Cells In Upper Layers Research Articles

A synchronous photoautotrophic-heterotrophic biofilm cultivation mode for Chlorella vulgaris biomass and lipid simultaneous accumulation

Microalgae biofilm was a porous structure formed by the accumulation of microalgae cells. The cells in upper layer of biofilm could receive light for photosynthetic growth and meanwhile removed the inorganic nitrogen and phosphorus etc. From wastewater to cleaner biomass. While with the increase of biofilm thickness, light could not reach the underlayer, forming a natural dark area in the bottom of the biofilm for microalgae heterotrophic growth. The naturally formed synchronous photoautotrophic-heterotrophic microalgae growth environment realized the synchronization of biofilm growth and lipid enrichment. The transmission of metabolic gases made the biofilm loose and porous (120.18% increase on the biofilm porosity), which provided more channels for the transmission of nutrients. Due to the synchronous heterotrophic enrichment in the bottom layer of biofilm, the maximum growth rate of biomass cultivated under synchronous photoautotrophic-heterotrophic biofilm was 3.04 times higher than that of photo-autotrophic biofilm and lipid yield increased by 2.22 times. The proportion of photosynthetic autotrophy of cells in the biofilm increased with the increase of light intensity which made an increase of 33.45% in the growth rate of biofilm but with a price of 43.88% reducing in the lipid content. While, the increase of heterotrophic proportion in the biofilm realized the simultaneous enhancement of biomass density and lipid content. The biomass productivity increased to 55.81 g m−2 d−1 and the lipid content increased to 46.79% at the same time. Thus, it solved the contradictory between the growth of microalgae and the accumulation of lipid, and provided a new idea for the application of changing wastewater resources to clean biofuel. It would have an important significant to the development of low-carbon renewable and clean energy.

Adipocyte Stem Cells Ameliorate Total Body Irradiation-Induced Hematopoietic Syndrome and Late Radiation Fibrosis

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at day 14 post irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell coculture revealed significant down regulation of profibrotic genes (TGF β, and Col 1-4) in irradiated cells. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Irradiated HFF revealed high expression of HGF receptor c-Met. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson’s Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation-induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. A single 1 x 106 ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.

Adipocyte Stem Cells Ameliorate Total Body Irradiation Induced Hematopoietic Syndrome and Late Radiation Fibrosis

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse or 3) human cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice (n=12) received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P. saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at days 1, 14, and 21 post-irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender-mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell ASCs demonstrated significant down regulation in lower level cells of pro-fibrotic genes (including collagen 1-4, and TGF β) in unirradiated acutely irradiated and irradiation- fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson’s Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. Single 1 x 106 ASCs injection day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.

Abstract 980: Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF)

Abstract Radiation therapy doses above 50Gy to head and neck, breast cancer and soft tissue sarcomas can result in the rare late complication of RIF, which can significantly impair function. Several published case studies suggest injection of fibrotic sites with autologous adipose tissue stem cells can ameliorate RIF. In this study, we elucidated the molecular mechanism in an in vitro co-cultivation system and in vivo using a mouse model of hind limb fibrosis. In vitro Transwell co-cultures contained a bottom layer of irradiated human foreskin fibroblasts (HFFs), mouse or human cell lines derived from clinical fibrosis biopsies, and an upper layer containing freshly prepared ASCs. We quantitated levels of fibrosis -related genes in the lower layer cells and regulatory cytokines in the upper layer cells using quantitative real-time (qRT) PCR. In vivo studies: Female C57BL/6 mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electron beam. The irradiated and contralateral unirradiated flank tissue was monitored for expression of fibrosis related genes at days 1 and 14 post irradiation (PI). Fibrosis was confirmed by histologic staining for collagen and range of limb motion measurements. Irradiated fibrotic sites were injected at day 28 with ASCs from luciferase+ GFP+ mice. RIF was uniformly detected at day 14 and amplified by day 28, and confirmed by histologic staining for collagen by Masson's Trichrome. At day 14 PI there was clear upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), collagen 1 (400 fold), collagen3 (500 fold) and collagen4 (500 fold) compared to nonirradiated contralateral hind limb tissue. Irradiation induced by day 28 a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p &amp;lt; 0.0001) degrees in the contralateral nonirradiated limb. A single ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). To measure possible systemic radiation mitigatory effects of ASCs PI, a single intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (P = 0.047). In transwell co-cultures ASCs demonstrated significant downregulation of profibrotic genes (including collagen 1-4, and TGF β) in both acutely irradiated and irradiation-fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was a key secreted mediator in co-culture models. Addition of human recombinant HGF to irradiated HFFs significantly downregulated profibrotic gene transcripts. Thus, HGF secreted by ASCs is involved in the reduction of RIF in vivo and with in vitro models of radiation fibrosis. Further studies should elucidate the mechanism of HGF and other secreted and cell contact regulators of adipose tissue-based cell therapy of radiation fibrosis. Citation Format: Asim Ejaz, Micheal Epperly, Renne Fisher, Xichen Zhang, Moriah Johngrass, Asher Schusterman, Lauren Kokai, Joel Greenberger, Peter Rubin. Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 980.

The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0

ABSTRACTPurpose: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.Methods: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.Results: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.Conclusions: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

POU-III Transcription Factors (Brn1, Brn2, and Oct6) Influence Neurogenesis, Molecular Identity, and Migratory Destination of Upper-Layer Cells of the Cerebral Cortex

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.

A new approach to solving Poisson system for free surface nonhydrostatic flow simulations

SUMMARY A nonhydrostatic finite volume model is presented to simulate free surface flow in a two-dimensional vertical plane. The algorithm is based on a projection method including the solution of the pressure Poisson equation (PPE). The model is developed in a Cartesian grid in which the size of all the cells in the computational domain, excluding those of the top layer, is constant in time. To simulate the variable water surface, the heights of the top layer cells are variable and proportional to the local water elevation. Taking the layout of the grid system into consideration, a new method is proposed to solve the PPE derived in Cartesian coordinates. In this method, the system of pressure equations is divided into two subsystems, namely a subsystem for the upper layer cells and another for the remaining cells. The coefficient matrix of the former is variable with respect to time, whereas that of the latter remains constant. Therefore, the coefficient matrix of the latter subsystem can be inversed once and saved throughout the simulation. The application of this procedure reduces the computational cost compared with other PPE solvers in certain conditions. The model is applied to simulate a series of numerical tests including strong vertical accelerations and is verified against analytical and experimental results, demonstrating satisfactory performance. Copyright © 2011 John Wiley & Sons, Ltd.

Keratinocyte Differentiation and Proliferation are Regulated by Adhesion to the Three-Dimensional Meshwork Structure of Type IV Collagen

We examined the behavior of human foreskin keratinocytes (HFKs) on reconstituted type IV collagen gel. HFKs survived for several days and the upper layer cells expressed a differentiation marker, involucrin. Apoptosis was induced after involucrin expression while cell proliferation was suppressed. On molecular type IV collagen, integrins shifted from α2β1 to α3β1 during HFK culture. On type IV collagen gel, HFKs initially expressed integrin α2β1, and later expressed integrin α3β1 in the presence of α2β1 did not disappear. Using synthetic peptides, we examined integrin α2-mediated adhesion to type IV collagen gel. Addition of synthetic peptide dose-dependently inhibited cell adhesion both on type IV collagen gel and on molecular type IV collagen. On type IV collagen gel, weaker phosphorylation of focal adhesion kinase, paxillin, and Akt was observed compared with the molecular forms. Based on these observations, we think type IV collagen gel is a novel culture substrate that mimics the physiological environment for HFKs.

The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

The Role of Neural Activity in Cortical Axon Branching

Axonal branching is an important process for establishing the final pattern of connections between a neuron and its target cells. Cortical connections between upper-layer cells in the neocortex have provided insights into the cellular mechanisms by which electrical activity regulates neural connectivity, including branch formation. Recent evidence further indicates that spontaneous firing and synaptic transmission contribute to axonal branching of cortical neurons through postsynaptic activation.

Epithelial–Mesenchymal Transition in Colonies of Rhesus Monkey Embryonic Stem Cells: A Model for Processes Involved in Gastrulation

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Contour integration in striate cortex. Classic cell responses or cooperative selection?

Psychophysics has established various rules of contour integration in gestalt perception. We tested the rule of good continuation by stimulating behaving monkeys with simple figures composed of Gabor patches, while recording from upper layer cells in visual cortex (V1). By decomposing these figures into their components and stimulating receptive field centers and surrounds separately with this stimulus set, we tested center-surround interaction for linearity. In pure-fixation tasks, the interaction was negative for the early strong evoked response, i.e., in this phase figural context rather inhibited the cells. However, in the following tonic response phase, a subgroup of neurons showed positive interaction during the whole stimulus presentation period of at least 1,000 ms. Attention to the figure in discrimination tasks only slightly improved this positive interaction between 150 and 300 ms. We interpret these results as selective cooperation and mutual facilitation of cortical V1 cells, thereby supporting the saliency of borders and contours in perception of visual scenes.

Altered interneuron development in the cerebral cortex of the flathead mutant.

One approach to defining mechanisms essential to neocortical development is to analyze the phenotype of novel spontaneous mutations that dramatically affect the generation and differentiation of different neocortical neurons. Previously we have shown that there is a large decrease in the total number of cortical neurons in the flathead mutant rat, and in this paper we show that the flathead (fh/fh) mutation causes an even larger decrease in the number of interneurons. The decrease in relative interneuron number is different in different cortical lamina and for different interneuron subtypes. Specifically, the percentage of GABA and calretinin- positive cells in upper layers of somatosensory cortex is not appreciably decreased in homozygous mutants, while other interneuron subtypes in somatosensory cortex and all GABA-positive interneuron types in entorhinal cortex are decreased. In addition, the soma and dendritic arbors of interneurons in flathead are greatly hypertrophied, while those of pyramidal neurons are not. Furthermore, we found that at embryonic day 14, flathead mutants display high levels of cell death throughout both the neocortical and ganglionic eminence (GE) proliferative zones with a larger increase in cell death in the GE than in the neocortical VZ. In addition, we provide evidence that there is widespread cytokinesis failure resulting in binucleate pyramidal cells and interneurons, and the number of binucleate interneurons is greater than the number of binucleate pyramidal neurons. Together, these results demonstrate that the fh mutation causes dramatic alterations in interneuron development, and suggest that the flathead mutation causes differential cytokinesis failure and cell death in different types of neocortical progenitors.

A neural network model for the self-organization of cortical grating cells.

A neural network model with incremental Hebbian learning of afferent and lateral synaptic couplings is proposed,which simulates the activity-dependent self-organization of grating cells in upper layers of striate cortex. These cells, found in areas V1 and V2 of the visual cortex of monkeys, respond vigorously and exclusively to bar gratings of a preferred orientation and periodicity. Response behavior to varying contrast and to an increasing number of bars in the grating show threshold and saturation effects. Their location with respect to the underlying orientation map and their nonlinear response behavior are investigated. The number of emerging grating cells is controlled in the model by the range and strength of the lateral coupling structure.

Extrastriate feedback to primary visual cortex in primates: a quantitative analysis of connectivity.

Knowledge-based or top-down influences on primary visual cortex (area V1) are believed to originate from information conveyed by extrastriate feedback axon connections. Understanding how this information is communicated to area V1 neurons relies in part on elucidating the quantitative as well as the qualitative nature of extrastriate pathway connectivity. A quantitative analysis of the connectivity based on anatomical data regarding the feedback pathway from extrastriate area V2 to area V1 in macaque monkey suggests (i) a total of around ten million or more area V2 axons project to area V1; (ii) the mean number of synaptic inputs from area V2 per upper-layer pyramidal cell in area V1 is less than 6% of all excitatory inputs; and (iii) the mean degree of convergence of area V2 afferents may be high, perhaps more than 100 afferent axons per cell. These results are consistent with empirical observations of the density of radial myelinated axons present in the upper layers in macaque area V1 and the proportion of excitatory extrastriate feedback synaptic inputs onto upper-layer neurons in rat visual cortex. Thus, in primate area V1, extrastriate feedback synapses onto upper-layer cells may, like geniculocortical afferent synapses onto layer IVC neurons, form only a small percentage of the total excitatory synaptic input.

Thalamic control of cat area-18 supragranular layers: simple cells, complex cells, and cells projecting to the lateral suprasylvian visual area.

The goal of this study was to determine the effects of inactivating layer A or the C layers of the cat lateral geniculate nucleus on the supragranular layers of area 18, including cells antidromically activated from the lateral suprasylvian visual area (LS). Isolated cells were visually driven via the contralateral eye while the retinotopically corresponding regions of layer A or, in some cases, the C layers were reversibly inactivated with injections of cobaltous chloride. Simple cells were frequently encountered and were on average more dependent upon layer A than were complex cells, a result qualitatively similar to that found previously in area 17 (Malpeli, 1983; Malpeli et al., 1986). However, the influence of the C layers on area 18 was much more apparent than for area 17. In area 18, as in area 17, the dependence of simple cells on particular geniculate layers appears to follow the terminal patterns of the major direct geniculate inputs. Those simple cells most dependent on layer A were located in lower layer 3. Simple cells in upper layer 3, like complex cells, showed little dependence on layer A, but were strongly dependent upon the C layers. All cells antidromically activated from LS were simple cells with rapidly conducting axons. They had, on average, the same moderately strong dependence on layer A as the patches of LS receiving area 18 input (Lee et al., 1997), supporting the conclusion that the influence of layer A in these patches is largely transmitted via association inputs from area 18. These results demonstrate that simple cells play a major role in association pathways.

Local-circuit neurones in the medial prefrontal cortex (areas 25, 32 and 24b) in the rat: morphology and quantitative distribution.

This paper is a light microscopical study describing the detailed morphology and quantitative distribution of local circuit neurones in areas 25, 32, and 24b of the medial prefrontal cortex (mPFC) in the rat. Cortical interneurones were identified immunocytochemically by their expression of calretinin (CR), parvalbumin (PV), and calbindin D-28k (CB) immunoreactivity. Neurones immunoreactive for gamma-aminobutyric acid (GABA) were also investigated, as were interneurones containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity. Several distinct classes of CR+, PV+, and CB+ neurones were identified; the most frequent were: bipolar/bitufted CR+ cells in upper layer 3; multipolar PV+ neurones in layers 3 and 5; and bitufted/multipolar CB+ neurones in lower layer 3. CB+ neurones resembling Martinotti and neurogliaform cells were also present in layers 5/6. The morphologies and depth distributions of each cell type were consistent across the three areas of mPFC studied. Seven classes of diaphorase-reactive mPFC neurone are described; these cells were composed about 0.8% of the total neurone population and had a peak distribution located in mid- to lower layer 5 in each area. In areas 32 and 25, three defined bands of diffuse NADPH diaphorase staining were located in layer 2 and in upper and deep layer 5. Diaphorase reactivity was very infrequently colocalised with either CR, PV, or CB immunoreactivities. The numerical densities of neurones (N(V), number of cells per mm3) in each layer were calculated stereologically. The mean total neuronal N(V) estimate for areas 25, 32, and 24b was 51,603 +/- 3,324 (mean +/- S.D.; n = 8). Significant interareal differences were detected. From cortical thickness data and neuronal N(V) estimates, the absolute number of neurones under 1 mm2 of cortical surface (N(C)) have been derived. The mean N(C) value for areas 25, 32, and 24b was 57,328 +/- 7,505 neurones. In immunolabelled Nissl-stained sections, CR+ neurones constituted an overall 4.0%, PV+ cells 5.6%, and CB+ 3.4% of the total neurone populations in mPFC. GABA+ cells represented a mean of 16.2% (14.8-17.2%) of neurones in areas 25, 32 and 24b. The absolute numbers of CR+, PV+, CB+, and GABA+ neurones within individual layers in a column of cortex under 1 mm2 of cortical surface (N(L)) have also been derived, with significant interareal differences in N(L) values being detected. The data provide the structural basis for a qualitative and quantitative definition of local cortical circuits in the rat mPFC.

The influence of input from the lower cortical layers on the orientation tuning of upper layer V1 cells in a primate.

The receptive fields of cells in the primary visual cortex (area 17 or V1) show clear orientation selectivity, unlike those of the lateral geniculate nucleus (LGN) cells which provide their visual input. The intrinsic circuitry of V1 cells is believed to be partly responsible for this selectivity. We investigated the influence of ascending projections from neurons in the lower layers (5 and 6) of V1 on the orientation selectivity of single neurons in the upper layers (2,3, and 4) by reversibly inactivating ("blocking") lower layer neural activity with iontophoretic application of gamma-aminobutyric acid (GABA) while recording from upper layer cells in the prosimian primate, Galago crassicaudatus. During lower layer blocking, the majority (20/28 = 71.4%) of upper layer neurons exhibited a change in the orientation of their preferred stimulus, a reduction in their orientation tuning, and/or an increase in their response amplitude. Twelve (42.9%) neurons exhibited shifts in their preferred orientation averaging 11 (+/- 4) deg. These neurons were located on average, 272 (+/- 120) microns tangential from the vertical axis of the pipette center. Eleven neurons (39.2%) exhibited an average reduced orientation tuning of 52.5%. Their average location was 230 +/- (115) microns away from the vertical axis of the pipette. Five (17.9%) neurons with average location 145 (+/- 75) microns from the vertical axis exhibited both effects. Two (7.1%) neurons that exhibited significant increases in response amplitude to stimulus angles within 10 deg of the peak excitatory stimulus without changes in orientation selectivity or tuning were located less than 100 microns from the vertical axis. The effects on the orientation tuning of cells were restricted in all cases to within +/- 30 deg of the preferred stimulus orientation. This suggests that layer blocking affects cells with preferred stimulus orientations similar to those of the recorded neurons. Only cells located within 500 microns tangential to the vertical axis of the injection site exhibited these effects. These results suggest that cells within layers 5 and 6 provide organized, orientation-tuned inhibition that sharpens the orientation tuning of cells in the upper cortical layers within the same, or closely neighboring, cell columns.

Two-dimensional receptive-field organization in striate cortical neurons of the cat.

The two-dimensional organization of receptive fields (RFs) of 44 cells in the cat visual cortex and four cells from the cat LGN was measured by stimulation with either dots or bars of light. The light bars were presented in different positions and orientations centered on the RFs. The RFs found were arbitrarily divided into four general types: Punctate, resembling DOG filters (11%); those resembling Gabor filters (9%); elongate (36%); and multipeaked-type (44%). Elongate RFs, usually found in simple cells, could show more than one excitatory band or bifurcation of excitatory regions. Although regions inhibitory to a given stimulus transition (e.g. ON) often coincided with regions excitatory to the opposite transition (e.g. OFF), this was by no means the rule. Measurements were highly repeatable and stable over periods of at least 1 h. A comparison between measurements made with dots and with bars showed reasonable matches in about 40% of the cases. In general, bar-based measurements revealed larger RFs with more structure, especially with respect to inhibitory regions. Inactivation of lower cortical layers (V-VI) by local GABA injection was found to reduce sharpness of detail and to increase both receptive-field size and noise in upper layer cells, suggesting vertically organized RF mechanisms. Across the population, some cells bore close resemblance to theoretically proposed filters, while others had a complexity that was clearly not generalizable, to the extent that they seemed more suited to detection of specific structures. We would speculate that the broadly varying forms of cat cortical receptive fields result from developmental processes akin to those that form ocular-dominance columns, but on a smaller scale.

A study on cell lineage, especially the germ cell line, in embryos of the teleost fish,Barbus conchonius

Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.