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.

Distribution of cones in human and monkey retina: individual variability and radial asymmetry.

The distribution of photoreceptors is known for only one complete human retina and for the cardinal meridians only in the macaque monkey retina. Cones can be mapped in computer-reconstructed whole mounts of human and monkey retina. A 2.9-fold range in maximum cone density in the foveas of young adult human eyes may contribute to individual differences in acuity. Cone distribution is radially asymmetrical about the fovea in both species, as previously described for the distribution of retinal ganglion cells and for lines of visual isosensitivity. Cone density was greater in the nasal than in the temporal peripheral retina, and this nasotemporal asymmetry was more pronounced in monkey than in human retina.

Stratified distribution of synapses in the inner plexiform layer of primate retina.

Distributions of bipolar (B) and amacrine (A) synapses and postsynaptic ganglion cell (G) dendritic profiles in the inner plexiform layer (IPL) were analyzed in EM montages of monkey central and human foveal and peripheral retinae. Synapses and profiles were counted and plotted for each 5% interval of IPL, with 0% at the inner edge of the inner nuclear layer and 100% at the outer edge of the ganglion cell layer. In monkey and human retinae, both A and B synapses occur throughout the IPL, but the ratio of A to B synapses varies from 2:1 to more than 6:1. In the monkey central retina, four bands of A conventional synapses are concentrated at 15, 35, 60, and 80% depth. In the human foveal slope, there are two main A bands at 45 and 85%, whereas in the human periphery, there are five bands at 15, 35, 60, 75, and 90%. In both species, A processes containing large dense-core vesicles are concentrated in three bands at 10-20, 50, and 80-90% depth, corresponding to previously described levels of peptides, dopamine, and GABA. B ribbon synapses are distributed fairly evenly throughout the IPL, with a suggestion of four broadly overlapping bands. Most B ribbons are presynaptic to one A and one G (B----A/G). In the human, there are significantly more B dyads with postsynaptic G's (B----A/G, B----G/G) in the fovea (91%) than in the periphery (66%), implying greater A cell processing peripherally. Also in the human, B terminals containing glycogenlike granules are concentrated in the outer half of the IPL, with agranular terminals in the inner half. Our results demonstrate multiple strata containing different types of synaptic contacts in primate IPL.

The structural organization of the inferior and lateral subdivisions of the Macaca monkey pulvinar.

Previous light microscopic studies of Macaca pulvinar have demonstrated that both the inferior and adjacent portion of the lateral pulvinar subdivisions are reciprocally connected to the entire occipital lobe, including striate cortex. They differ in that inferior but not lateral pulvinar receives a projection from the superficial layers of the superior colliculus. In this study, the internal organization of these two subdivisions in compared by relating light microscopic Golgi morphology to the synaptic organization observed by electron microscopy. The Golgi impregnated neurons in inferior and lateral pulvinar are typical of other thalamic nuclei and are not qualitatively different in the two subdivisions. Projections neurons (PN) vary in cell body (15--40 micrometers) and dendritic tree (150--600 micrometers) diameters but bear the same varieties of dendritic appendages; spine-like, hair-like, and knot-like. Local circuit neurons (LCN) have smaller cell body diameters (10--20 micrometers) but can have very large dendritic field diameters (150--600 micrometers). They are best distinguished from PNs by their elaborate dendritic appendages, which have been identified as pre-synaptic dendrites in the EM. LCN axons are infrequently seen. In the EM both subdivisions contain four types of synaptic terminals. RS and RL terminal both contain round symaptic vesicles and make asymmetric synaptic contacts, but are subdivided on the basis of small (RS = 0.09 micrometers) versus large (RL = 2.2 micrometers) cross sectional diameters and organelle content. RLs contact larger caliber dendrites and frequently form synaptic complexes with presynaptic dendrites of LCNs, while RSs contact fine caliber dendrites and rarely take part in synaptic complexes. F terminal and P boutons both contain flat and pleomorphis vesicles and make symmetric synaptic contacts. They are characterized by vesicle number and cytoplasmic density. Fs are infrequently observed in pulvinar compared to P boutons and are of uncertain origin. P boutons can be equated with LCN dendritic appendages and have been identified as pre-synaptic dendrites. The quantitative distribution of each type is very similar in both subdivisions, avveraging RS 85%, RL 5%, F 0.3%, P 8% and unidentified 2%.

The morphology and distribution of striate cortex terminals in the inferior and lateral subdivisions of the Macaca monkey pulvinar.

The origin of the various types of axon terminals in Macaca pulvinar remains uncertain because of the contradictory results obtained in EM degeneration studies. We have used EM-autoradiography to determine the morphology of terminals in the inferior and lateral pulvinar which originate from neurons in visual cortex. After injections of H3 proline into area 17, both the small diameter (RS) and the large diameter (RL) terminals containing round vesicles and making asymmetric contacts are labeled in the two pulvinar subdivisions. Labeled and unlabeled terminals are intermixed within the pulvinar focus which suggests that the dendrites of the same pulvinar neuron receive overlapping inputs from several cortical areas. Because only 5% of the pulvinar terminals are RLs (Ogren and Hendrickson, '79), and this small number of RLs originates from at least two visual cortical areas plus the superior colliculus (Partlow et al., '77), superior colliculus input to inferior pulvinar is small compared to the combined RS and RL cortical input. Together the findings from this study and the preceding paper (Ogren and Henderickson, '79), show that while pulvinar is typical of other thalamic nuclei in the structure of its neurons and synapses, it differs in that the input from subcortical structures is minimal. It is suggested that inferior and lateral pulvinar function principally as integrators of visula cortical information.

Neuronal and synaptic structure of the dorsal lateral geniculate nucleus in normal and monocularly deprived Macaca monkeys.

The dorsal lateral geniculate nucleus (dLGN) of Macaca monkeys was studied by Golgi and quantitative electron microscopic (EM) methods to determine if differences in neuronal morphology exist which might correlate with the known physiological separation of X-type cells into the parvocellular and Y-type cells into the magnocellular laminae. Monocularly lid-sutured Macaca monkeys were also studied by quantitative EM methods to compare the synaptic organization within laminae innervated by the deprived and nondeprived retinae. We have divided our sample of Golgi-impregnated neurons into three groups: Types A, B, and C. Type A neurons comprise the majority of the projections cells and are quite heterogeneous in their overall morphology. Type B neurons have long dendrites with multiple appendages; some have a locally ramifying beaded axon Type C neurons are characterized by dendrites which are mainly restricted to the interlaminar zones. We found Type A and B neurons in both the parvocellular and magnocellular laminae. The cell bodies of Type C neurons lay within the interlaminar zones or the parvocellular laminae. All three types contributed dendrites to the interlaminar zones. No significant differences in Golgi morphology other than overall size were found in parvocellular or magnocellular laminae that would explain the previously demonstrated electrophysiological differences. Terminal profiles and synapses in the parvocellular, magnocellular, and interlaminar zones were classified and counted using quantitative EM methods. RSD and F terminals were most numerous in all three zones. RLP terminals were rare in the interlaminar zones. A new type of terminal, RMD was found in the magnocellular interlaminar zones. The laminar and interlaminar zones have the same overall synaptic density, but differed in types of synaptic terminals. The only quantitative difference between any of these regions was in the magnocellular laminae where the counts showed 70% more F terminals per unit area. The same quantitative methods were applied to the laminar and interlaminar zones of dLGNs from monocularly lid-sutured monkeys. We found no qualitative or quantitative difference between the synaptology of zones receiving input from the deprived retina compared to the open eye retina, nor between any regions of the dLGN in deprived monkeys compared to normal monkeys.

Development redistribution of photoreceptors across the Macaca nemestrina (pigtail macaque) retina.

Redistributions of monkey cones and rods during the first year after birth include a fivefold increase in peak foveal cone density from 43,000 to 210,000 cones/mm2, a decrease in the diameter of the rod-sparse area, and a two- to threefold decrease in peripheral photoreceptor density. Two weeks before birth, higher cone density is already apparent in the future fovea, as are the nasotemporal asymmetry in cone distribution, a higher density "cone streak" along the horizontal meridian, a large rod-sparse central fovea, and a ring of high rod density. Despite the early appearance of these basic patterns, photoreceptor distribution is not mature until 1 to 5 years postnatally. Total cones varied from 4 million at birth to 3.1 million in the average adult. The two oldest eyes had fewer cones, suggesting up to a 25% loss late in development. There were 60 to 70 million rods in the adult macaque retina and little evidence of postnatal changes in number. Neither of these small changes is sufficient to account for the reduction in peripheral photoreceptor density and both are in the wrong direction to explain increasing foveal density, ruling out a major role for either photoreceptor death or generation. Retinal area increased by a factor of 2.4 from 2 weeks before birth to adulthood. In contrast, the posterior pole of the retina was dimensionally stable throughout this period, with the distance between the fovea and optic disc varying nonsystematically from 3.37 to 4.05 mm. Retinal coverage of the globe was also stable at 48-60%. Thus postnatal growth can be ruled out as a factor in the density changes occurring in central retina. Adult retinas have a higher proportion of both cones and rods in midperiphery, whereas young retinas have a higher proportion of photoreceptors in far periphery. It appears that photoreceptors are radially redistributed from peripheral toward central retina during postnatal development, resulting in the marked increase in foveal cone density and the decrease in the eccentricity of the rod ring. Up to 13 weeks postnatally, midperipheral growth of the retina is substantial and increases with eccentricity. At later ages, expansion continues only in the very far periphery. Retinal growth appears sufficient to explain the decreases in peripheral rod and cone density with age. These and previous data strongly suggest that differentiated photoreceptors, with complex cytology and synaptic contacts, migrate toward the foveal center, explaining the increase in foveal photoreceptor density.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomical consequences of long-term monocular eyelid closure on lateral geniculate nucleus and striate cortex in squirrel monkey.

The effects of long-term monocular deprivation on the geniculostriate system in squirrel monkeys were studied with neuroanatomical methods. Four neonates were visually deprived by monocular eyelid suture during their first 10 days of life and survived from 9 to 40 months. In the lateral geniculate nucleus (LGN), deprivation resulted in severe cell size changes. Neurons in the deprived laminae were smaller compared to those in the undeprived laminae. Deprivation left the reciprocal connections between LGN and striate cortex intact: After horseradish peroxidase (HRP) injections into striate cortex, retrogradely transported enzyme labeled a wedge of neurons in deprived and undeprived LGN laminae; anterogradely transported HRP filled preterminal and terminal axons in this wedge. Following 3H-proline injections into the deprived eye for transneuronal transport, autoradiography showed in the ipsilateral striate cortex a silver grain distribution over most of layer IVc similar to that in normal squirrel monkeys, except for a small strip in the anterior calcarine fissure. Here, a few, irregularly spaced "patches" of higher grain density occurred deep in layer IVc. Layer IVc of contralateral area 17 was also uniformly labeled over most of its extent, except for a very few and inconspicuous accumulations of slightly increased silver grains. After visual stimulation of the deprived eye, the 14C-2-deoxyglucose method showed in the contralateral striate cortex some alternating "patches" of higher uptake superimposed on the heavy labeling in layer IVc. Layer IVc in the ipsilateral cortex was more uniformly labeled. Regularly spaced arrays of labeled "puffs" in layers II/III were present in both hemispheres. Cytochrome oxidase staining showed no change in the distribution pattern of the enzyme in the deprived monkeys from the basic pattern of normal adults. No changes in cell sizes were found in layer IVc in cresyl-violet-acetate-stained sections. These results lead to the conclusion that in area 17 of squirrel monkeys there is no distinct segregation of inputs from the two eyes into anatomically discrete ocular dominance columns and they support the view of a predominantly binocular organization of area 17.

Quantitative analysis of synaptogenesis in the inner plexiform layer of macaque monkey fovea.

Synaptogenesis has been tracked by using quantitative electron microscopic methods in the inner plexiform layer (IPL) of the developing Macaca monkey fovea from fetal day (Fd) 55 to Fd132. Vesicle-containing profiles were classified according to whether (1) they contained a ribbon indicating that they originated from a bipolar cell, or (2) the profile formed a junction. Group 2 was further subdivided by morphological characteristics into (2a) amacrine, (2b) bipolar, or (2c) unknown profiles. Ribbon-containing bipolar profiles are clearly identifiable at Fd55 when they occur at a density of 0.9/100 microns2. Bipolar synapses increase rapidly to 4.7/100 microns2 by Fd88, similar to their density at Fd132. Identifiable amacrine profiles forming a junction are rare at Fd55-68. By Fd88, amacrine synaptic density has jumped to 6.7/100 microns2 and continues to increase to 9.5/100 microns2 at Fd132. These quantitative data strongly suggest that, at the Macaca fovea, bipolar synaptogenesis both begins and ends before amacrine synaptogenesis. The large number of immature amacrine synaptic profiles and densities at Fd132 suggests that amacrine synapses continue to form after Fd132. This study confirms that cone-dominated monkey fovea has a different sequence of synaptogenesis than the rod-dominated peripheral retina (Nishimura and Rakic, [1985] J. Comp. Neurol 241:420-434). The data support the concept that synaptic developmental sequence is determined by the type of photoreceptor which dominates a particular retinal region or species. Bipolar ribbon synapses are observed in the outer half of the IPL at Fd55, are present in the inner IPL at Fd60, and then, with increasing age, are found throughout the IPL. This pattern strongly suggests that vertical OFF bipolar pathways form earlier than ON pathways in the IPL. In contrast, amacrine profiles are found throughout the IPL at the youngest ages, with an adult-like banding pattern present by Fd132.

Photoreceptor topography of the retina in the adult pigtail macaque (Macaca nemestrina).

In spite of the crucial role retinal photoreceptors play in mapping optical images into a pattern of neural excitation, there are no complete studies of photoreceptor topography in any primate retina. We have measured the spatial density and inner segment areas of cones and rods across the whole mounted retinas of three adult pigtail macaques (Macaca nemestrina) and constructed maps of photoreceptor density and inner segment diameter. These retinas contain an average of 3.1 million cones (2.8-3.3 million), with an average peak foveal cone density of 210,000 cones/mm2 (190,000-260,000 cones/mm2). Cone density falls steeply with increasing eccentricity, to 100,000 cones/mm2 at 200 microns from the fovea, and to 50,000 cones/mm2 at 750 microns. Imposed on this gradient is a "streak" of higher cone density along the horizontal meridian. At equivalent eccentricities, cone density is higher in nasal and inferior retina. Cone inner segments increase in diameter from 2.3 microns at the foveal center to 11 microns in far temporal retina and 10 microns in far nasal retina. These retinas contain an average of 60.1 million rods (44.9-75.3 million). Rod density is zero within 20 microns of the foveal center, increases to the crest of a "rod ring" at the eccentricity of the optic disk, and then declines. Central rod topography is asymmetric, with higher densities in superior retina. Density along the crest of the rod ring peaks in superior retina at 177,000 rods/mm2, dips as low as 120,000 rods/mm2 along the horizontal meridian, and increases to about 150,000 rods/mm2 in inferior retina. Far peripheral rod topography is relatively symmetric around the fovea. Rod inner segment diameter ranged from 1.5 microns in the fovea to 4 microns at the temporal edge and 3.4 microns at the nasal edge of the retina. At eccentricities exceeding 6 mm, rod inner segment diameter was greater temporally than nasally. Cone inner segments cover 85-90% of the central fovea, with extrareceptor space accounting for the remainder. Cone coverage declines with increasing eccentricity to 20% at the temporal edge and 35% at the nasal edge of the retina. In contrast, rod coverage increases from zero at the foveal center to a maximum of 65% in temporal retina and 50% in nasal retina. The photoreceptor topography of the pigtail macaque is qualitatively similar to that of other macaques and to humans. Photoreceptor topography is formed by a complex interaction between regional changes in cone and rod density and inner segment diameter.

Calcium-binding proteins as markers for subpopulations of GABAergic neurons in monkey striate cortex.

Recent studies have shown that the presence of immunoreactivity for parvalbumin (PV-IR) and calbindin-D 28k (Cal-IR) can be used as markers for certain types of gamma-aminobutyric acid (GABA) immunoreactive interneurons in monkey cerebral cortex. Little quantitative information is available regarding the features that distinguish these two subpopulations, however. Therefore, in this study we localized PV-IR and Cal-IR neurons in Macaca monkey striate cortex and analyzed quantitatively their laminar distribution, cell morphology, and co-localization with GABA by double-labeling immunocytochemistry. PV-IR was found in nonpyramidal cells in all layers of the cortex, although PV-IR cells in layer 1 were rare. In contrast, Cal-IR was found mainly in nonpyramidal cells in two bands corresponding to layers 2-3 and 5-6. We found very few double-labeled PV-IR/Cal-IR cells but confirmed that almost all PV-IR and Cal-IR cells are GABAergic. Overall, 74% of GABA neurons in striate cortex displayed PV-IR compared to only 12% that displayed Cal-IR and 14% that were GABA-IR only. Quantitative analysis indicated that the relative proportion of GABA cells that displayed PV-IR or Cal-IR showed conspicuous laminar differences, which were often complementary. Cell size measurements indicated that PV-IR/GABA cells in layers 2-3 and 5-6 were significantly larger than Cal-IR/GABA cells. Analysis of the size, shape, and orientation of stained cell bodies and proximal dendrites further demonstrated that each subpopulation contained several different types of smooth stellate cells, suggesting that Cal-IR and PV-IR are found in functionally and morphologically heterogeneous subpopulations of GABA neurons. There was a thick bundle of PV-IR axons in the white matter underlying the striate but not prestriate cortex. PV-IR punctate labeling matched the cytochrome oxidase staining pattern in layers 4A and 4C, suggesting that PV-IR is present in geniculocortical afferents as well as intrinsic neurons. Cal-IR neuropil staining was high in layers 1, 2, 4B, and 5, where cytochrome oxidase staining is relatively low. We did not find a preferential localization of either PV-IR or Cal-IR cell bodies in any cytochrome oxidase compartments in layers 2-3 of the cortex. These findings indicate that PV and Cal are distributed into different neuronal circuits.

The neuroanatomical organization of pathways between the dorsal lateral geniculate nucleus and visual cortex in Old World and New World primates.

Pathways between the dorsal lateral geniculate nucleus (dLGN) and visual cortex in Old World (Macaca, Papio, Erythrocebus, Cercopithecus) and New World (Saimiri, Cebus) primates were studied after injections of horseradish peroxidase and H3 or S35 amino acids into the dLGN or visual cortex. Trans-synaptic autoradiography was also used to study these pathways after an injection of H3 proline-fucose into one eye. The subsequent autoradiographs of visual cortex showed that Old World primates have separate eye inputs (ocular dominance columns) in the striate cortex, whereas New World monkeys have overlapping or non-separated eye inputs. In both primate groups the geniculocortical input to layer IVA formed a pattern which resembled a honeycomb in tangential sections, unlike the solidly labeled layer IVC. Also common to the two primate groups was a projection from dLGN to layer VI. There was no dLGN projection to any prestriate area in any of the primates. However, after an injection limited to the prestriate cortex of Macaca, light autoradiographic labeling was seen in the interlaminar zones and the magnocellular and S laminae, demonstrating a prestriate-dLGN pathway. Our results indicate that the primate visual system differs significantly from the cat in having no dLGN projection to area 18. There are also signficant differences between primates in the level at which the possibility of binocularity (of an excitatory nature) first occurs in the striate cortex because in the species studied thus far with neuroanatomical methods, Old World primates have ocular dominance columns in layer IV but most New World monkeys lack them.

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.

Localization of glycine-containing neurons in the Macaca monkey retina.

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars. Synaptic labeling was seen throughout the inner plexiform layer (IPL) but with a preferential distribution in the inner half. Bands of labeled puncta occurred in S2, S4, and S5. Both light and postembedding electron microscopic (EM) immunocytochemistry identified different types of amacrine and bipolar cell bodies and their synaptic terminals. The most heavily labeled Gly+ cell bodies typically were amacrine cells having a single, thick, basal dendrite extending deep into the IPL and, at the EM level, electron-dense cytoplasm and prominent nuclear infoldings. This cell type may be homologous with the Gly2 cell in human retina (Marc and Liu: J. Comp. Neurol. 232:241-260, '85) and the AII/Gly2 of cat retina (Famiglietti and Kolb: Brain Res. 84:293-300, '75; Pourcho and Goebel: J. Comp. Neurol. 233:473-480, '85a). Gly+ amacrines synapse most frequently onto Gly- amacrines and both Gly- and Gly+ bipolars. Gly+ bipolar cells appeared to be cone bipolars because their labeled dendrites could be traced only to cone pedicles. The pattern of these labeled dendritic trees indicated that both diffuse and midget types of biopolars were Gly+. The EM distribution of labeled synapses showed Gly+ amacrine synapses throughout the IPL, but these composed only 11-23% of the amacrine population. Most of the Gly+ bipolar terminals were in the inner IPL, where 70% of all bipolar terminals were labeled. These findings are consistent with previous data from cats and humans and suggest that both amacrine and bipolar cells contribute to glycine-mediated neurotransmission in the monkey retina.

Ultrastructural localization of immunoreactivity in the developing piriform cortex.

The purpose of this study was to determine the ultrastructural basis for the immunoreactivity patterns in synaptic structures during development in layers I and II of the piriform cortex (PC) of rats. Antisera to cholecystokinin (CCK) and glutamic acid decarboxylase (GAD) were used at several different postnatal days (PN) and in adults to describe the distribution, characteristics, and relative frequency of labeled profiles--especially axons and terminals--with emphasis on details of the synaptic contacts. GAD-positive terminals occur from PN 2 to adulthood but only form contacts in deeper sublayers (Ib and II) initially. Contacts increase in layer I after PN 6 and are reduced in layer II after PN 21 when the GAD-labeled terminals and synapses take on adult features with flattened vesicles and symmetric contacts. CCK-labeled terminals are present in deeper sublayers at PN 2 but are few and rarely form contacts. Both terminals and contacts increase between PN 2 and 9, taking on distinctive shapes and vesicle morphology by PN 13. At PN 21 and older, CCK terminals have mainly flattened vesicles and mostly form symmetric contacts onto dendrites and somata in deeper layers (Ib and II). Superficial sublayer Ia has very few CCK-labeled synapses and axons. Thus immunoreactivity occurs in terminals prior to synapse formation; labeling of the presynaptic specializations precedes subsequent maturation; synaptic vesicle morphology and membrane specializations are similar for the vast majority of both CCK and GAD terminals; inhibitory (GABA) synapses are established sooner than the possibly excitatory CCK synapses; a deep to superficial gradient of synaptogenesis is associated with GAD-positive terminals in the PC; and the labeling patterns may be related to critical developmental or synaptogenic periods.

Ultrastructure of synaptic remodeling in piriform cortex of adult rats after neonatal olfactory bulb removal: an immunocytochemical study.

The purpose of this investigation was to study possible remodeling in synaptic structures of the piriform cortex (PC) of adult rats following neonatal deafferentation by removal of the olfactory bulb (OB) at birth. Emphasis was placed on possible qualitative changes in the ultrastructure and immunocytochemical localization of cholecystokinin (CCK, a possible excitatory neurotransmitter or modulator) and glutamic acid decarboxylase (GAD, precursor enzyme to the inhibitory transmitter GABA) in axons, terminals, and synaptic complexes. Light microscopic results in normal adult material show that GAD-positive terminals form a dense band subjacent to the lateral olfactory tract (LOT), become less dense in deeper Ib, and are rare in layer II. Following deafferentation, GAD-positive terminals appear denser and more homogeneously distributed throughout layer I and are also more prevalent in layer II. Ultrastructural results of normals and controls indicate GAD-positive terminals normally contain pleomorphic or flattened vesicles and form symmetric contacts onto dendritic shafts and branches throughout layer I. In deafferented layer I not only do there appear to be greater numbers of symmetric GAD-positive contacts, but in contrast to normals, asymmetric contacts mainly onto spines are now present. Light microscopic results from deafferented material also show an apparent proliferation with spread or sprouting of CCK-positive fibers or axonlike structures mainly into layer Ia, whereas these fibers are normally observed only in the LOT and are generally few in number. Also in normals the few CCK-positive terminals in the area subjacent to the LOT contain flattened or pleomorphic vesicles and form symmetric contacts. Deafferentation results in CCK-positive terminals throughout layer I with a greater frequency of synaptic contacts which now also include a few asymmetric contacts onto spines. The findings clearly show modifications in synaptic patterns of immunocytochemical-labeled terminals that might be compatible with the process of atypical reinnervation of deafferented postsynaptic sites and possible ingrowth of new axons.

Anatomical organization of primate visual cortex area VII.

The Golgi Rapid and Kopsch techniques have been used to provide material for an examination of the internal neuronal organization of cortical area VII of the Macaca monkey. The afferent and efferent relationships of area VII, as shown by axoplasmic transport tracing techniques in our own material and in previous studies in other laboratories, are reviewed. Comparison is made between the internal organisation of VI and VII cortex in terms of (1) the marked different in spiny and nonspiny neurons populations of granular layer 4, (2) the difference in relationship of lamina 6 pyramidal neurons to the overlying layers with a shift away from any relationship to granular layer 4 in VII, and (3) differences in the organization of VI lamina 4B and VII lamina 3B--both similarly placed, fiber-rich bands in the two cortical areas. The extrinsic relationships of VI and VII with the lateral geniculate nucleus, superior colliculus, pulvinar, peristriate cortex, cortical area STS, and with each other are compared in terms of laminar locations of efferent neurons and afferent axon terminal fields. It is hoped that this anatomical survey will provide a better foundation for physiological explorations of the region.

Human photoreceptor topography.

We have measured the spatial density of cones and rods in eight whole-mounted human retinas, obtained from seven individuals between 27 and 44 years of age, and constructed maps of photoreceptor density and between-individual variability. The average human retina contains 4.6 million cones (4.08-5.29 million). Peak foveal cone density averages 199,000 cones/mm2 and is highly variable between individuals (100,000-324,000 cones/mm2). The point of highest density may be found in an area as large as 0.032 deg2. Cone density falls steeply with increasing eccentricity and is an order of magnitude lower 1 mm away from the foveal center. Superimposed on this gradient is a streak of high cone density along the horizontal meridian. At equivalent eccentricities, cone density is 40-45% higher in nasal compared to temporal retina and slightly higher in midperipheral inferior compared to superior retina. Cone density also increases slightly in far nasal retina. The average human retina contains 92 million rods (77.9-107.3 million). In the fovea, the average horizontal diameter of the rod-free zone is 0.350 mm (1.25 degrees). Foveal rod density increases most rapidly superiorly and least rapidly nasally. The highest rod densities are located along an elliptical ring at the eccentricity of the optic disk and extending into nasal retina with the point of highest density typically in superior retina (5/6 eyes). Rod densities decrease by 15-25% where the ring crosses the horizontal meridian. Rod density declines slowly from the rod ring to the far periphery and is highest in nasal and superior retina. Individual variability in photoreceptor density differs with retinal region and is similar for both cones and rods. Variability is highest near the fovea, reaches a minimum in the midperiphery, and then increases with eccentricity to the ora serrata. The total number of foveal cones is similar for eyes with widely varying peak cone density, consistent with the idea that the variability reflects differences in the lateral migration of photoreceptors during development. Two fellow eyes had cone and rod numbers within 8% and similar but not identical photoreceptor topography.

Development of the calcium-binding protein parvalbumin and calbindin in monkey striate cortex.

The development of immunoreactivity for the calcium-binding proteins parvalbumin (PV) and calbindin-D28K (Cal) was studied in Macaca nemestrina striate cortex from fetal (F) 60 days to postnatal (P) 5 + years. We correlated changes in PV and Cal staining patterns with the well-documented developmental sequence for primate striate cortex neuron generation and maturation, synaptogenesis, and thalamocortical axon interactions in an attempt to deduce a functional role for these proteins. Our major findings is that Cal and PV have diametrically opposed developmental patterns except in layer 1. At F60 days both are present only in neurons of layer 1 and the number of labeled cell bodies and processes increases up to F125 days. Almost all Cal+ and PV+ cells in layer 1 disappear by P12 weeks. Cal is present by F113 days in pyramidal and stellate neurons, particularly layers 4-6. The numbers and staining density of cells in layers 2-6 increases up to birth and then both decline by P9-12 weeks. Supragranular layers show a second increase in Cal labeling from P20-36 weeks, and then there is a slow decline to the adult pattern which is reached by P1-2 years. Cell bodies in layers 4A, 4C alpha, and deep 4C beta are heavily Cal+ during pre- and early post-natal periods, but upper 4C beta remains unlabeled. PV is not seen until F155-162 days in layers 2-6. Large stellate and a few pyramidal cells appear first in layers 5/6 and 4C alpha, but PV+ stellate neurons are found in all layers except 4C beta by P6 weeks. Layer 4C beta contains a few PV+ cell bodies at P3 weeks, and light neuropile staining at P6 weeks, but then PV labeling rapidly increases so that by P12 weeks the density of 4C beta exceeds that of 4C alpha. Striate cortex has an adult pattern of cell number and neuropile density by P20 weeks. These developmental patterns suggest that the highest density of Cal cell body staining does not correlate with synaptogenesis, or the postnatal critical period of visually driven, binocular interactions. Rather Cal appears when lateral geniculate axons arrive in cortex, persists over the entire span of thalamocortical interactions, and disappears during the decline of cortical plasticity. The appearance of PV is highly correlated with the onset of complex visually driven activity at birth, while both the number of PV+ cell bodies and the density of PV+ neuropile reach adult levels coincident with the completion of thalamocortical connections.

The origin of efferent pathways from the primary visual cortex, area 17, of the macaque monkey as shown by retrograde transport of horseradish peroxidase.

The retrograde transport of horseradish peroxidase has been used to identify efferent cells in area 17 of the macaque. Cells projecting to the lateral geniculate nucleus are small to medium sized pyramidal neurons with somata in lamina 6 and the adjacent white matter. The projection to the parvocellular division arises preferentially from the upper half of lamina 6, while that to the magnocellular division arises preferentially from the lower part of the lamina. The projection to both superior colliculus and inferior pulvinar arises from all sizes of pyramidal neurons lying in lamina 58 (Lund and Boothe, '75); at least pyramidal neurons of lamina 5B send collateral axon branches to both destinations. Injections with extensive spread of horseradish peroxidase show that many cells of lamina 4B and the large pyramidal neurons of upper lamina 6 also project extrinsically but their terminal sites have not been identified. Other studies have indicated that cells of laminae 2 and 3 project to areas 18 and 19. Therefore every lamina of the visual cortex, with the exception of those receiving a direct thalamic input, contains cells projecting extrinsically. Further, each lamina projects to a different destination and from Golgi studies can be shown to contain cells with specific patterns of dendritic branching which relate to the distribution of thalamic afferents and to the patterns of intracortical connections. These findings emphasise the significance of the horizontal organisation of the cortex with relation to the flow of information through it and contrast with the current concept of columnar organisation shown in physiological studies.