Cun-Jian Dong

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.

Contribution to the kinetics and amplitude of the electroretinogram b-wave by third-order retinal neurons in the rabbit retina

The ERG b-wave is widely believed to reflect mainly light-induced activity of on-center bipolar cells and Müller cells. Third-order retinal neurons are thought to contribute negligibly to generation of the b-wave. Here we show that pharmacological agents which affect predominantly third-order neurons alter significantly both the kinetics and amplitude of the b-wave. Our results support the notion that changes in the amplitude and kinetics of light-induced membrane depolarization in third-order neurons produce similar changes in the amplitude and kinetics of the b-wave. We conclude that activity in third-order neurons makes a significant contribution to b-wave generation. Our results also provide evidence that spiking activity of third-order neurons truncates the a-wave by accelerating the onset of the b-wave.

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.

GABAc feedback pathway modulates the amplitude and kinetics of ERG b-wave in a mammalian retina in vivo.

The electroretinogram b-wave is generally believed to reflect mainly light-induced activity of ON-center bipolar cells and Muller cells. Recently, there is increasing evidence that third-order retinal neurons can also contribute significantly to the b-wave. In a previous study (Vis. Res. 40 (2000) 579) we proposed that the GABAc feedback from amacrine cells to bipolar cells can affect both the amplitude and kinetics of the b-wave. Here we show that blocking this feedback has profound effects on b-wave amplitude and kinetics. These results demonstrate that feedback to bipolar cells is an important mechanism through which amacrine cells contribute to b-wave generation. Our results also provide functional evidence that the feedback may be involved in temporal processing in the mammalian retina.

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.

Presynaptic Inhibition by α2 Receptor/Adenylate Cyclase/PDE4 Complex at Retinal Rod Bipolar Synapse

G-protein-coupled receptor (GPCR)-mediated presynaptic inhibition is a fundamental mechanism regulating synaptic transmission in the CNS. The classical GPCR-mediated presynaptic inhibition in the CNS is produced by direct interactions between the Gβγ subunits of the G-protein and presynaptic Ca2+ channels, K+ channels, or synaptic proteins that affect transmitter release. This mode of action is shared by well known GPCRs such as the α2, GABAB, and CB1 receptors. We report that the α2 receptor-mediated inhibition of presynaptic Ca2+ channel and transmitter release in rat retinal rod bipolar cells depends on the Gα subunit via a Gα–adenylate cyclase–cAMP cascade and requires participation of the type 4 phosphodiesterase (PDE4), a new role for phosphodiesterase in neural signaling. By using the Gα instead of the Gβγ subunits, this mechanism is able to use a cyclase/PDE enzyme pair to dynamically control a cyclic nucleotide second messenger (i.e., cAMP) for the regulation of synaptic transmission, an operating strategy that shows remarkable similarity to that of dynamic control of cGMP and transmitter release from photoreceptors by the guanylate cyclase/PDE6 pair in phototransduction. Our results demonstrate a new paradigm of GPCR-mediated presynaptic inhibition in the CNS and add a new regulatory mechanism at a critical presynaptic site in the visual pathway that controls the transmission of scotopic information. They also provide a presynaptic mechanism that could contribute to neuroprotection of retinal ganglion cells by α2 agonists, such as brimonidine, in animal models of glaucoma and retinal ischemia and in glaucoma patients.

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