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.

27Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma, where elevated 28 intraocular pressure (ocular hypertension, OHT) stresses retinal ganglion cell (RGC) axons as they exit 29 the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy, 30 transport inhibition, and gliosis. Importantly, many of these changes appear to occur prior to irreversible 31 neuronal loss, making them promising points for early diagnosis of glaucoma. It is unknown whether 32OHT has similarly early effects on the function of RGC output to the brain. To test this possibility, we 33 elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. 5 weeks post-34injection, bead-injected eyes showed a modest RGC loss in the peripheral retina, as evidenced by RBPMS 35 antibody staining. Additionally, we observed reduced dendritic complexity and lower spontaneous spike 36 rate of On-RGCs, targeted for patch clamp recording and dye filling using a Opn4-cre reporter mouse 37 line. To determine the influence of OHT on retinal projections to the brain, we expressed 38Channelrhodopsin-2 (ChR2) in melanopsin-expressing retinal ganglion cells by crossing the Opn4-cre 39 mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical 40relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 41 460 nm light. The use of a Opn4-cre reporter system allowed for expression of ChR2 in a narrow subset 42of RGCs responsible for image-forming vision in mice. Five weeks following OHT induction, paired 43 pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability 44at retinogeniculate synapses was elevated. Additionally, miniature synaptic current frequency was slightly 45 reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons, 46assessed using a Sholl analysis, showed a reduced complexity. Strikingly, these changes occurred prior to 47 major loss of RGCs labeled with the Opn4-Cre mouse, as indicated by immunofluorescence staining of 48ChR2-expressing retinal neurons. Thus, OHT leads to pre-and post-synaptic functional and structural 49 changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport 50 changes, these retinogeniculate synaptic changes are among the earliest signs of glaucoma. 51 52

Within the nervous system, homeostatic mechanisms stabilize network activity following disruption by injury, disease, or degeneration. Vision loss and optic nerve injury in age-related diseases such as glaucoma might trigger homeostatic responses in direct retinal projection targets in the brain in adulthood. We tested this possibility using patch-clamp electrophysiology, optogenetics, and single-cell dendritic analysis to probe the effects of optic nerve injury and vision loss on dLGN thalamocortical (TC) relay neurons and their synaptic inputs following bilateral enucleation. Using vGlut2 immunostaining, we found that retinal axon terminals in the dLGN degenerated over several days post-enucleation, which corresponded with the loss of retinogeniculate (RG) synaptic function as assessed with optogenetic stimulation of ganglion cell axons in acute brain slices. Analysis of TC neuron dendritic structure from single-cell dye fills revealed a gradual loss of dendrites proximal to the soma, where TC neurons receive the bulk of RG inputs. Surprisingly, there was little change to the frequency of miniature post-synaptic currents (mEPSCs), even two weeks post-enucleation, although we did find an increase in the relative proportion of mEPSCs with slower kinetics, hinting at a possible enhancement of corticogeniculate input. Whole-cell current clamp recordings showed that enucleation enhanced TC neuron action potential firing and input resistance, consistent with homeostatic scaling of intrinsic neuronal excitability following perturbation of synaptic inputs. Our findings show that degeneration of the retinal axons/optic nerve and loss of RG synaptic inputs induces structural and functional changes in TC neurons consistent with compensatory homeostatic plasticity in the dLGN.