Background in retinal research Background in retinal research
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Following the elegant work of Regehr and Armstrong it has become clear that certain neurons in the cerebellum (Purkinje cells) and hippocampus (e.g.CA3) are not isopotential, i.e. different regions are at different potentials depending upon their inputs and history of activity. This property can be studied using low resistance patch-clamp electrodes to minimise the attenuation of the dendritic signal through space-clamping artefacts. When the soma is voltage-clamped and depolarized through brief command potentials, the regenerative current spike (representative of the action potential in unclamped cells) which is due to the opening of voltage-gated sodium channels that underlie the action potential could be seen to separate into distinct population peaks. Regehr and Armstrong logically inferred that because the threshold potential in the soma/axon hillock would result in an all-or-none spike composed of excitable sodium channels, then the excitable sodium channels must be present exclusively in the axon and soma. However, if the spike were split, then excitable sodium and/or calcium channels must therefore also be present in the dendrites, supporting the classical convictions of Sugimori and Llinas.
In our 'flat-mount' recordings from retinal ganglion cells, smaller regenerative inward currents were elicited before that of the main spike, with a latency that became shorter as the somatic threshold potential was approached. These initial investigations suggest that the dendrites of retinal ganglion cells are anisopotential. This has far-reaching implications for the integration and assimilation of synaptic information in the receptive field of the ganglion cell.
In addition the action of the NMDA-receptor antagonist eliprodil upon
ganglion cells was tested using a fast piezo-driven perfusion system in
recordings of ganglion cells from slices. This technique demonstrqted thqt it is
possible to record fast receptor-operated synaptic channel currents in the slice
preparation.
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Bipolar cells are interneurons in the retina that relay visual information from photoreceptors to ganglion cells. The On-bipolar cell, which depolarizes in response to light, expresses a metabotropic L-glutamate receptor whose activation produces membrane hyperpolarization and a decrease in membrane conductance (Shiells et al ., 1981; Nawy and Copenhagen, 1987). This receptor has a high affinity for the L-glutamate receptor agonist L-2-amino-4-phosphono-butyrate (L-APB) (Slaughter and Miller, 1981). On the basis of previous studies (Nawy and Jahr, 1990; Shiells and Falk, 1990; Nawy and Jahr, 1991; Yamashita and Wassle, 1991; de la Villa et al., 1995), a model has been proposed to explain how L-glutamate hyperpolarizes the On- bipolar cell membrane.
The mGluR6 receptor is positively coupled via a G-protein to a cGMP phosphodiesterase (PDE). The binding of L-glutamate to the APB receptor activates the PDE and produces hydrolysis of cGMP. As a result of the fall in cGMP concentration, a cGMP-dependent cation channel closes and hyperpolarizes the cell. In the absence of agonist , some of the cGMP-dependent channels are constitutively open and continuously depolarize the cell due to an influx of cations through the channel (Yamashita and Wassle, 1991). Under voltage-clamp, this depolarization is observed as a tonic inward current. When exogenous cGMP is applied through the recording pipette, the inward current becomes larger as cGMP diffuses into the cell and opens cGMP-dependent cation channels (Nawy and Jahr, 1990; de la Villa et al., 1995). The APB receptor pathway is reminiscent of the transduction cascade expressed by photoreceptors (reviewed in Lagnado and Baylor, 1992), the cells to which the bipolar cell is developmentally most closely related. This current is voltage-independent (Shiells and Falk, 1994) and responses to APB do not desensitize in either whole-cell or perforated-patch recordings where high access resistances are obtained (Nawy and Jahr, 1990; Shiells and Falk, 1990; Yamashita and Wassle, 1991; de la Villa et al., 1995).
A gene encoding a rat APB receptor that is expressed by On-bipolar cells has recently been cloned and classified as mGluR6 (Nakajima et al., 1993). It is closely related to the mGluR4, mGluR7 and mGluR8 metabotropic receptors that have a high affinity for APB (Okamoto et al., 1994; Duvoisin et al, 1995). Behavioral and electrophysiological experiments with mice that do not express the mGluR6 receptor reveal a profound visual impairment (Masu et al.,1995). More detailed behavioral studies in goldfish (Bilotta et al, 1995) and monkey (Schiller, 1992; Dolan and Schiller, 1994), using intra-ocular application of APB to saturate the receptor, show profound deficits in basic visual performance such as spatial contrast or in the detection of incremental changes in background light. These experiments firmly establish an important role for the APB receptor in vision. More subtle physiological modulation of this receptor by endogenous mechanisms would provide a means for fine-tuning of spatial contrast and absolute sensitivity in response to changes in ambient light levels. There is increasing evidence for modulatory roles of dopamine, nitric oxide and neuroactive peptides in regulating synaptic transmission between retinal neurons, allowing the retina to maintain its sensitivity over a wide range of light intensities by trading high absolute sensitivity in dim light for greater spatial and temporal resolution at higher light intensities. It is not yet clear how light, or other environmental cues might control the release of these modulatory substances. The intracellular targets and mechanisms of action of these compounds are not well defined either. In addition the pathways for terminating the responses of the beta-adrenergic receptor and rhodopsin have been extensively studied. Receptor kinases that are activated by free beta-gamma subunits are believed to phosphorylate the C-termini of these receptors allowing the subsequent binding of proteins of the arrestin family. This sequence of events terminates receptor activity in these systems, but so far evidence for the role of arrestin-like proteins or G-protein receptor kinases in the L-APB receptor pathway in the On-bipolar cell has not been forthcoming.
In this study we attempted to define functional sites within the APB receptor
pathway that are modulated by kinases. These sites that provide a potential
mechanism for the regulation of the On-bipolar cell synaptic response.
Specifically, we report that responses to exogenous APB are profoundly altered
by inhibitors of CaMKII and PKA, and by alkaline phosphatase. On this basis we
propose that both CaMKII and PKA regulate the APB receptor pathway, and that
intra-or extracellular ligands that activate these kinases may have important
roles in controlling the sensitivity of this synapse and the visual system as a
whole. In addition we have provided preliminary evidence for a G-protein
receptor kinase that terminates the activity of the agonist-stimulated receptor.
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The brief was to develop a viable rat retinal slice culture system in
agarose. In two separate sets of cultures morphology was retained in 0.5%
agarose culture after 4-5 days in culture, and only a few dead cells were
observed in any neuronal layer as judged by ethidium homodimer staining. The
positive control (calcein-AM) stained all cell layers brightly. In contrast
slices grown upon filter supports lost all morphological integrity and laminar
structure within 2 days. In conclusion this system provides an ideal method for
the live-dead ratio imaging system (Molecular Probes).
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The electrophysiological characterization of cultured P10 bipolar and ganglion cells was performed. Western blotting and whole-cell recordings were used to show that P10 cultured rat on-bipolar cells expressed the components of the L-APB-receptor cascade. In addition the AMPA/kainate currents were recorded from P0 retinal cultures.
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