William A. Hare

α2 Adrenergic Modulation of NMDA Receptor Function as a Major Mechanism of RGC Protection in Experimental Glaucoma and Retinal Excitotoxicity

PURPOSE alpha2 Agonists, such as brimonidine, have been shown to protect retinal ganglion cells (RGCs) in animal models of glaucoma and acute retinal ischemia. In this study, the authors investigated the neural mechanism that may underlie alpha2 neuroprotection of RGCs. METHODS The authors used in situ RGCs in the isolated rat retina to investigate possible interactions between alpha2 and N-methyl-D-aspartate (NMDA) receptors and rat glaucoma or rabbit retinal NMDA excitotoxicity models to verify in vitro findings under in vivo conditions. RESULTS Application of NMDA elicited a robust intracellular Ca(2+) signal and inward current in individual in situ RGCs voltage clamped at -70 mV. NMDA-elicited responses were blocked by D-AP5 (D-2-amino-5-phosphonopentanoic acid), a selective NMDA receptor antagonist. Brimonidine pretreatment also significantly reduced NMDA-elicited whole-cell currents and cytosolic Ca(2+) signals in RGCs. This suppressive action of brimonidine was blocked by alpha2 antagonists, cAMP analogs, an adenylate cyclase activator, and a cAMP-specific phosphodiesterase (PDE4) inhibitor, indicating that this brimonidine effect is mediated by the alpha2 receptor through a reduction of intracellular cAMP production. Brimonidine or NMDA receptor blockers protected RGCs in rat glaucoma and rabbit retinal NMDA excitotoxicity models. The brimonidine neuroprotective effect was abolished by an alpha2 antagonist or a PDE4 inhibitor in both in vivo models. CONCLUSIONS The results demonstrate alpha2 modulation of NMDA receptor function as an important mechanism for neuroprotection. These results suggest a new therapeutic approach based on neuromodulation, instead of direct inhibition, of the NMDA receptor for the treatment of glaucoma and other CNS disorders associated with NMDA receptor overactivation.

Origins of the electroretinogram oscillatory potentials in the rabbit retina.

The electroretinogram (ERG) oscillatory potential (OP) is a high-frequency, low-amplitude potential that is superimposed on the rising phase of the b-wave. It provides noninvasive evaluation of inner retina function in vivo and is a useful tool in basic research as well as in the clinic. While the OP is widely believed to be generated mainly by activity of the inner retina, the exact underlying neural mechanisms are not well understood. We have investigated the retinal mechanisms that underlie OP generation in Dutch-belted rabbits. The OP was isolated by band-filtering (100-1000 Hz) ERG signals. We used pharmacological agents that block specific transmitter receptors or voltage-gated channels in order to examine contributions of various retinal mechanisms to OP generation. Our results show that the OP elicited by a bright brief flash can be classified into early, intermediate, and late subgroups that are likely generated mainly by photoreceptors, action-potential-independent, and action-potential-dependent mechanisms in the ON pathway of the inner retina, respectively. ON bipolar cells themselves make only a small direct contribution to OP generation, as do horizontal cells and neurons in the OFF pathway.

Experimental Glutamatergic Excitotoxicity in Rabbit Retinal Ganglion Cells: Block by Memantine

PURPOSE Excessive activity of NMDA-type (N-methyl-d-aspartate) glutamatergic channels has been implicated as a mechanism for neuronal injury in neurologic disorders, including glaucoma, and retinal disease. This study was designed to characterize the retinal response to experimental manipulations that mimic features of glutamatergic excitotoxic insult and also to determine whether memantine, an NMDA-type glutamatergic channel blocker, is effective in reversing experimental excitotoxicity. METHODS Recordings of the electroretinogram (ERG) and spiking activity of single retinal ganglion cells (RGCs) were made from rabbit retinas. Excitotoxic insult was induced by either (1) application of NMDA, a selective NMDA receptor agonist; (2) application of TBOA (dl-threo-beta-benzyloxyaspartic acid), a selective inhibitor of glutamate transporters, or (3) perfusion with magnesium-free medium. For each condition, memantine was coapplied to determine its efficacy for reversal of experimental excitotoxicity. Memantine was also applied in isolation to characterize any effect on retinal responses to light stimuli. RESULTS All three experimental manipulations were associated with an increase in the tonic level of RGC spiking activity, a reduction in RGC spike amplitude, and, in some cells, block of spike generation. Experimental excitotoxicity had little or no effect on ERG responses. Coapplication of memantine was associated with recovery of RGC tonic spiking activity and spike amplitude toward control levels. Application of memantine in isolation was associated with a dose-dependent effect on the timing of ERG and RGC-OFF responses. CONCLUSIONS Memantine was effective in reversing acute experimental excitotoxicity at concentrations that have little effect on retinal light signaling.

Effects of bicarbonate versus HEPES buffering on measured properties of neurons in the salamander retina

Electrophysiological studies of the isolated retina involve perfusing the tissue with a physiological Ringers. Organic pH buffers such as HEPES have become increasingly popular in recent years because for many purposes they offer a convenient and reliable alternative to the more traditional bicarbonate/CO2. In this paper, however, we report that important functional properties of rods, bipolar cells, and horizontal cells in the salamander, Ambystoma tigrinum, are sensitive to the choice of buffer and, in the case of horizontal cells, that sensitivity is acute. In bicarbonate/CO2 Ringers, the dark potential of the horizontal cell was typically near -50 mV and saturating light caused it to hyperpolarize to about -75 mV. On switching to HEPES-buffered Ringers at the same pH, horizontal cells depolarized in darkness to about -20 mV, close to the chloride equilibrium potential, and the kinetics of their light responses changed. The cone-driven components of light responses increased in size relative to rod-driven components. Saturating lights still hyperpolarized the cells to -75 mV, however. Horizontal cells, being coupled via gap junctions, form a syncytium and syncytial length constants, measured in bicarbonate/CO2 Ringers, were generally in the range 150-225 microm. On switching to HEPES-buffered Ringers, length constants increased substantially to 250-330 microm. All these changes were reversible. We discuss our findings within the context of the cells ability to regulate its internal pH.

Nimodipine Enhancement of α2 Adrenergic Modulation of NMDA Receptor via a Mechanism Independent of Ca2+ Channel Blocking

PURPOSE To further understand alpha2 receptor signaling in the retina and the mechanisms that mediate ocular beneficial effects of brimonidine (an alpha2 agonist) and nimodipine (an L-type Ca(2+) channel blocker). METHODS The authors used in situ retinal ganglion cells (RGCs) in the isolated rat retina to characterize alpha2 modulation of NMDA receptor function and a rabbit retinal NMDA excitotoxicity model to verify in vitro findings under in vivo conditions. Electrophysiological (whole-cell patch clamp) recordings and Ca(2+) imaging were used to characterize NMDA receptor function and to verify the effect of various Ca(2+) channel blockers. In vivo drug application in rabbits was achieved by intravitreal injections. RESULTS Application of NMDA elicited a robust whole-cell inward current in individual in situ RGCs voltage clamped at -70 mV. Pretreatment with brimonidine significantly reduced NMDA-elicited currents in RGCs. This suppressive effect of brimonidine was substantially enhanced by background addition of nimodipine or isradipine, but not by diltiazem, verapamil, or cadmium. This effect of nimodipine was blocked by either a selective alpha2 antagonist, a cyclic adenosine monophosphate (cAMP) analogue, or an adenylate cyclase activator, indicating that nimodipine acts through the alpha2 receptor-G(alphai)-coupled pathway. Brimonidine protects RGCs in the rabbit excitotoxicity model. This brimonidine protection is also enhanced significantly by application of nimodipine but not of diltiazem. CONCLUSIONS These in vitro and in vivo findings demonstrate a novel neural mechanism involving nimodipine enhancement of alpha2 signaling in RGCs. This nimodipine effect appears to be independent of its classic L-type Ca(2+) channel-blocking action.

Signal transfer from photoreceptors to bipolar cells in the retina of the tiger salamander

Under conditions of dark-adaptation, in response to weak stimuli, the distal retina behaves as a linear system. During the process of voltage transfer from the rods to the bipolar cells the information encoded in the rod responses is spatially filtered. The spatial filtering is determined by the spatial properties of the receptive field of the bipolar cell. These, in turn, depend upon the spatial properties of three syncytia, those of the receptors, the horizontal cells and the bipolar cells themselves. The response of the bipolar cell to these weak stimuli is a linear difference of two components; a component generated by the receptive field center and a component generated by the receptive field surround. The receptive field surround is misnamed since it extends throughout the receptive field center and contributes to the response of the bipolar cell to stimuli located anywhere within the receptive field. The receptive field surround has the spatial properties that would be expected if it were generated by an input from the horizontal cells to the receptive field center of the bipolar cell. The cellular pathway mediating this input remains unclear though we have evidence that it involves, at least in part, a feedforward pathway from horizontal cells to bipolar cells. If a feedback pathway also exists it is not mediated by the GABAA synapse on the synaptic terminals of the cones.

The Effect of Light Level and Small Pupils on Presbyopic Reading Performance

Purpose To examine the impact of small pupils and light levels on reading performance of distance-corrected presbyopes. To determine whether small pupils would enable presbyopes to read at near even at low light levels. Methods To establish the lower range of text luminances, we quantified the space-averaged luminance of text in nine different artificially lit interior environments, and examined the impact of the text characters on space-averaged luminance of electronic and printed displays. Distance and near reading speeds of 20 presbyopes (ages 40-60 years) were measured while viewing through artificial pupils (diameters 1-4.5 mm), natural pupils, or with a multifocal contact lens. Space-averaged text luminance levels varied from 0.14 to 140 cd/m2 (including the range of measured environmental text luminances). Results Adding black text to a white computer display or paper reduces luminance by approximately 15% to 31%, and the lowest encountered environmental text luminance was approximately 2 to 3 cd/m2. For both distance and near reading performance, the 2- to 3-mm small pupil yielded the best overall reading acuity for space-averaged text light levels ≥ 2 cd/m2. The 2- to 3-mm artificial pupils and the multifocal contact lenses both enabled maximum or near-maximum reading speeds for 0.5 logMAR characters at distance and near, but with natural pupils, reading speeds were significantly reduced at near. Conclusions Although photon noise at low luminance reduces the visual benefits of small pupils, the benefits of 2- to 3-mm artificial pupils are sufficient to enable >80% of distance-corrected presbyopes to read proficiently at near, even at the lowest text luminances found in interior environments.

Neural Mechanisms Underlying Brimonidine’s Protection of Retinal Ganglion Cells in Experimental Glaucoma

Glaucoma is a neurodegenerative disease characterized by a progressive loss of retinal ganglion cells (RGCs), the output neurons of the retina. Elevated intraocular pressure (IOP) has long been recognized as a major risk factor for human glaucoma (Kass et al., 1980; Quigley et al., 1994; Tsai & Kanner, 2005). Indeed, in animal models of glaucoma, ranging from rodents (Johnson & Tomarev, 2010) to primates (Gaasterland & Kupfer, 1974; Hare et al., 2004), elevated IOP produced either biophysically (Gaasterland & Kupfer, 1974; WoldeMussie et al., 2001) or genetically (Anderson et al., 2001; Ju et al., 2009) can lead to RGC degeneration similar to that found in human glaucoma (Quigley, 2005). A common and effective treatment for glaucoma is the use of IOP lowering topical drugs that act at a variety of cellular targets, such as the α2 and β adrenergic receptors (Tsai & Kanner, 2005). However, in many patients, the disease continues to progress despite successful IOP reduction with topical drugs (Heijl et al., 2002; Vasudevan et al., 2011). Brimonidine, a selective α2 receptor agonist, is the active ingredient in one class of topical IOP lowering drugs, such as Alphagan® and Alphagn-P®. Brimonidine has been shown to protect RGCs in experimental glaucoma (WoldeMussie et al., 2001; Dong et al., 2008), retinal ischemia (Donello et al., 2001; Lai et al., 2002), optic nerve injury (Yoles et a., 1999), and retinal excitotoxicity (Dong et al., 2008). In experimental glaucoma, brimonidine’s neuroprotective effect appears to be independent of its IOP lowering action (Dong et al., 2008; Hernandez et al., 2008). More recently, in a randomized, double-masked, multicenter clinical trial, brimonidine has been shown to be more effective in slowing disease progression (visual field loss), compared with timolol (a β blocker), despite the fact that the mean treated IOP was similar in both treatment groups at all time points (Krupin 2011). These clinical data suggest that brimonidine may have a direct RGC protective effect that is independent of its IOP lowering action in human low-pressure glaucoma, similar to that found in experimental glaucoma (Dong et al., 2008; Hernandez et al., 2008). In this chapter, we will summarize the results of our recent studies on the mechanisms that underlie brimonidine’s protection of RGCs in experimental glaucoma and retinal excitotoxicity. We will first describe the properties of RGCs and the ex vivo and in vivo models used in our studies on the mechanisms of RGC injury and protection. Then we will