Early-Stage Ocular Hypertension Alters Retinal Ganglion Cell Synaptic Transmission in the Visual Thalamus

Abstract

Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma, where elevated intraocular pressure (ocular hypertension, OHT) stresses retinal ganglion cell (RGC) axons as they exit the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy, transport inhibition, and gliosis. Importantly, many of these changes appear to occur prior to irreversible neuronal loss, making them promising points for early diagnosis of glaucoma. It is unknown whether OHT has similarly early effects on the function of RGC output to the brain. To test this possibility, we elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. Five weeks post-injection, bead-injected eyes showed a modest RGC loss in the peripheral retina, as evidenced by RBPMS antibody staining. Additionally, we observed reduced dendritic complexity and lower spontaneous spike rate of On-αRGCs, targeted for patch clamp recording and dye filling using a Opn4-Cre reporter mouse line. To determine the influence of OHT on retinal projections to the brain, we expressed Channelrhodopsin-2 (ChR2) in melanopsin-expressing RGCs by crossing the Opn4-Cre mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 460 nm light. The use of a Opn4-Cre reporter system allowed for expression of ChR2 in a narrow subset of RGCs responsible for image-forming vision in mice. Five weeks following OHT induction, paired pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability at retinogeniculate synapses was elevated. Additionally, miniature synaptic current frequency was slightly reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons, assessed using a Sholl analysis, showed a reduced complexity. Strikingly, these changes occurred prior to major loss of RGCs labeled with the Opn4-Cre mouse, as indicated by immunofluorescence staining of ChR2-expressing retinal neurons. Thus, OHT leads to pre- and post-synaptic functional and structural changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport changes, these retinogeniculate synaptic changes are among the earliest signs of glaucoma.

Neuronal and synaptic plasticity in the visual thalamus in mouse models of glaucoma

“…A strain-matched control line that contains a wild allele of the Gpnmb gene and does not develop elevated eye pressure or glaucoma was used as a control (DBA/2J Gpnmb+ /SjJ ; D2-control; Jax# 007048) (Howell et al, 2007). For microbead injection experiments, we used mice produced as a cross of Opn4 Cre/Cre (Ecker et al, 2010) and Ai32 (Jax #024109) (Madisen et al, 2012) lines (Opn4-Cre;Ai32) (Bhandari et al, 2019). Mouse were housed in a 12/12 hour light/dark cycle and provided with food and water ad libitum .…”

“…To induce ocular hypertension, fluorescently-tagged polystyrene microspheres (10 micron, Invitrogen F8836) were bilaterally injected into the anterior chambers of Opn4 Cre ;Ai32 mice at ~6-8 weeks of age (Sappington et al, 2010; Calkins et al, 2018; Bhandari et al, 2019). This procedure was performed under isoflurane anesthesia and following instillation of anesthetic eye drops (0.5% proparacaine, Akorn, Lake Forest, IL).…”

“…Intraocular pressure (IOP) was monitored using a Tonolab rebound tonometer (iCare, Vantaa, Finland) in mice lightly anesthetized with isoflurane, as we have described previously (Bhandari et al, 2019). IOP was measured approximately monthly in D2 and D2-control mice beginning around 2-4 months of age and was measured prior to and at 2 days, 1, 2, and 4 weeks post-injection for microbead-injected mice.…”

“…250-micron thick coronal brain slices containing the dLGN were acutely prepared using the “protected recovery” method (Ting et al, 2014, 2018), as we have described previously (Bhandari et al, 2019; Van Hook, 2020). Following euthanasia by CO 2 asphyxiation and cervical dislocation, brains were submerged in a slush of artificial cerebrospinal fluid (aCSF) comprised of (in mM) 128 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 24 NaHCO 3 , 12.5 glucose, 2 CaCl 2 , and 2 MgSO 4 and continuously bubbled with a mixture of 5% CO 2 and 95% O 2 .…”

“…However, glaucoma-associated changes to visual function are likely the result of more than RGC and axonal degeneration, as cell loss is preceded by numerous changes in the structure and function of RGC-associated circuits. Within the retina, for instance, pressure elevation upregulates Na + channel expression and spiking behavior of RGCs, alters the size and complexity of their dendritic fields, changes receptive field properties, leads to changes in post-synaptic receptor composition, and alters the responses of upstream retinal circuits (Della Santina et al, 2013, 2013; Frankfort et al, 2013; Wang et al, 2014; Pang et al, 2015; Ou et al, 2016; Bhandari et al, 2019; McGrady et al, 2020; Sladek and Nawy, 2020). Within the optic projection and at RGC output sites in the brain, eye pressure and glaucomatous injury lead to alterations in optic nerve glial function and energy demands (Baltan et al, 2010; Inman and Harun-Or-Rashid, 2017; Jassim et al, 2019; Cooper et al, 2020), RGC axon terminal swelling and atrophy (Smith et al, 2016), changes in axon terminal mitochondrial health (Smith et al, 2016), altered synaptic vesicle release properties (Bhandari et al, 2019), and post-synaptic neuronal dendritic remodeling and somatic atrophy (Gupta and Yücel, 2003; Gupta et al, 2007, 2009, Liu et al, 2011, 2014; Bhandari et al, 2019).…”