Dr. Steele’s Project Illustrators and Descriptors:
Calcium dynamics in rod photoreceptor terminals:
We previously published both immunocytochemical and physiological evidence for a concentration of L-type calcium channels in the membrane of the presynaptic terminals of rod photoreceptors acutely dissociatedfrom tiger salamander retina.
(See Steele et al., 2005: http://jn.physiology.org/cgi/content/full/94/6/4544
Click on the icon below to watch the changes in intracellular calcium, recorded as changes in fura-2 fluorescence in response to brief depolarizing puffs of high extracellular K+. To accentuate the morphology of the cell, the images have been inverted such that brightness corresponds to low intracellular Ca2+ levels and dimming corresponds to an increase in intracellular Ca2+ images. Note that the presynaptic terminal compartment dims in response to puffs of high K+, indicating a large increase in intracellular Ca2+ levels, while the other compartments of the rod do not change significantly.
High resolution ratiometric calcium imaging combined with recordings of whole cell ionic currents can be a powerful tool for dissecting the effects of various pharmacologic reagents on the functioning of rod photoreceptors and thus their potential to disturb the normal visual signal transmission. We previously used such an approach to demonstrate the ability of fluoxetine, the active ingredient of the commercially available antidepressant Prozac, to diminish depolarization-elicited increases in intracellular calcium levels and associated calcium-dependent ionic currents in both the somatic and presynaptic terminal compartments of rod photoreceptors, thus likely disturbing both the primary processing of the visual signal within the photoreceptor and its transmission via synaptic transmission to second order neurons of the retina. These data are consistent with visual side effects reported by some patients undergoing a regimen of high doses of the drug. (See Steele et al., 2005:http://www.molvis.org/molvis/v11/a137/)
In our published studies of rod photoreceptor physiology, we have stressed the importance of using fully intact rod photoreceptors possessing axons and presynaptic terminals in physiological studies of these cells, an important point often overlooked in many previously published studies. Specifically, we provided evidence that a cell with no obvious axon and terminal may have reabsorbed the terminal structure. Following reabsorption, the membrane of the terminal compartment, and the proteins it contains, are consequently contiguous with the membrane of the somatic compartment and can therefore contribute to and alter the normal physiological properties of this compartment. In the report, we provided immunocytochemical evidence of this reabsorption. Below we provide time lapse imaging of this reabsorption process occurring in a cell where the extended axon and terminal is initially attached to the coverslip. Obviously, this process can and does progress on a much faster time scale when the terminal is not attached to the coverslip.
The pore-forming L-type calcium channel Cav1.4 (α1F) subunit has been shown by other labs to be highly expressed in the presynaptic terminals of retinal rod photoreceptors and is thought to the predominant mediator of transmission of visual signals from rod photoreceptors to second order neurons in the retina. Humans with loss-of-function mutations in the CACNA1F gene encoding this calcium channel subunit exhibit congenital stationary night blindness (CSNB2) and greatly reduced electroretinogram (ERG) b-wave amplitudes. Mice with loss-of-function mutations in the cacna1f gene also show greatly reduced b-wave amplitudes and morphologic disturbances of synapses between the photoreceptors and second order retinal neurons. The remaining photoreceptor function in these patients and mutant mice has been attributed to rod-cone coupling and cone-mediated transmission by a distinct isoform of L-type calcium channel subunit, Cav1.3 (α1D). However, we recently reported unambiguous in situ hybridization data, demonstrating the presence of abundant Cav1.3(α1D) mRNA in murine rod photoreceptors. These data support a more parsimonious explanation of the residual b-wave amplitude: the signal is mediated, albeit very inefficiently, by Cav1.3(α1D) calcium channel subunits. These calcium channels subunit proteins may be expressed at significantly lower amounts than Cav1.4 (α1F) or have profoundly different conduction properties. Our observation of abundant mRNA in most retinal cell types suggests that elimination or mutation of the gene encoding this calcium channel subunit may also have severe visual consequence.
(See Xiao et al., 2007: http://www.molvis.org/molvis/v13/a83/)
Müller cell changes and Diabetic Retinopathy:
To study the in vivo effects of diabetes on Müller cell expression, localization, and function, we are utilizing streptozotocin (STZ) injection to selectively destroy the insulin-secreting beta pancreatic cells of young Sprague Dawley rats, thus rendering them in a perpetually hyperglycemic (fasting blood glucose ~400 mg/dL) state 1-2 days after injection. Müller cells, which can be easily identified by their distinct morphological features are acutely isolated from dissected retinas via digestion with the proteolytic enzyme papain followed by gentle tritutration. Cells isolated by this method from normal and diabetic retinas are plated onto glass-bottomed dishes for comparison of immunocytochemical staining and functional profiles. Current studies are focusing on changes in P2X and P2Y purinergic receptor expression and functional profiles.
In order to determine whether changes in Müller cell expression or functional profiles are intrinsic responses to diabetic conditions, we employ an immortalized rat Müller cell line (kindly provided by Dr. Vijay Sarthy; See Sarthy et al.,1998: http://www.iovs.org/cgi/reprint/39/1/212) and compare the properties of these cells cultured under normal (euglycemic, euinsulinemic) to those of cells cultured under diabetic (hyperglycemic, hypoinsulinemic) conditions. Preliminary data demonstrating the presence and functioning of P2X and P2Y purinergic receptors in immortalized and native rat Müller cells are currently being prepared for publication.
Retinal Ischemia in the MCAO Stroke Model:
Middle cerebral artery occlusion (MCAO) is the most widely utilized experimental model for producing and studying unilateral transient focal strokes in the brains of rodents. Because of the close proximity of the ophthalmic artery to the middle cerebral artery, we suspected MCAO might also affect the retina and that retinal damage could be a confounding factor for the observed motor dysfunction following stroke in this model. Initial semiquantitative studies of retinal wholemounts following intravenous injection of fluorescent microspheres demonstrated a near total unilateral diminishment in retinal perfusion of ischemic retinas by MCAO compared to contralateral controls.
Cresyl violet (Nsill) staining was used to reveal retinal degeneration resulting from transient (30 or 60 minutes) MCAO followed by reperfusion. No signs of degeneration were obvious following 30 minutes MCAO followed by reperfusion for 2 hours or 2 days. However, abundant pyknotic nuclei were observed in both the inner nuclear layer (>30%) and the ganglion cell layers (>50%) of ischemic retinas, but not in contralateral controls, following 60 minutes MCAO and a short reperfusion period of 2 hours.
We used TUNEL staining to determine if pyknotic nuclei revealed by cresyl violet staining represented cells undergoing apoptosis. Only very few cells appeared to be undergoing apoptosis following 60 minutes MCAO and 1 day of reperfusion, suggesting that most acute damage is mediated via non-apoptotic mechanisms.
These preliminary data have just been accepted (November 28, 2007) for publication in Stroke, the Journal of the American Stroke Association, a Division of the American Heart Association.
Future studies using this model will be aimed at determining the underlying cellular and molecular bases for the retinal damage, testing the efficacy of “neuroprotective” reagents at ameliorating or preventing retinal damage via these mechanisms, and determining the contribution of this retinal damage to motor dysfunction observed after a stroke in this model.
