Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

Abstract

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal 1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells -the output cells of the retina 3,4 .A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes 5,6 , we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon -a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release 7,8 -and how it contributes to the unique signaling properties of this first retinal synapse. Video LinkThe video component of this article can be found at https://www.jove.com/video/50007/ Protocol Retinal Slices Preparation1. Assemble the chamber (design illustrated in Figure 1). Place two beads of vacuum grease, spaced ~8-10 mm apart, across the recording chamber to form a channel for superfusate and in which to embed the retinal slices. Add a second bead of grease a few millimeters farther out beyond each of these two beads of grease to act as a levee and limit spillover. Place a small triangular piece of KimWipe at the end of the chamber to ensure fluid contact with the reference electrode. 2. Press a piece of nitrocellulose membrane (~ 5 x 10 mm; 0.8 μm pores) flat against the glass microscope slide into two small beads of vacuum gre...

Weak endogenous Ca2+buffering supports sustained synaptic transmission by distinct mechanisms in rod and cone photoreceptors in salamander retina

“…They were then decapitated, quickly pithed, and enucleated. Retinas were mounted on a nitrocellulose membrane (type AAWP, 0.8  μ m pores; Millipore) and cut into 125  μ m vertical slices using a razor blade tissue chopper (Stoelting), as described previously (Van Hook and Thoreson 2013). Slices were rotated 90 degrees to view the retinal layers and mounted in a recording chamber that was positioned on an upright fixed-stage microscope (Nikon E600FN) equipped with a 60× water immersion objective.…”

“…Cell identity was confirmed based on physiological criteria (Van Hook and Thoreson 2013) and, in some cases, by imaging of fluorescent dyes included in the pipette solution. Patch pipettes were pulled from borosilicate glass (1.2 mm OD, 0.9 mm ID, with an internal filament; World Precision Instruments).…”

Synaptic Ribbon Active Zones in Cone Photoreceptors Operate Independently from One Another

“…The eyes were enucleated, the retina isolated, and vertical retinal slices (125 μm thickness) were prepared as described in detail elsewhere (Van Hook and Thoreson, 2013). …”

Very long-chain fatty acids support synaptic structure and function in the mammalian retina

“…Recordings were performed under standard conditions as described previously (Bennett et al, 2014;Van Hook and Thoreson, 2013). There was no significant difference in I Ca between WT and KO mice, indicating that loss of presynaptic I Ca are not responsible for any decreases in synaptic transmission in KO mice (Figs.…”