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The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1andProk2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16–19).Prok1mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediatorsIl6,Il1b,Tnf,Cxcl2andCxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression ofProk1or its receptor (Prokr1) in fetal membranes. These results suggest that althoughProk1exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.

Cytoplasmic lipid droplets (LDs) are dynamic cellular organelles; their accumulation is associated with several cellular processes, such as cell proliferation, apoptosis and necrosis. 1H Nuclear Magnetic Resonance (NMR) spectroscopy detects resonances from lipids present in cytoplasmic (LDs); an understanding of the relationship between LD characteristics and NMR lipid signals is important. In this study, five nervous system cancer cell lines were investigated. Nile red staining was used to measure the diameter of LDs. High-resolution magic angle spinning NMR (HR-MAS) was performed on harvested cell pellets to quantify the patterns of lipid signals. LDs were present in all five cell lines with different morphology. An average LD diameter of approximately 0.2 μm was found in all cell types. Diameter of the largest LDs varied across the cell lines. The intensity of NMR lipid signals varied greatly between cell types, and a good correlation was found between total volume of LDs and the proton NMR lipid signal intensity at 0.9 and 1.3 ppm. The correlation implied that little NMR signal is detected from LDs of diameters less than approximately 0.34 μm, most likely due to restriction of rotational motion of the lipids.

Improved non-invasive imaging biomarkers of treatment response contribute to optimising cancer management and metabolites detected by proton magnetic resonance spectroscopy ((1)H MRS) show promise in this area. Understanding (1)H MRS changes occurring in cells during cell stress and cell death in vitro should aid the selection of pertinent biomarkers for clinical use.BT4C glioma cells in culture were exposed to either 50 μM cis-dichlorodiammineplatinum II (cisplatin) or starvation by culture in phosphate buffered saline. High resolution magic angle spinning (1)H MRS was performed on cells using a Varian 600 MHz nanoprobe and metabolites were quantified by a time domain fitting method. Cell viability was assessed by trypan blue, HE, 4',6-diamino-2-phenylindole (DAPI), DNA laddering and annexin V-FITC labelled flow cytometry; propidium iodide flow cytometry was used to assess the cell cycle phase.With cisplatin exposure, cells initially accumulated in the G1 stage of the cell cycle with low numbers of apoptotic and necrotic cells and this was associated with decreases in phosphocholine, succinate, alanine, taurine, glycine and glutamate and increases in lactate and glycerophosphocholine (GPC). Starvation, leading to necrotic cell death within 6-18 h, caused decreases in succinate, alanine, glycine, and glutamate and increases in GPC. Principal component analysis revealed two patterns of metabolite changes, one common to both types of cell stress and another specific for necrosis secondary to cell starvation.(1)H MRS reveals alterations in multiple metabolites during cell cycle arrest and cell death which may provide early biomarker profiles of treatment efficacy in vivo.

Using both NADPH diaphorase and anti-nNOS antibodies, we have identified—from retinal flatmounts—neuronal types in the inner retina of the chicken that are likely to be nitrergic. The two methods gave similar results and yielded a total of 15 types of neurons, comprising 9 amacrine cells, 5 ganglion cells, and 1 centrifugal midbrain neuron. Six of these 15 cell types are ubiquitously distributed, comprising 3 amacrine cells, 2 displaced ganglion cells, and a presumed orthotopic ganglion cell. The remaining nine cell types are regionally restricted within the retina. As previously reported, efferent fibers of midbrain neurons and their postsynaptic partners, the unusual axon-bearing target amacrine cells, are entirely confined to the ventral retina. Also confined to the ventral retina, though with somewhat different distributions, are the “bullwhip” amacrine cells thought to be involved in eye growth, an orthotopic ganglion cell, and two types of large axon-bearing amacrine cells whose dendrites and axons lie in stratum 1 of the inner plexiform layer (IPL). Intracellular fills of these two cell types showed that only a minority of otherwise morphologically indistinguishable neurons are nitrergic. Two amacrine cells that branch throughout the IPL are confined to an equatorial band, and one small-field orthotopic ganglion cell that branches in the proximal IPL is entirely dorsal. These findings suggest that the retina uses different processing on different regions of the visual image, though the benefit of this is presently obscure.

Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.

The visual system of birds includes an efferent projection from a visual area, the isthmo-optic nucleus in the midbrain, back to the retina. Using a combination of anterograde labeling of efferent fibers, reconstruction of dye-filled neurons, NADPH-diaphorase staining, and transmission electron microscopy, we have examined the distribution of efferent fibers and their synaptic structures in the chicken retina. We show that efferent fibers terminate strictly within the ventral retina. In two completely mapped retinas, only 2 fibers from a total of 15,359 terminated in the dorsal retina. The major synapse made by each efferent fiber is with a single efferent target amacrine cell (TC). This synapse consists of 5–25 boutons of 2 μm diameter, each with multiple active zones, pressed into the TC soma or synapsing with a basketwork of rudimentary TC dendrites in the inner nuclear layer (INL). This basketwork, which is sheathed by Muller cell processes, defines a private neuropil in the INL within which TCs were also seen to receive input from retinal neurons. In addition to the major synapse, efferent fibers typically produce several very thin processes that terminate nearby in single small boutons and for which the soma of a local amacrine cell is one of the likely postsynaptic partners. A minority of efferent fibers also give rise to a thicker process, terminating in a strongly diaphorase-positive ball about 5 μm in diameter.

GABAergic amacrine cells, cultured from embryonic chick retina, display spontaneous mini frequencies ranging from 0–4.6 Hz as a result of the release of quanta of transmitter from both synapses and autapses. We show here that at least part of this variation originates from differences in the degree to which endocannabinoids, endogenously generated within the culture, are present at terminals presynaptic to individual cells. Though all cells examined scored positive for cannabinoid receptor type I (CB1R), only those showing a low initial rate of spontaneous minis responded to CB1R agonists with an increase in mini frequency, caused by a Gi/o-mediated reduction in [cAMP]. Cells displaying a high initial rate of spontaneous minis, on the other hand, were unaffected by CB1R agonists, but they did show a rate decrease with CB1R antagonists. Such a regulation of spontaneous transmitter release by endocannabinoids might be important in network maintenance in amacrine cells and other inhibitory interneurons.

It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.

Miniature postsynaptic currents (minis) in cultured retinal amacrine cells, as in other central neurons, show large variations in amplitude. To understand the origin of this variability, we have exploited a novel form of synapse in which pre- and postsynaptic receptors sample the same quantum of transmitter. At these synapses, mini amplitudes measured simultaneously in the 2 cells show a strong correlation, accounting for, on average, more than half of the variance in amplitude. Two pieces of evidence support the conclusion that variations in the amount of transmitter in different quanta underlie this correlation. First, diazepam, which enhances GABA binding, increases mini amplitude, implying therefore that transmitter concentration is not saturating. Second, we show that amplitude distributions from all cells, even those with a small number of release sites, have the same shape, implying that most or all variance is intrinsic to each release site.

As Cajal discovered in the late 19th century, the bird retina receives a substantial input from the brain. Approximately 10,000 fibers originating in a small midbrain nucleus, the isthmo-optic nucleus (ION), terminate in each retina. The input to the ION is chiefly from the optic tectum which, in the bird, is the primary recipient of retinal input. These neural elements constitute a closed loop, the centrifugal visual system (CVS), beginning and ending in the retina, that delivers positive feedback to active ganglion cells. Several features of the system are puzzling. All fibers from the ION terminate in the ventral retina and an unusual axon-bearing amacrine cell, the target cell, is the postsynaptic partner of these fibers. While the rest of the CVS is orderly and retinotopic, target cell axons project seemingly at random, mostly to distant parts of the retina. We review here the most significant features of the anatomy and physiology of the CVS with a view to understanding its function. We suggest that many of the facts about this system, including some that are otherwise difficult to explain, can be accommodated within the hypothesis that the images of shadows cast on the ground or on objects in the environment, initiate a rapid and parallel search of the sky for a possible aerial predator. If a predator is located, shadow and predator would be temporarily linked together and tracked by the CVS.

Biomarkers of early response to treatment have the potential to improve cancer therapy by allowing treatment to be tailored to the individual. Alterations in lipids detected by in vivo MRS have been suggested as noninvasive biomarkers of cell stress and early indicators of cell death. An improved understanding of the relationship between MRS lipids and cell stress in vitro would aid in the translation of this technique into clinical use. Rat BT4C glioma cells were treated with 50 µ m cis-dichlorodiammineplatinum II (cisplatin), a commonly used chemotherapeutic agent, and harvested at several time points up to 72 h. High-resolution magic angle spinning (1) H MRS of cells was then performed on a 600-MHz NMR spectrometer. The metabolites were quantified using a time domain fitting method, TARQUIN. Increases were detected in saturated and polyunsaturated fatty acid resonances early during the exposure to cisplatin. The fatty acid CH(2) /CH(3) ratio was unaltered by treatment after allowing for contributions of macromolecules. Polyunsaturated fatty acids increased on treatment, with the group -CH=CH-CH(2) -CH=CH- accounting for all the unsaturated fatty acid signals. Transmission electron microscopy, in addition to Nile red and 4',6-diamino-2-phenylindole co-staining, revealed that the lipid increase was associated with cytoplasmic neutral lipid droplets. Small numbers of apoptotic and necrotic cells were detected by trypan blue, annexin V-fluorescein isothiocyanate-labelled flow cytometry and DNA laddering after up to 48 h of cisplatin exposure. Propidium iodide flow cytometry revealed that cells accumulated in the G1 stage of the cell growth cycle. In conclusion, an increase in the size of the lipid droplets is detected in morphologically viable cells during cisplatin exposure. (1) H MRS can detect lipid alterations during cell cycle arrest and progression of cell death, and has the potential to provide a noninvasive biomarker of treatment efficacy in vivo.

The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain, we have been able to identify their terminals in retinal slices and make patch-clamp recordings from TCs. TCs generate Na+-based action potentials (APs) triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate, and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl− transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain.

Vertebrate photoreceptors are unusual neurons in that they are capable of continuous calcium-mediated release of neurotransmitter (Trifonov, 1968; Hagins et al., 1970). In this study, we have examined the development and characteristics of calcium currents in chick cone cells placed in culture on embryonic day 8. Cone cells were identified by their lectin-binding properties, rhodopsin-like immunoreactivity, and the presence of an oil droplet. Using the whole-cell patch-clamp method, we have seen calcium currents in these cells after three days in culture, slightly before the appearance of synapses (Gleason & Wilson, 1989). Because cone calcium currents are blocked by cadmium and nifedipine but are enhanced by Bay K 8644, they most closely resemble L-type current (Nowycky et al., 1985). An unexpected feature of these currents is that their gating ranges varied widely between cells so that some cells showed the foot of their activation range at —70 mV and others as positive as —25 mV. Calcium imaging of fura-2 loaded cells was used to confirm the time course of calcium current development and describe the distribution of cytosolic calcium. As expected, depolarization of young cells failed to increase cytosolic calcium but in older cells an increase of threefold to fourfold was usually observed. Both at rest and during depolarization, most cone cells showed regional differences in internal calcium concentration. In the most mature cones, depolarization strongly elevated cytosolic calcium at the terminal end of the cell while producing a lesser change around the oil droplet and the ellipsoid region, suggesting that calcium channels are localized to the terminal.

Management of brain tumours in children would benefit from improved non-invasive diagnosis, characterisation and prognostic biomarkers. Metabolite profiles derived from in-vivo MRS have been shown to provide such information. Studies indicate that using optimum a priori information on metabolite contents in the construction of linear combination (LC) models of MR spectra leads to improved metabolite profile estimation. Glycine (Gly) is usually neglected in such models due to strong overlap with myo-inositol (mI) and a low concentration in normal brain. However, biological studies indicate that Gly is abundant in high-grade brain tumours. This study aimed to investigate the quantitation of Gly in paediatric brain tumours using MRS analysed by LCModel™, and its potential as a non-invasive biomarker of malignancy. Single-voxel MRS was performed using PRESS (TR 1500 ms, TE 30 ms/135 ms) on a 1.5 T scanner. Forty-seven cases (18 high grade (HG), 17 low grade (LG), 12 ungraded) were retrospectively selected if both short-TE and long-TE MRS (n = 33) or short-TE MRS and high-resolution magic-angle spinning (HRMAS) of matched surgical samples (n = 15) were available. The inclusion of Gly in LCModel™ analyses led to significantly reduced fit residues for both short-TE and long-TE MRS (p

The prostaglandin F2α (PGF2α) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF2α signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF2α increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF2α in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF2α-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF2α-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis. [Cancer Res 2009;69(14):5726–33]

Contains fulltext : 81322.pdf (Publisher’s version ) (Open Access) The retinas of birds receive a substantial efferent, or centrifugal, input from a midbrain nucleus. The function of this input is presently unclear, but previous work in the pigeon has shown that efferent input is excluded from the area centralis, suggesting that the functions of the area centralis and the efferent system are incompatible. Using an antibody specific to rods, we have identified the area centralis in another species, the chicken, and mapped the distribution of the unique amacrine cells that are the postsynaptic partners of efferent fibers. Efferent target amacrine cells are found within the chicken area centralis and their density is continuous across the border of the area centralis. In contrast to the pigeon retina then, we conclude that the chicken area centralis receives efferent input. We suggest that the difference between the two species is attributable to the presence of a fovea within the area centralis of the pigeon and its absence from that of the chicken.

Cells from the embryonic quail retina were immortalized with the v-mil oncogene and cloned by limiting dilution. Their phenotype was examined using the whole-cell patch clamp method. Three membrane currents, IK(IR), INa and IK, were found at different frequencies within a sample of 170 cells drawn from a large clone. Nearly all combinations of these three markers were found and the frequency of combinations showed that the markers assorted independently. Examination of clones of less than 10 cells showed that heterogeneity originates with a high probability within clones, arguing that chromosomal mutation, for example, is unlikely to account for phenotypic diversity. A possible explanation is that phenotypic differences between cells might reflect the local exchange of instructive signals. If so, then the genes for the three phenotypic markers are controlled independently.

When mapped with a small spot of light, the central receptive fields of bipolar cells in the salamander retina are much larger than the extent of bipolar cell dendrites. Furthermore responses of bipolar cells to distant spots of light are considerably delayed relative to proximal spots. Using quantitative modelling, electrical coupling between bipolar cells is examined and rejected as a sufficient explanation of the data. An active process appears to shape signal waveform as signals spread laterally in the bipolar cell layer. Chemical synaptic coupling between bipolar cells is considered and shown to be inconsistent with the data. It is suggested that local, transient negative feedback from amacrine cells is involved in shaping bipolar cell signals.

Background: Particulate air pollution (PM{sub 10}) consists of a mixture of components, including nanoparticles and metals. Studies from our laboratory have demonstrated that transition metals can potentiate the ability of nanoparticles to induce lung inflammation and that the zinc content of PM{sub 10} was largely responsible for their potential to induce inflammation. These results are also relevant to zinc-containing engineered nanoparticles. Objectives: To investigate the potential of ZnCl{sub 2} and FeCl{sub 3} to interact with nanoparticle carbon black in cell-free and biological systems to generate ROS, express pro-inflammatory mediators and cytotoxic ability. Methods: ROS production was examined using DCFH-DA. J774 cells were treated for 4 h with 14 nm CB and/or ZnCl{sub 2} before measuring TNF-{alpha} by ELISA. Cytoskeletal changes were investigated using confocal microscopy. Flow cytometry was used to examine apoptotic/necrotic cells and phagocytic ability. Results: In a cell-free system the particles generated significant ROS, whereas ZnCl{sub 2} did not. Treatment of cells with 100 {mu}M ZnCl{sub 2}, but not FeCl{sub 3}, increased TNF-{alpha}. Treatment with 14 nm CB alone induced TNF-{alpha}, which was synergistically enhanced by ZnCl{sub 2}. No interactions were observed in cells treated with 14 nm CB and FeCl{sub 3}. Cytoskeletal changes were observed with increasingmore » concentrations of ZnCl{sub 2}. These results were confirmed by flow cytometry indicating that ZnCl{sub 2} induced markers of apoptosis and necrosis. The phagocytic ability of cells was also significantly decreased. Nanoparticle carbon black alone did not induce changes in apoptosis/necrosis or the phagocytosis activity of the cells. Conclusion: Despite an inability to induce ROS production, ZnCl{sub 2} stimulated TNF-{alpha} production which was synergistically enhanced by 14 nm carbon black. The ability of zinc to induce morphological changes and cell death was not altered by nanoparticle treatment.« less

The Ca2+ that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca2+channels. Using electrophysiology and Ca2+ imaging, we show that, in amacrine cell dendrites, at least some of the Ca2+ that triggers transmitter release comes from endoplasmic reticulum Ca2+ stores. We show that both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca2+] during the brief depolarization of a dendrite. Only the Ca2+ released through IP3Rs, however, seems to promote the release of transmitter. Antagonists for the IP3R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca2+ from internal stores, enhanced both spontaneous and evoked transmitter release.

Both the ultrafine particle and transition metal components of particulate air pollution (PM(10)) have been hypothesized to be important factors in determining toxicity and potential adverse health effects. In this study we aimed to investigate interactions between transition metal salts and a surrogate environmental particle-ultrafine carbon black (ufCB). In all experimental systems employed, the ufCB was found to be more reactive than its fine counterpart (CB). Incubation of ufCB with the reactive oxygen species (ROS)-sensitive probe dichlorofluorescin in the absence of cells generated significantly more ROS than CB. With addition of either cupric sulfate (CuSO(4)), ferrous sulfate (FeSO(4)), or ferric chloride (FeCl(3)), the ROS generation in the presence of ufCB was enhanced in a potentiative manner. In Mono Mac 6 macrophages, ufCB again produced more ROS than CB. However, addition of iron salts had no additive effect over and above that induced in the macrophages by ufCB. In the mouse macrophage cell line J774, ufCB decreased the cellular content of GSH and ATP. Addition of iron further decreased both GSH and ATP and a potentiative interaction between ufCB and FeSO(4) was observed, but only at the highest iron concentrations tested. A concentration-dependent increase in tumor necrosis factor-alpha production by J774 cells was also observed following exposure to ufCB, which was not further enhanced by the addition of iron. J774 cells were also found to sequester or chelate iron without inducing toxicity. In the rat lung ufCB induced a significant neutrophil influx and this inflammatory effect was potentiativelly enhanced by the addition of FeCl(3) (100 microM). These findings suggest that (1) ultrafine particles and metals interact by chemical potentiation in a cell-free environment to generate ROS, (2) potentiation between ultrafine particles and metal salts is not observed in the presence of macrophages as iron is sequestered or chelated by the cells, (3) in the lung, ultrafine particles and iron salts interact in a potentiative manner to generate inflammation.

We have previously shown that disabling forward-mode Na+-Ca2+exchange in amacrine cells greatly prolongs the depolarization-induced release of transmitter. To investigate the mechanism for this, we imaged [Ca2+]iin segments of dendrites during depolarization. Removal of [Na+]oproduced no immediate effect on resting [Ca2+]ibut did prolong [Ca2+]itransients induced by brief depolarization in both voltage-clamped and unclamped cells. In some cells, depolarization gave rise to stable patterns of higher and lower [Ca2+] over micrometer-length scales that collapsed once [Na+]owas restored. Prolongation of [Ca2+]itransients by removal of [Na+]ois not due to reverse mode operation of Na+-Ca2+exchange but is instead a consequence of Ca2+release from endoplasmic reticulum (ER) stores over which Na+-Ca2+exchange normally exercises control. Even in normal [Na+]o, hotspots for [Ca2+] could be seen following depolarization, that are attributable to local Ca2+-induced Ca2+release. Hotspots were seen to be labile, probably reflecting the state of local stores or their Ca2+release channels. When ER stores were emptied of Ca2+by thapsigargin, [Ca2+] transients in dendrites were greatly reduced and unaffected by the removal of [Na+]oimplying that even when Na+-Ca2+exchange is working normally, the majority of the [Ca2+]iincrease by depolarization is due to internal release rather than influx across the plasma membrane. Na+-Ca2+exchange has an important role in controlling [Ca2+] dynamics in amacrine cell dendrites chiefly by moderating the positive feedback of the Ca2+amplifier.

Studies into the effects of ultrafine particles in the lung have shown adverse effects considered to be due in part to the particle size. Air pollution particles (PM(10)) are associated with exacerbations of respiratory disease and deaths from cardiovascular causes in epidemiological studies and the ultrafine fraction of PM(10) has been hypothesized to play an important role. The aim of the present study was to investigate proinflammatory responses to various sizes of polystyrene particles as a simple model of particles of varying size including ultrafine. In the animal model, we demonstrated that there was a significantly greater neutrophil influx into the rat lung after instillation of 64-nm polystyrene particles compared with 202- and 535-nm particles and this was mirrored in other parameters of lung inflammation, such as increased protein and lactate dehydrogenase in bronchoalveolar lavage. When surface area instilled was plotted against inflammation, these two variables were directly proportional and the line passed through zero. This suggests that surface area drives inflammation in the short term and that ultrafine particles cause a greater inflammatory response because of the greater surface area they possess. In vitro, we measured the changes in intracellular calcium concentration in mono mac 6 cells in view of the potential role of calcium as a signaling molecule. Calcium changes after particle exposure may be important in leading to proinflammatory gene expression such as chemokines. We demonstrated that only ultrafine polystyrene particles induced a significant increase in cytosolic calcium ion concentration. Experiments using dichlorofluorescin diacetate demonstrated greater oxidant activity of the ultrafine particles, which may explain their activity in these assays. There were significant increases in IL-8 gene expression in A549 epithelial cells after treatment with the ultrafine particles but not particles of other sizes. These findings suggest that ultrafine particles composed of low-toxicity material such as polystyrene have proinflammatory activity as a consequence of their large surface area. This supports a role for such particles in the adverse health effects of PM(10).

OBJECTIVES—In July 1995 the Soufriere Hills volcano on the island of Montserrat began to erupt. Preliminary reports showed that the ash contained a substantial respirable component and a large percentage of the toxic silica polymorph, cristobalite. In this study the cytotoxicity of three respirable Montserrat volcanic ash (MVA) samples was investigated: M1 from a single explosive event, M2 accumulated ash predominantly derived from pyroclastic flows, and M3 from a single pyroclastic flow. These were compared with the relatively inert dust TiO2 and the known toxic quartz dust, DQ12. METHODS—Surface area of the particles was measured with the Brunauer, Emmet, and Teller (BET) adsorption method and cristobalite content of MVA was determined by x ray diffraction (XRD). After exposure to particles, the metabolic competence of the epithelial cell line A549 was assessed to determine cytotoxic effects. The ability of the particles to induce sheep blood erythrocyte haemolysis was used to assess surface reactivity. RESULTS—Treatment with either MVA, quartz, or titanium dioxide decreased A549 epithelial cell metabolic competence as measured by ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). On addition of mannitol, the cytotoxic effect was significantly less with M1, quartz, and TiO2. All MVA samples induced a dose dependent increase in haemolysis, which, although less than the haemolysis induced by quartz, was significantly greater than that induced by TiO2. Addition of mannitol and superoxide dismutase (SOD) significantly reduced the haemolytic activity only of M1, but not M2 or M3, the samples derived from predominantly pyroclastic flow events. CONCLUSIONS—Neither the cristobalite content nor the surface area of the MVA samples correlated with observed in vitro reactivity. A role for reactive oxygen species could only be shown in the cytotoxicity of M1, which was the only sample derived from a purely explosive event. These results suggest that in general the bioreactivity of MVA samples in vitro is low compared with pure quartz, but that the bioreactivity and mechanisms of biological interaction may vary according to the ash source. Keywords: volcanic ash; cristobalite; surface reactivity

The concept of a safe, immunocontraceptive vaccine using the zona pellucida glycoprotein 3 (ZP3) as an immunogen has been marred by the appearance of ovarian dysfunction in several species. However, careful selection of epitopes on mouse ZP3 have demonstrated that it is possible to segregate contraceptive bone marrow-derived (B)-cell epitopes from the cytotoxic thymus-derived (T)-cell epitopes thought to be responsible for inducing ovarian disease. B-cell epitopes on marmoset ZP3 (mstZP3) were identified by epitope mapping studies. Using a panel of polyclonal antibodies against recombinant mstZP3, an immunodominant epitope mstZP3(301-320) was identified. A chimeric peptide was co-linearly synthesized incorporating this sequence with a promiscuous tetanus toxoid T-helper cell epitope. Using the common marmoset (Callithrix jacchus) as an animal model, we have compared the consequences of active immunization with homologous recombinant mstZP3 and mstZP3(301-320) chimeric peptide vaccine. Long-term infertility was achieved using mstZP3 but at the expense of ovarian function. In contrast, no disruption to ovarian function was observed following mstZP3(301-320) immunization. Antibodies to this peptide immunolocalized to the zona pellucida of both marmoset and human ovarian sections and inhibited human sperm-zona binding by approximately 60% in vitro. However, in-vivo studies indicated that targeting a single ZP3 epitope was insufficient to reliably and consistently achieve a contraceptive effect.

PROBLEM: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. METHOD OF STUDY: Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3 45-64 , hZP3 93-110 , hZP3 172-190 and hZP3 341-360 ) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3 45-64 and hZP3 172-190 recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. RESULTS: No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. CONCLUSION: Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.

Frerking, Matthew, Salvador Borges, and Martin Wilson. Are some minis multiquantal? J. Neurophysiol. 78: 1293–1304, 1997. The amplitude distribution of miniature postsynaptic currents (minis) in many central neurons has a large variance and positive skew, but the sources of this variance and skew are unresolved. Recently it has been proposed that spontaneous Ca2+ influx into a presynaptic bouton with multiple release sites could cause spontaneous multiquantal minis by synchronizing release at all sites in the bouton, accounting for both the large variance and skew of the mini distribution. We tested this hypothesis by evoking minis with internally perfused, buffered Ca2+ and the secretagogue α-latrotoxin, both in the absence of external Ca2+. With these manipulations, the synchronized release model predicts that the mini distribution should collapse to a Gaussian distribution with a reduced coefficient of variation. Contrary to this expectation, we find that mini amplitude distributions under these conditions retain a large variance and positive skew and are indistinguishable from amplitude distributions of depolarization-evoked minis, strongly suggesting that minis are uniquantal.

A fundamental issue in synaptic physiology is whether the postsynaptic response to a quantum of transmitter is limited by the number of receptors available. Fierce debate over the past few years has yielded no consensus. The majority of evidence suggests that the degree of receptor occupancy is likely to be sensitive to a number of factors, including the detailed anatomy of the synaptic cleft and the time course of transmitter clearance, and is probably different from one synapse to the next.

The strength of synaptic connections between two neurons is characterized by the number of release sites (N) on the presynaptic cell, the probability (p) of transmitter release at those sites in response to a stimulus, and the average size (A) of the postsynaptic response from each site. Quantal analysis can determine N, p, and A, but the large variance in the amplitudes of minis at central synapses is predicted to obscure quantal peaks and render quantal analysis unusable. Recently it has been suggested that the variance in mini amplitude is generated by differences between release sites, rather than by quantum-to-quantum fluctuations at identical sites, and that this form of variance in mini amplitude reduces the amount of variance expected in quantal peaks. Using simulations, we examine the possibility of resolving quantal peaks assuming either form of variance in mini amplitude. We find that individual quantal peaks are resolvable in neither case, provided that the uniquantal distribution is similar to the mini distribution. Because this lack of resolution compromises the utility of quantal analysis, we develop a general description that can solve N and p, given the statistical parameters of the mini distribution and the evoked distribution. We find that this description is relatively insensitive to the source of variance in mini amplitude.

The peptide, neurotensin, is found in a class of amacrine cells synapsing chiefly withother amacrine cells in the chicken retina (Li & Lam, 1990; Watt et al., 1991). Toinvestigate the possible effects of neurotensin, we have used Ca2+imaging to measure cytosolic Ca2+concentrations in cultured chick amacrine cells. Following a delay of about 2 min, neurotensin (300 nM) induced oscillations in Ca2+concentration that typically had a period of 2 min and peak values of about 300 nM when averaged over the cell body. The phospholipase C inhibitors U-73, 112 and 4′-bromophenacyl bromide terminated oscillations induced by neurotensin but the protein kinase inhibitors H7 and staurosporine did not inhibit oscillations, increasing their frequency instead. In the absence of external Ca2+, neurotensin induced only a single Ca2+transient, much briefer than when external Ca2+was present. Together these results suggest that neurotensin activates phospholipase C, thereby producing IP3that triggers Ca2+release from an internal store. Although this released Ca2+contributes to periodic Ca2+peaks, the majority of cytosolic Ca2+, even in the first peak, comes from Ca2+influx across the plasmalemma.

Do graded potential synapses work the same way as action potential synapses? Recent work emphasizes the differences and suggests that graded potential synapses are not all the same either.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Calcium influx into cultured retinal amacrine cells is followed by a small, slow, inward current that we show here results from the operation of electrogenic Na-Ca exchange. The activity of the exchanger is shown to correlate with the magnitude of the Ca2+ load and to depend on both the Ca2+ and Na+ gradients. Li+ is unable to substitute for Na+ and in the absence of Na+, slow tail currents are almost entirely suppressed. A rapid change in [K+]o does not affect the activity of the exchanger, suggesting that only Na+ and Ca2+ are transported. The ratio of charge entering as Ca2+ current to the charge entering as exchange current is highly variable between cells. We suggest that variability results from a variable fraction of Ca2+ load, we estimate typically 40%, being removed by a process other than Na-Ca exchange. This process is likely to involve internal buffering or sequestration since inhibition of the plasmalemmal Ca(2+)-ATPase does not increase the fraction of Ca2+ expelled by the exchanger. Ca2+ loading performed in the absence of Na+o generates smaller exchange charge the longer the delay in returning Na+o to the neuron. About 30% of exchange charge is lost for a delay of 1 sec.

Cultured retinal amacrine cells show quantal GABAergic synaptic transmission. Voltage clamping pre- and post-synaptic cells of an isolated pair has allowed us to examine the entry and removal of Ca2+ at synaptic terminals. Brief presynaptic Ca2+ currents elicit an initial postsynaptic current that probably reflects the roughly synchronous exocytosis of docked vesicles. Prolonged Ca2+ currents elicit an additional second phase of release whose time course can greatly exceed that of the presynaptic voltage step. The time course of this second phase reflects a sustained increase in cytosolic Ca2+ and is matched closely by the activity of the presynaptic Na-Ca exchanger, as revealed by an exchange current. Eliminating the activity of the exchanger by removal of external Na+ prolongs this second phase of transmission greatly. Because transmitter release at these synapses outlasts Ca+ channel opening, Na-Ca exchange plays a significant role in shaping transmission.

We have examined synaptic transmission between isolated pairs of chick GABAergic amacrine cells, maintained in sparse culture and identified by their binding of an amacrine cell-selective antibody. Using the perforated-patch method to whole-cell clamp both cells of a pair, postsynaptic currents were examined for step depolarizations of the “presynaptic” cell. Synaptic transmission, frequently reciprocal, was calcium dependent and reversibly blocked by bicuculline. Post-synaptic currents, excluding those due to ohmic electrical coupling, were elicited only for presynaptic voltage steps positive to about -40 mV and were always very noisy, suggesting that they were summed from relatively small numbers of quanta. Postsynaptic currents continued well after the termination of the 100 msec presynaptic voltage step when the step was to -10 mV, or positive to this value. This result is interpreted to imply that presynaptic calcium concentration remains elevated after the membrane is returned to its holding potential. When presynaptic voltages were kept low or else presynaptic voltage was uncontrolled, spontaneous quantal events mediated by GABAA receptors could often be seen. Quanta rose quickly (less than 4 msec) and decayed with a mean time constant of 19.3 msec. The amplitude distributions of quantal currents were positively skewed, sometimes showing rare quanta of exceptionally large amplitude. Peak conductance per quantum was about 300 pS, corresponding to the simultaneous opening of only 17 GABAA channels and corresponding to a net flux of only 32 x 10(3) Cl- ions per millivolt of driving force. Estimates of the maximum sustained release rate at individual release sites suggest an upper bound of between 19 and 42 quanta per second.

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve paris of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (IK) and inward rectifier (IK(IR)) potassium currents. Despite qualitative and quantitative variability in IK and IK(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between –110 and +50 mV. We estimated IK(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated IK(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed IK(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis. © 1993 John Wiley & Sons, Inc.

1. Horizontal cells, identified by their morphology, were isolated from the salamander retina and examined in whole cell patch clamp. 2. All cells showed large outward currents activating positive to about -50 mV, and a minority of cells showed fast, tetrodotoxin-suppressible Na+ currents. Slow inward currents that might shape the light responses were never observed. 3. All cells showed conductance increases to both gamma-aminobutyric acid (GABA) and glycine that were completely blocked by bicuculline and strychnine, respectively. No cross-blocking by these antagonists was observed. Partial replacements of Cl- with large, impermeant anions indicated that both GABA- and glycine-evoked currents were carried by Cl- ions. 4. Responses to both GABA and glycine desensitized strongly with time constants of approximately 2 s. 5. Responses to glutamate were not enhanced by glycine. Similarly, responses to GABA were not enhanced by glutamate. 6. GABA-mediated synaptic interactions between horizontal cells may account for the changes in the kinetics of horizontal cell light responses seen when glycine is applied to the intact retina.

The conduction of calcium ions through glutamate-gated channels is important in the induction of long-term potentiation and may trigger other cellular changes. In retinal bipolar cells, which lack the N-methyl-D-aspartate (NMDA) type of glutamate-gated channel, calcium permeability through non-NMDA channels was examined. Changes in extracellular calcium concentration unexpectedly affected the reversal potential for glutamate-induced currents in a manner consistent with these channels being highly permeable to calcium. External magnesium ions promote desensitization of these non-NMDA channels in a voltage-independent way. Thus, in addition to non-NMDA channels that conduct only sodium and potassium, there is a class that is also permeable to calcium.

Using the whole-cell patch clamp technique, we have examined the voltage-gated currents present in adult chicken cone cells. When calcium and calcium-gated currents are blocked, hyperpolarizing voltage steps elicit slowly increasing inward currents as has been shown for photoreceptors in other species. Unlike the case for other species, chicken cones appear to lack the inward-rectifying cationic current Ih that contributes to the shaping of the photovoltage. Instead of Ih, these cones possess an anionic inward-rectifying current that in kinetics, activation range and probably function is remarkably similar to Ih. This anion channel is unusual in that both nitrate and acetate are more permeant than chloride ions.

For ion channels that are opened by neurotransmitters, analysis of current noise has given valuable information on the kinetics of synaptic channel gating. In depolarizing bipolar cells of the vertebrate retina, we have recently characterized a synaptic current for which the neurotransmitter glutamate closes channels, and for which the channel open probability is low even in the absence of glutamate. We present here predictions for the current noise spectrum expected for various models of glutamate's action on the ion channels. Comparison of these theoretical predictions with experimental data allows us to rule out several simple kinetic schemes for the action of glutamate, and to conclude that the channels closed by glutamate must be able to exist in at least four different states.

1. Whole-cell patch clamping was used to study the membrane properties of isolated bipolar cells and the currents evoked in them by putative retinal neurotransmitters. 2. Isolated bipolar cells show an approximately ohmic response to voltage steps over most of the physiological response range, with an average input resistance of 1.3 G omega and resting potential of -35 mV. These values are underestimates because of the shunting effect of the seal between the patch electrode and the cell membrane. Depolarization beyond -30 mV produces rapid activation (10-100 ms) of an outward current (carried largely by potassium ions), which then inactivates slowly (0.5-2 s). 3. Of five candidates for the photoreceptor transmitter, four (aspartate, N-acetylhistidine, cadaverine, putrescine) had no effect on bipolar cells. The fifth substance, L-glutamate, opened ionic channels with a mean reversal potential of -12 mV in some cells (presumed hyperpolarizing bipolar cells), and closed channels with a mean reversal potential of -13 mV in other cells (presumed depolarizing bipolar cells). 4. The conductance increase induced by glutamate in presumed hyperpolarizing bipolar cells was associated with an increase in membrane current noise. Noise analysis suggested a single-channel conductance for the glutamate-gated channel of 5.4 pS. The power spectrum of the noise increase required the sum of two Lorentzian curves to fit it, suggesting that the channel can exist in three states. 5. The conductance decrease induced by glutamate in presumed depolarizing bipolar cells was associated with a decrease in membrane current noise that could be described as the sum of two Lorentzian spectra, and which suggested a single-channel conductance of 11 pS. The noise decrease implies that the channels closed by glutamate are not all open in the absence of the transmitter. 6. GABA (gamma-aminobutyric acid) and glycine, transmitters believed to mediate lateral inhibition in the retina, open chloride channels in isolated bipolar cells, and increase the membrane current noise. Noise analysis suggested that the channels gated by GABA and glycine have conductances of 4.4 and 7.5 pS respectively. The noise spectra required the sum of two Lorentzian curves to fit them. 7. By whole-cell patch clamping cells in retinal slices, the synaptic transmitter released by photoreceptors was shown to close channels with an extrapolated reversal potential around -3 mV in depolarizing bipolar cells.(ABSTRACT TRUNCATED AT 400 WORDS)

A simple design for a micromanipulator having fine movements in 3 dimensions is described. An inexpensive and easily executed method of construction for this device is given.

1. The properties of isolated single cones were studied using the voltage-clamp technique, with two micro-electrodes inserted under visual control. 2. Single cones had input resistances, when impaled with two electrodes, of up to 270 MΩ. This is probably lower than the true membrane resistance, because of damage by the impaling electrodes. The cone capacitance was about 85 pF. 3. The cone membrane contains a time-dependent current, IB, controlled by voltage, and a separate photosensitive current. 4. The gated current, IB, is an inward current with a reversal potential around -25 mV. It is activated by hyperpolarization over the range -30 to -80 mV, and at constant voltage obeys first order (exponential) kinetics. The gating time constant is typically 50 ms at the resting potential of -45 mV, rises to 170 ms at -70 mV, and decreases for further hyperpolarization. 5. The spectral sensitivity curve of the cone light response peaks at 620 nm wave-length, and is narrower than the nomogram for vitamin A2-based pigments. The light responses of isolated cones are spectrally univariant. 6. Voltage-clamped photocurrents were recorded at various membrane potentials, for light steps of various intensities. The photocurrent reversed at around -8 mV. The time course of the photocurrent, for a given intensity, was approximately independent of voltage (although its magnitude was voltage-dependent). The shape of the peak current—voltage relation of the light-sensitive current was independent of light intensity (although its magnitude was intensity-dependent). 7. These results can be explained if: (a) light simply changes the number of photosensitive channels open, without altering the properties of an open channel; (b) the reactions controlling the production of internal transmitter, the binding of internal transmitter to the photosensitive channels, and the closing and opening of the channels are unaffected by the electric field in the cone membrane, even though at least some of these reactions take place in the membrane. 8. IB plays only a small role in shaping the cone voltage response to light.

By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.

The retina occupies a special place in neuroscience. It is, after all, still the only part of the vertebrate brain where we can reasonably claim to know what it is for and how it does its job. In fact, this year is just about the thirtieth anniversary of the work of Werblin and Dowling 1969xWerblin, F.S. and Dowling, J.E. J. Neurophysiol. 1969; 32: 339–355PubMedSee all ReferencesWerblin and Dowling 1969 and Kaneko 1970xKaneko, A. J. Physiol. Lond. 1970; 207: 623–633See all ReferencesKaneko 1970 that gave us our model of how the retina works. The model derives from their surveys of the receptive fields of all the main retinal neuron types, plus a little knowledge about the synaptic connections as well as some plausible guesses. In the model, photoreceptors connect to bipolar cells and bipolar cells connect to ganglion cells in a vertical, straight-through pathway. Two horizontal layers of lateral interneurons modify these signals. In the outer retina, horizontal cells modify the spatial properties of the neural image by influencing the photoreceptor to bipolar step, while in the inner retina amacrine cells modify the temporal properties of the neural image by acting at the bipolar to ganglion cell step. At the level of bipolar cells, the neural image is split into two parallel pathways and remains split in ganglion cells and indeed long after signals leave the retina. One category of bipolar cells, the ON bipolar cells, depolarize to light and the other category, OFF bipolar cells, depolarize to darkness. The two categories of bipolar cells have their own halves of the inner synaptic layer. OFF bipolars contact OFF ganglion cells in the upper half, sublamina a, and ON bipolar cells contact ON ganglion cells in the lower half, sublamina b (Famiglietti and Kolb 1976xFamiglietti, E.V. and Kolb, H. Science. 1976; 194: 193–195Crossref | PubMedSee all ReferencesFamiglietti and Kolb 1976). This standard model of the retina is appealingly elegant with its symmetry and division of labor. Regrettably though, evolution is an inveterate tinkerer with no regard for simplicity, symmetry, or elegance, and so perhaps it is not surprising that discoveries of exceptions to the beautiful plan were not long in coming.One of the quirkiest exceptions is in the mammalian retina where rod photoreceptors have their own special bipolar cell, yet these bipolars don't make direct contact with any ganglion cells. Instead, they synapse onto a particular amacrine cell, the AII amacrine, which makes a glycinergic synapse onto cone OFF bipolars and is electrically coupled to cone ON bipolar cells, which do make contact with ganglion cells. These are not the only routes by which rod signals reach ganglion cells. We have known for a long time that some rod signals leak into cones via electrical junctions (Nelson 1977xNelson, R. J. Comp. Neurol. 1977; 172: 109–135Crossref | PubMedSee all ReferencesNelson 1977), but this is not the whole story either. Using transgenic mice without any conesSoucy et al. 1998xSoucy, E., Wang, Y., Nirenberg, S., Nathans, J., and Meister, M. Neuron. 1998; 21: 481–493Abstract | Full Text | Full Text PDF | PubMed | Scopus (189)See all ReferencesSoucy et al. 1998 showed that when transmission to the rod bipolar is blocked, rod signals nevertheless still got through to ganglion cells. Careful EM reconstructions, by Peichl's group, of the rodent retina, immunogold labeled for AMPA receptor subunits, shows that the novel pathway probably involves a rare synapse from rods to cone OFF bipolars (Hack et al. 1999xHack, I., Peichl, L., and Brandstatter, J.H. Proc. Natl. Acad. Sci. USA. 1999; 96: 14130–14135Crossref | PubMed | Scopus (122)See all ReferencesHack et al. 1999).In this issue of Neuron, DeVries further erodes the standard model, this time with respect to the location in which separate temporal channels are set up (DeVries 2000xDeVries, S.H. Neuron. 2000; 28: 847–856Abstract | Full Text | Full Text PDF | PubMed | Scopus (247)See all ReferencesDeVries 2000). In the standard model this, of course, is the inner retina. DeVries uses the ground squirrel retina, which has almost all cone photoreceptors, like our own fovea. Each cone photoreceptor makes synaptic contact with six types of bipolar cell, three of which are ON cells and three of which are of the OFF type. The ON bipolar cells depolarize when photoreceptors hyperpolarize in response to light, and to achieve this sign inversion, they employ a metabotropic receptor for the cone transmitter, glutamate. OFF bipolars are simpler, using common ionotropic AMPA/KA receptors. So how are the three kinds of OFF bipolar cells different from each other? What DeVries shows is that on careful examination, the three types, which he shows are anatomically recognizable when filled with dye from the patch pipet (see DeVries, Figure 1), each have characteristic receptors. Two of them are KA preferring and one is an AMPA receptor. To examine the kinetics of these receptors, DeVries uses the pipet to gently lift the cell body and dendrites of a bipolar cell from a slice into a flowing stream that allows rapid solution changes. The b2 bipolar cell, the one with AMPA receptors, recovers quickly from desensitization following the application of glutamate, but the b3 bipolar, with a KA receptor, takes more than 1 s for full recovery. The b7 bipolar cell has a different kind of KA receptor and it has intermediate kinetics. By simultaneously voltage-clamping a cone and a connected bipolar cell, DeVries shows that the recovery of the AMPA receptor from desensitization is not a rate-limiting step in the transmission from cone to b2 bipolar cell. Instead, some presynaptic step, like replenishing the supply of synaptic vesicles in the cone terminal, is limiting. In the case of the b3 bipolar cell, though, the KA receptor determines the dynamics of recovery. To drive home the point that a cone's signal has different temporal representations in different bipolar cells, DeVries, in a tour de force, records simultaneously from a b2 and a b3 bipolar cell while driving both of them with the same cone.In the life of a ground squirrel, cone cells don't experience constant saturating intensities of light interspersed with the occasional flash of darkness, the equivalent of the voltage regime applied to cones in the DeVries study. Mostly, their membrane potentials jog around in the middle of their voltage range. What effect will receptor kinetics have under these circumstances? A quantitative answer will have to wait for a more sophisticated experiment, but the gist is already clear. b3 cells will show sustained responses, and the b2 cells will be transient. Very probably the transient b2 cells will make contact with the transient Y type ganglion cells, and the b3 cells will drive the slow X type ganglion cells. For the ground squirrel retina, this is a plausible suggestion only since the properties of the ganglion cells are not known, but a recent study by Awatramani and Slaughter 2000xAwatramani, G.B. and Slaughter, M.M. J. Neurosci. 2000; 20: 7087–7095PubMedSee all ReferencesAwatramani and Slaughter 2000 lends support to this idea. Here they show that in the salamander retina, some morphological classes of ON bipolar cells are transient, while others have sustained responses to light. Again, this is due to the properties of the receptor for the photoreceptor transmitter, but the point here is that, as you would suppose, sustained bipolars drive sustained ganglion cells, whereas transient bipolars drive transient ganglion cells.If parallel temporal pathways are set up in the outer retina, does this mean that the inner retina is not involved? The evidence (for example8xNirenberg, S. and Meister, M. Neuron. 1997; 18: 637–650Abstract | Full Text | Full Text PDF | PubMedSee all References, 3xDong, C.J. and Werblin, F.S. J. Neurophysiol. 1998; 79: 2171–2180PubMedSee all References) argues that, on the contrary, amacrine cells contribute plenty of temporal tweaking. Like the case of the passage of rod signals to ganglion cells in mammals, we can expect that multiple mechanisms will be contributing.So the division of labor is not as clean as we thought. Parallel temporal pathways are established in the outer retina after all, and once again, the facts spoil a beautiful picture. But perhaps there could be a silver lining to this cloud. It may yet turn out, in a reprise of the discovery that ON and OFF pathways have their own sublaminae in the inner synaptic layer, that the different temporal inputs to the inner synaptic layer are layered in some intelligible pattern.

The properties of synapses between retinal neurons make an essential contribution to early visual processing. Light produces a graded hyperpolarization in photoreceptors, up to 25 mV in amplitude1, and it is conventionally assumed that all of this response range is available for coding visual information. We report here, however, that the rod output synapse rectifies strongly, so that only potential changes within 5 m V of the rod dark potential are transmitted effectively to postsynaptic horizontal cells. This finding is consistent with the voltage-dependence of the calcium current presumed to control neurotransmitter release from rods2. It suggests functional roles for the strong electrical coupling of adjacent rods3 and the weak electrical coupling of adjacent rods and cones4. The existence of photoreceptor coupling resolves the apparent paradox that rods have a 25 mV response range, while signals greater than 5 mV in amplitude are clipped during synaptic transmission. We predict that the strengths of rod-rod and rod-cone coupling are quantitatively linked to the relationship between the rod response range and the synapse operating range.

A new method is described for obtaining isolated rods and cones by physical dissociation of the retina. The method gives a large yield of isolated photoreceptors, without any changes in membrane properties that enzymatic dissociation techniques might produce. Photovoltages and photocurrents recorded from rods isolated in this way are presented.

Rod bipolar cells in the mammalian retina receive synaptic input from many noisy rod photoreceptors. When photons are scarce, linear addition of inputs would swamp signals with noise. A nonlinearity at the synapse optimizes the signal to noise ratio.

A quantitative description of the electrical properties of the photoreceptor layer in the salamander retina was obtained from earlier data on the characteristics of isolated rods and cones and on rod-rod coupling, and from new data on rod-cone and cone-cone coupling and on the rod photocurrent. Injecting -1 nA current into a rod elicits hyperpolarizations of about 20 mV in an adjacent rod and 4 mV in an adjacent cone. Responses of more distant receptors are smaller. Injecting -1 nA into a cone elicits hyperpolarizations of about 4 mV in an adjacent rod and 0.4 mV in a nearby cone. Depolarizing current evokes smaller responses. Assuming, in agreement with anatomical evidence, that each rod is electrically coupled to four rods and to four cones around it, and that there is no direct electrical coupling between cones, we found these results could be predicted from the properties of isolated rods and cones if adjacent rods are coupled by a resistance of 300 M omega and adjacent rods and cones are coupled by a resistance of 5000 M omega. The small cone-cone coupling seen is due to coupling via intervening rods. The two halves of double cones are not electrically coupled. The spectral sensitivity of both halves is a maximum around 620 nm wave-length. The rod photocurrent has been characterized by voltage-clamping rods isolated from the retina. In agreement with Bader, MacLeish & Schwartz (1979) we found the time course of the photocurrent to be approximately independent of voltage between -35 and -85 mV. The voltage responses of rods, single cones and double cones isolated from the retina obey the principle of univariance. Responses of receptors in the retina do not obey univariance. The main deviations from univariance observed can be explained if adjacent rods and cones are coupled by a resistance of 5000 M omega. Our data demonstrate that rod-cone coupling is relatively weak. We simplified our description of the photoreceptor network, by omitting cones, to investigate the spatiotemporal processing that the rod network is capable of. Computer simulations predict, as is found experimentally, that the rod voltage response to a large spot of bright light should show a much more pronounced initial transient hyperpolarization than the response to a small spot of light of the same intensity. This difference is produced by the combination of electrical coupling of the rods with the existence of a voltage-gated current, IA, in the rod membrane.(ABSTRACT TRUNCATED AT 400 WORDS)