Biomedical subjects

P Mobbs

Publications and source records attributed to P Mobbs.

At least 19 recordsLinked to original sources

PubMed2001

Early activation of Ca(2+)-permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons.

Calcium entry through Ca(2+)-permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca(2+)-indicator Calcium Green 1-AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca(2+)] in embryonic chick retina from day 6 (E6) onwards. This Ca(2+) increase is due to entry through AMPA-preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N-methyl-D-aspartic acid (NMDA) receptor antagonist AP5, the voltage-gated Ca(2+) channel blockers diltiazem or nifedipine, or by the substitution of Na+ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca(2+) influx through L-type voltage-gated Ca(2+) channels with diltiazem and nifedipine prevented the effect of 10-100 microM kainate but not that of 500 microM kainate. In addition, joro spider toxin-3, a blocker of Ca(2+)-conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2001

GABAb receptors regulate chick retinal calcium waves.

Correlated spiking activity and associated Ca(2+) waves in the developing retina are important in determining the connectivity of the visual system. Here, we show that GABA, via GABA(B) receptors, regulates the temporal characteristics of Ca(2+) waves occurring before synapse formation in the embryonic chick retina. Blocking ionotropic GABA receptors did no affect these Ca(2+) transients. However, when these receptors were blocked, GABA abolished the transients, as did the GABA(B) agonist baclofen. The action of baclofen was prevented by the GABA(B) antagonist p-3-aminopropyl-p-diethoxymethyl phosphoric acid (CGP35348). CGP35348 alone increased the duration of the transients, showing that GABA(B) receptors are tonically activated by endogenous GABA. Blocking the GABA transporter GAT-1 with 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid (SKF89976A) reduced the frequency of the transients. This reduction was prevented by CGP35348 and thus resulted from activation of GABA(B) receptors by an increase in external [GABA]. The effect of GABA(B) receptor activation persisted in the presence of activators and blockers of the cAMP-PKA pathway. Immunocytochemistry showed GABA(B) receptors and GAT-1 transporters on ganglion and amacrine cells from the earliest times when Ca(2+) waves occur (embryonic day 8). Patch-clamp recordings showed that K(+) channels on ganglion cell layer neurons are not modulated by GABA(B) receptors, whereas Ca(2+) channels are; however, Ca(2+) channel blockade with omega-conotoxin-GVIA or nimodipine did not prevent Ca(2+) waves. Thus, the regulation of Ca(2+) waves by GABA(B) receptors occurs independently of N- and L-type Ca(2+) channels and does not involve K(+) channels of the ganglion cell layer. GABA(B) receptors are likely to be of key importance in regulating retinal development.

Animals↗

PubMed2001

Use of pIRES vectors to express EGFP and connexin constructs in studies of the role of gap junctional communication in the early development of the chick retina and brain.

Control of cell proliferation is vital for the normal development of the neural retina. Gap junctional communication has been implicated in the control of retinal cell proliferation. We have previously shown that the expression of the gap junction protein Connexin 43 closely correlates with the first wave of cell proliferation in the retina. Preventing its expression using antisense oligonucleotides in the developing eye and surrounding tissues, produces a reduction in cell number and the formation of a small eye. In order to examine this in more detail we have developed a new means of manipulating connexin expression in the developing chick embryo. We have generated pIRES vectors which use cyclomegalovirus (CMV) to promote the expression of a green fluorescent protein (EGFP) and either wild type Cx43 or a dominant negative form ofthis connexin. Following injection ofthese constructs into the ventricles ofthe stage 10-11 chick embryo they can be incorporated into one side of the chick brain or optic vesicle using an electroporation technique, leaving the other side as a control. EGFP expression can be seen on the electroporated side of the chick brain within 24 hours. Expression of the dominant negative construct in cultures of chick limb bud mesenchyme results in total block of cascade blue transfer when injected into transfected cells. Expression of both wild type and dominant negative constructs in the developing chick retina perturbs the normal development of the eye.

Animals↗

PubMed1999

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.

Annexin A6↗

PubMed1999

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.

Animals↗

PubMed1998

Spontaneous Ca2+ transients and their transmission in the developing chick retina.

The development of the central nervous system is dependent on spontaneous action potentials and changes in [Ca2+]i occurring in neurons [1-4]. In the mammalian retina, waves of spontaneous electrical activity spread between retinal neurons, raising [Ca2+]i as they pass [5-7]. In the ferret retina, the first spontaneous Ca2+ waves have been reported at postnatal day 2 and are thought to result from the Ca2+ influx associated with bursts of action potentials seen in ganglion cells at this time [5-7]. These waves depend on depolarisation produced by voltage-gated sodium channels, but their initiation and/or propagation also depends upon nicotinic cholinergic synaptic transmission between amacrine cells and ganglion cells [8]. Here, we report contrasting results for the chick retina where Ca2+ transients are seen at times before retinal synapse formation but when there are extensive networks of gap junctions. These Ca2+ transients do not require nicotinic cholinergic transmission but are modulated by acetylcholine (ACh), dopamine and glycine. Furthermore, they propagate into the depth of the retina, suggesting that they are not restricted to ganglion and amacrine cells. The transients are abolished by the gap-junctional blocker octanol. Thus, the Ca2+ transients seen early in chick retinal development are triggered and propagate in the absence of synapses by a mechanism that involves several neurotransmitters and gap junctions.

Acetylcholine↗

PubMed1998

Modulation by zinc of the glutamate transporters in glial cells and cones isolated from the tiger salamander retina.

1. Zinc may be released from some presynaptic glutamatergic neurons, including hippocampal mossy fibres and retinal photoreceptors. We whole-cell-clamped glial (Müller) cells isolated from the salamander retina to investigate the effect of zinc on glutamate transporters in these cells. Glutamate-evoked currents in these cells are generated largely by carriers homologous to the mammalian GLAST/EAAT1 transporter. 2. Zinc inhibited both glutamate uptake into the cells, and glutamate release by reversal of the uptake process. The IC50 for inhibition of uptake (< 1 microM) was similar to or below the values for zinc modulating NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and GABA receptors, and 100-fold less than the calculated value for the rise in extracellular zinc concentration evoked by depolarization with potassium in area CA3 of the hippocampus. 3. Although zinc altered the apparent affinity of the transporter for glutamate and Na+, it did not act simply by binding competitively to the glutamate-, Na(+)-, K(+)- or H(+)-binding sites on the transporter. Zinc inhibited both forward and reversed glutamate transport from the outside of the cell membrane, but not from the inside. The inhibitory action of zinc on uptake was voltage independent, indicating a zinc-binding site outside the membrane field. 4. As well as inhibiting glutamate transport, zinc potentiated activation of the anion conductance in the Müller cell glutamate transporter. However, zinc reduced the current mediated by the anion conductance in the cone synaptic terminal glutamate transporter (homologous to the mammalian EAAT5), indicating that zinc has different actions on different glutamate transporter subtypes. 5. By acting on glutamate transporters, zinc may have a neuromodulatory role during synaptic transmission and a neuroprotective role during transient ischaemia.

ATP-Binding Cassette Transporters↗

PubMed1998

Cell coupling in the retina: patterns and purpose.

Gap junction channels (electrical synapses) are a major component of the central nervous system mediating both electrical and metabolic coupling between neurons and glia. Their roles are as diverse as the cell types in which they are expressed and only some of these are reviewed here. In the adult the plastic nature of the gap junction channel allows for changes in the writing of the retinal circuitry that optimize visual processing to suit ambient lighting conditions. Gap junctional communication has been proposed to play a key role in embryonic development in general and in particular during the development of the retina where its roles may include control of neurogenesis, cell specification, synaptogenesis, and the synchronization of the spontaneous electrical activity required for the sharpening of central visual projections. Here we review gap junctional coupling within the retina and present data correlating gap junction expression with development events in the chick retina.

Animals↗

PubMed1997

Retinal processing: visionary transgenics.

Two recent reports in which transgene techniques were used to label specific cell classes in the mouse retina have opened the way to new methods of studying retinal signal processing.

Action Potentials↗

PubMed1996

Developmental expression and self-regulation of Ca2+ entry via AMPA/KA receptors in the embryonic chick retina.

Excessive activation of glutamate receptors in the late embryonic and adult retina leads to excitotoxic cell death through an increase in intracellular calcium concentration. Here we use the cobalt-staining technique of Pruss et al. to investigate the developmental expression of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid/kainate (AMPA/KA) receptors in the embryonic chick retina, and the effects of AMPA/KA receptor activation on cell survival and AMPA/KA receptor expression. Ca(2+)-permeable AMPA/KA receptors are present in the retina as early as embryonic day 6 (E6). While sustained activation of these receptors with KA led to massive cell death in explant and dissociated cultures of the chick retina late in development, continuous application of high doses of KA from early times was not excitotoxic. Cell survival in KA is correlated with both a reduction in cobalt staining and the KA-evoked membrane current, and thus with a reduction in the Ca2+ entry into cells via AMPA/KA receptors. The effects of KA could be blocked by the non-N-methyl-D-aspartic acid (NMDA) receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), but not by the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP5) nor the L-type Ca2+ channel blockers diltiazem and nifedipine. The action of AP5 was mimicked by exposure to glutamate but not by the metabotropic receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid. Thus exposure of retinal neurons to glutamate early in development may protect them from its excitotoxic actions later on.

Animals↗

PubMed1995

Retinal development. Waves are swell.

Waves of spontaneous electrical activity and calcium transients occur in the retina during its development. Recent work raises the question of how these waves are produced and propagated.

Animals↗

PubMed1995

Radial tunnel syndrome. A retrospective review of 30 decompressions of the radial nerve.

Radial tunnel syndrome results from compression of the radial nerve by the free edge of the supinator muscle or closely related structures in the vicinity of the elbow joint. Despite numerous reports on the surgical management of this disorder, it remains largely unrecognized and often neglected. The symptoms of radial tunnel syndrome can resemble those of tennis elbow, chronic wrist pain or tenosynovitis. Reliable objective criteria are not available to differentiate between these pathologies. These difficulties are discussed in relation to 29 patients who underwent 30 primary explorations and proximal decompressions of the radial nerve. Excellent or good results were obtained in 70%, fair results in 13% and poor results in 17% of patients. The results can be satisfactory despite the prolonged duration of symptoms. We believe that a diagnosis of radial tunnel syndrome should always be born in mind when dealing with patients with forearm and wrist pain that has not responded to more conventional treatment. Patients with occupations requiring repetitive manual tasks seem to be particularly at risk of developing radial tunnel syndrome and it is also interesting to note that 66% of patients with on-going medico-legal claims had successful outcomes following surgery.

Adolescent↗

PubMed1994

Voltage-gated currents in rabbit retinal astrocytes.

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was -70 +/- 4.8 mV (mean +/- SEM; n = 54), and the input resistance and cell capacitance were 558 +/- 3.6 M omega and 19.5 +/- 1.8 pF respectively. Depolarization to potentials positive to -50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na+ in the bathing solution, and reduced by 50% in the presence of 1 microM tetrodotoxin. The time-to-peak of the Na+ current was more than twice that for the Na+ current found in retinal neurons. The glial Na+ current was half-inactivated at -55 mV. A transient component of the outward K+ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between -150 and -50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.

4-Aminopyridine↗

PubMed1994

Neurotransmitter transporters.

In the past year, our knowledge of neurotransmitter transporters has increased significantly. Recently, new members of two families of plasma membrane uptake carriers have been cloned, and the stoichiometries, physiological function and mechanisms of modulation of some of these transporters are now better understood. These developments highlight the possible role of neurotransmitter transporters in disease states, in the development of the nervous system, and as targets for therapeutic drugs.

Animals↗

PubMed1992

The spatial relationship between Müller cell processes and the photoreceptor output synapse.

Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Müller) cells. The anatomical distribution of Müller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Müller cells wrap around the synaptic terminals of both rods and cones and come within 1-3 microns of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.

Ambystoma↗

PubMed1992

Signal shaping by voltage-gated currents in retinal ganglion cells.

The role of voltage-gated currents in information processing by retinal ganglion cells was assessed by comparing the light-evoked current and voltage responses of identified ganglion cells with those produced by current injection. These experiments show that the light-evoked signal is clipped at the bipolar-to-ganglion cell synapse because the synaptic current evoked by illumination with bright light is greater than that which the cell can convert into a change in action potential frequency. Ganglion cell responses to injected current fell into 2 classes: those producing sustained spiking responses and those producing transient responses. Further, this division is correlated with the light response of the cells; those producing transient responses to exogenous current produced transient responses to light, while those with sustained responses to current injection produced sustained responses to light flashes. The voltage-gated currents present in the ganglion cell membrane contribute to information processing in the retina by clipping the light-evoked signal and by producing transcience in the output of the retina.

Action Potentials↗

PubMed1992

Development of functional calcium channels in cultured avian photoreceptors.

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.

Animals↗

PubMed1992

Transmitter-operated channels in rabbit retinal astrocytes studied in situ by whole-cell patch clamping.

Glutamate and GABA open ion channels in the membranes of astrocytes found on the vitreal surface of the rabbit retinal visual streak. The glutamate-operated channels are opened by kainate, quisqualate, and AMPA, but not by NMDA, aspartate, or the metabotropic agonist 1-aminocyclopentane-1 S,3R-dicarboxylic acid. The effects of glutamate and its analogs can be blocked by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione. The conductance increase evoked by 10 microM glutamate, a concentration of this transmitter near to that found in the vitreous humor that bathes these cells, was equivalent to 22% of the cell's resting conductance. The conductance increase evoked by 1 microM GABA, a concentration near that found in the vitreous, was equivalent to 131% of the cell's resting conductance. The effects of GABA can be blocked by bicuculline. These data show that GABA, and non-NMDA-type glutamate receptors play an important part in determining the resting potential of visual streak astrocytes in situ and that these channels may be of general importance for the functions of astrocytes in vivo.

Animals↗