining. The response to growth factors was assayed by 3H-thymidine incorporation. The synthesis of matrix macromolecules was studied by metabolic labeling with 3H-proline and identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis-immunoblotting. RESULTS: Under these culture conditions, migrating cells emerged from capillary fragments after 1 to 2 days and formed large colonies by 1 week. Cells exhibited a mean doubling time of 44.5 hours during the first 3 to 5 days of culture and 23 hours at 6 to 8 days in culture, and they formed a confluent monolayer by 12 to 14 days. These cells demonstrated uptake of ac-LDL, expressed von Willebrand factor and the cell adhesion protein CD31, and did not contain smooth muscle alpha-actin. Before purification, 92% of the cells in primary cultures were identified as EC. The EC could be maintained in vitro for more than 1 month without the addition of growth factors; however, basic fibroblast growth factor and vascular endothelial growth factor each stimulated cell replication. Secreted extracellular proteins included fibronectin, collagen types I and IV, laminin, and SPARC (secreted protein, acidic, and rich in cysteine). CONCLUSIONS: This study is the first description of the culture and propagation of purified retinal EC from Macaca monkey, a widely accepted model for the human retina. These cultures will be highly relevant to studies of abnormal vascular disease in the human eye.

Normal and pathological mechanisms in retinal vascular development.

Angiogenesis is a complex biologic process that occurs normally in development and in turnover and remodeling of mature vascular networks. Pathological angiogenesis and neovascularization occur in association with retinal and ocular ischemic diseases, in retinopathy of prematurity and other developmental disorders, and in tumor growth and metastasis. We describe current understanding of cellular and molecular mechanisms of retinal vascular development, highlighting aspects that relate to eye diseases, that provide sites of therapeutic intervention in ophthalmology and that are potential avenues for research.

Immunohistochemical characterization of developing and mature primate retinal blood vessels.

PURPOSE: To characterize developing retinal blood vessels with vascular markers and to relate the histochemical profile of maturing vessels to morphologic stages in retinal vascular development. METHODS: Vessels were examined in frozen and paraffin-embedded retinas and in wholemounts of Macaca monkeys ranging in age from fetal day 75 (F75) to adulthood. Endothelial cells were visualized immunohistochemically using antisera to von Willebrand's factor and CD31 with lectins Ulex europaeus, Bandeiraea simplicifolia, peanut agglutinin, Ricinis communis, and wheat germ agglutinin, and by ATPase and ADPase enzymatic histochemistry. Antibodies to vascular basement membrane and matrix markers laminin, fibronectin, and collagen types I and VIII, and antisera recognizing cell cycle-specific nuclear proteins (cyclin, Ki-67, Mib-1) also were used. RESULTS: Newly formed and mature vessels were reactive with reagents specific for CD31, von Willebrand's factor, types I and VIII collagens, laminin, fibronectin, U. europaeus, R. communis, and peanut agglutinin. Wheat germ agglutinin labeled vessels only after pretreatment with neuraminidase. All vascular markers appeared simultaneously, but some were distributed differentially between capillaries and larger vessels, along the central-peripheral extent of a vascular plexus, and among different vascular laminae. Markers of vessels failed to label spindle-shaped presumed vascular precursor cells lying peripheral to the advancing vessels during development. Spindle cells exhibited cyclin, Ki-67, and Mib-1 immunoreactivity. CONCLUSIONS: Immature and mature vitread and sclerad vessels displayed histochemical profiles that were qualitatively similar but that had subtle quantitative differences. Results do not support identification of spindle-shaped cells as vascular precursors in the developing monkey retina and are discussed in relation to mechanisms of retinal vascularization.

Synaptic development in macaque monkey retina and its implications for other developmental sequences.

New and existing data are presented regarding synaptic development in primate retina with the aims to identify the sequence in which individual cell types form synapses in the inner plexiform (IPL) and outer plexiform (OPL) layers; to compare synaptic development sequences in cone-dominated fovea and rod-dominated peripheral retina; to compare synaptic formation with other aspects of cell differentiation; and to explore the possible roles for synapses in development. The first synapses are formed in the foveal IPL by bipolar axons at fetal day 55, followed at fetal day 60 by cone ribbon synapses. Amacrine synapses in the foveal IPL only appear in significant numbers at fetal day 88. In peripheral retina amacrine synapses are formed at fetal day 78, bipolar at 99, and photoreceptors at 105. Thus, the fovea forms the first synapses and the IPL matures before the OPL across the retina, but the fovea has a different bipolar/amacrine sequence than peripheral retina. Foveal synapses are present before many photoreceptor-specific proteins such as opsins can be detected, suggesting that some phenotypic information from the inner retina could influence the direction of photoreceptor development. The early synaptic development in the fovea may serve an important mechanical role during subsequent cell migrations that form the mature foveal pit and tightly packed cone foveola.

The appearance of rod opsin during monkey retinal development.

PURPOSE: To determine the temporal and spatial pattern of rod opsin appearance in Macaca monkey retina. METHODS: Frozen sections from fetal day (Fd) 55 to adulthood (birth = Fd168) containing the entire horizontal meridian were stained using Rho4D2 monoclonal antiserum visualized with immunofluorescent labeling. At Fd66, Fd79, and Fd89, retinal samples taken at known eccentricities were studied from the opposite eye using standard electron microscope methods. RESULTS: Rod opsin was detected at Fd66 in or near the fovea, and a second focus appeared at Fd75 to Fd77 near the optic disc in the nasal rod ring. The earliest opsin appeared in the apical stubs, which resembled the apical connecting cilium in the electron microscope. Staining of the entire cell body membrane, including the synaptic spherule, was present 4 to 7 days later. Opsin expression had a nasal bias with rods at the nasal ora labeled at Fd140, whereas temporal ora was not labeled until Fd155. Cell body labeling disappeared by Fd132 across central retina but persisted into the first postnatal year in far peripheral retina. Outer segment (OS) length measurements showed that rods in the rod ring had the longest OS between Fd115 and postnatal week 9. Rod OS at all retinal eccentricites continued to elongate between 11 months of age and adulthood. CONCLUSIONS: Rod opsin expression follows a foveal-to-peripheral gradient beginning at Fd66 and ending near birth. Rod opsin is detected first in the connecting cilium and slightly later in the entire cell membrane, and then cell membrane labeling disappears as the heavily labeled OS elongates. Although the first OS appear on rods near the fovea, these OS still are short at birth and do not reach adult length until after 2 years of age. The longest OS at birth are found on rods at the rod ring, suggesting that this region could have higher scotopic sensitivity than central retina at birth.

Developmental changes in calretinin expression in GABAergic and nonGABAergic neurons in monkey striate cortex.

The development of the calcium-binding protein calretinin (CaR) and its co-localization with GABA was studied in the striate cortex of Macaca monkeys from fetal day (Fd) 45 to adult. At Fd45, early neurons resembling Cajal-Retzius cells are stained in the marginal zone (MZ). At Fd55 the MZ is filled with CaR+ Cajal-Retzius cells and their processes, and scattered CaR+ cells are also found in deep cortical plate (CP), intermediate zone (IZ), and subventricular zone (SVZ). At Fd66, a band of CaR+ fibers appears in the IZ, corresponding to the location of the geniculocortical axons. This fiber band labels heavily until Fd130 but then ceases to be immunoreactive by postnatal (P) 16 weeks. At Fd85-101, the number of CaR+ cells in the CP, SVZ, and ventricular zone (VZ) reaches its highest cell density. After Fd130, CaR+ cells are concentrated in layer II and upper layer III, and this distribution changes little into adulthood. After mid-gestation, there is a progressive loss of CaR+ cell bodies and processes in the MZ, and these are rare in the adult cortex. Just before birth, a weakly stained CaR+ cell band appears in layer IVA at the border between layer IVA and IVB, but this band disappears immediately after birth. Another CaR+ cell band appears transiently in upper layer V just below the border with layers IV at P6 months. These results suggest that CaR is expressed early in fetal development in the cell populations that are immunoreactive for CaR in the adult. However, developmental events related to cortical maturation during late prenatal and early postnatal stages result in transient expression of CaR in neurons that are not immunoreactive for CaR in the adult. CaR-immunoreactivity is colocalized with GABA in almost all CaR+ cells with the exception of Cajal-Retzius cells in the MZ and some large cells observed at Fd70-101 in the VZ. The band of CaR+ fibers in the IZ is GABA-. At Fd90, almost all (> 96%) CaR+ cells are GABA+ in the CP and the first developed layers V/VI. This percentage declines later, so that on average 80% of CaR+ cells are GABA+ in adult cortex. At Fd135, 53% of GABA+ neurons located in layers II/III are CaR+; this percentage declines to 37% in the adult. These double-label patterns suggest that early in fetal development the majority of GABA+ cells stain for CaR and that expression of CaR may be related to the migration of these neurons into the cortical plate. Once they attain their final position in the cortex many GABA+ cells loose CaR-immunoreactivity, so that in postnatal life only a minority of GABA+ neurons contain this calcium-binding protein.

Transient co-localization of calretinin, parvalbumin, and calbindin-D28K in developing visual cortex of monkey.

This paper reports a double-labelling immunocytochemical study of the three calcium-binding proteins calretinin, parvalbumin, and calbindin-D28k in developing and adult Macaca primary visual cortex. In adult visual cortex, each protein marks a subset of GABAergic neurons with a characteristic laminar distribution and virtually no co-localization was found between these three proteins, suggesting that each calcium-binding protein may serve as a marker for one or more cortical subcircuits. The immature visual cortex, immunostained using identical techniques was then analysed to determine if each calcium-binding protein could serve as a developmental marker for these circuits. The Cajal-Retzius cells of layer 1 contained all three proteins during development. Calbindin-D28k and calretinin were co-localized starting at Fd (foetal day) 45 and after Fd125, parvalbumin also was present in the same Cajal-Retzius cells. All three proteins continued to be expressed until the Cajal-Retzius disappeared postnatally. In layers 2-6 calbindin-D28k and calretinin were never co-localized. In contrast, parvalbumin and calretinin were found in neurons of deep layer 3 from Fd 155 to postnatal (P6) weeks with a few persisting even later. Before birth almost all PV+ neurons in layers 4-6 were CaB+, but by P3 weeks only a few PV+/CaB+ neurons remained in layer 4C and these completely disappeared by P6 weeks. Co-localization in layer 4 neurons overlaps the period of ocular dominance segregation, suggesting that the onset of cortical maturity coincides with segregation of calcium-binding proteins within the GABA interneurons.