Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Using Looming Visual Stimuli to Evaluate Mouse Vision

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina ...

Wayne State University School of Medicine

Inhibition is mediated by two classes of ionotropic receptors in the retina, GABA(A) and GABA(C) receptors. We used the GABA transport blocker NO-711 to examine the role of GABA transporters in shaping synaptic responses mediated by these two receptors in the salamander retinal slice preparation. Focal applications (puffs) of GABA onto GABA(C) receptors on bipolar cells terminals or GABA(A) receptors on ganglion cells elicited currents that were enhanced by NO-711, demonstrating the presence of transporters in the inner plexiform layer (IPL). IPSCs were evoked in bipolar and ganglion cells by puffing kainate into the IPL. NO-711 enhanced the IPSCs only in bipolar cells, suggesting that, when GABA uptake was blocked, the GABA(C) receptors were more strongly activated by spillover transmission than the GABA(A) receptors on ganglion cells. NO-711 enhanced the light-evoked IPSCs mediated by GABA(C) receptors on bipolar cell axon terminals, which resulted in reduced transmission between bipolar and ganglion cells. NO-711 also shifted the intensity-response relationship of the ganglion cell, reducing its sensitivity to light. Surround illumination has been shown by others to produce similar shifts in ganglion cell light sensitivity. Our results show that GABA transporters limit the extent of inhibitory transmission at the inner retina during light-evoked signal processing.

GABA transporters regulate inhibition in the retina by limiting GABA(C) receptor activation.

To test the novel hypothesis that the K+ efflux mediated by NMDA receptors might be regulated differently than the influx of Ca2+ and Na+ through the same receptor channels, NMDA receptor whole-cell currents carried concurrently or individually by Ca2+, Na+ and K+ were analysed in cultured mouse cortical neurons. In contrast to the NMDA inward current carried by Ca2+ and Na+, the NMDA receptor outward K+ current or NMDA-K current, recorded either in the presence or absence of extracellular Ca2+ and Na+, and at different or the same membrane potentials, showed much less sensitivity to alterations in intracellular Ca2+ concentration and underwent little rundown. In line with a selective regulation of the NMDA receptor K+ permeability, the ratio of the NMDA inward/outward currents decreased, and the reversal potential of composite NMDA currents recorded in physiological solutions shifted by -8.5 mV after repeated activation of NMDA receptors. Moreover, a depolarizing pre-pulse of a few seconds or a burst of brief depolarizing pulses selectively augmented the subsequent NMDA-K current, but not the NMDA inward current. On the other hand, a hyperpolarizing pre-pulse showed the opposite effect of reducing the NMDA-K current. The voltage- and activity-dependent regulation of the NMDA-K current did not require the existence of extracellular Ca2+ or Ca2+ influx; it was, however, affected by the duration of the pre-pulse and was subject to a time-dependent decay. The burst of excitatory activity revealed a lasting upregulation of the NMDA-K current even 5 s after termination of the pre-pulses. Our data reveal a selective regulation of the NMDA receptor K+ permeability and represent a novel model of voltage- and excitatory activity-dependent plasticity at the receptor level.

Ca2+-independent, but voltage- and activity-dependent regulation of the NMDA receptor outward K+ current in mouse cortical neurons.

Retinal bipolar cells are slow potential neurons that respond to photoreceptor inputs with graded potentials and do not fire action potentials. We found that transient ON bipolar cells recorded in retinal slices possess voltage-gated sodium channels located on either their dendrites or somas. The sodium currents in these neurons did not generate spikes but enhanced voltage responses evoked by visual stimulation, which selectively boosted transmission to transient ganglion cells. In contrast, sodium currents were not found in sustained ON bipolar cells, and light responses in sustained bipolar cells and ganglion cells were not affected by TTX. The presence of sodium channels in transient ON bipolar cells contributed to the separation of transient and sustained signals by selectively enhancing the responses of ON transient ganglion cells to light. Our results suggest that bipolar cell sodium channels augment transient signals and contribute to the temporal segregation of visual information.

Sodium channels in transient retinal bipolar cells enhance visual responses in ganglion cells.

Illumination of the receptive-field surround reduces the sensitivity of a retinal ganglion cell to centre illumination. The steady, antagonistic receptive-field surround of retinal ganglion cells is classically attributed to the signalling of horizontal cells in the outer plexiform layer (OPL). However, amacrine cell signalling in the inner plexiform layer (IPL) also contributes to the steady receptive-field surround of the ganglion cell. We examined the contributions of these two forms of presynaptic lateral inhibition to ganglion cell light sensitivity by measuring the effects of surround illumination on EPSCs evoked by centre illumination. GABA(C) receptor antagonists reduced inhibition attributed to dim surround illumination, suggesting that this inhibition was mediated by signalling to bipolar cell axon terminals. Brighter surround illumination further reduced the light sensitivity of the ganglion cell. The bright surround effects on the EPSCs were insensitive to GABA receptor blockers. Perturbing outer retinal signalling with either carbenoxolone or cobalt blocked the effects of the bright surround illumination, but not the effects of dim surround illumination. We found that the light sensitivities of presynaptic, inhibitory pathways in the IPL and OPL were different. GABA(C) receptor blockers reduced dim surround inhibition, suggesting it was mediated in the IPL. By contrast, carbenoxolone and cobalt reduced bright surround, suggesting it was mediated by horizontal cells in the OPL. Direct amacrine cell input to ganglion cells, mediated by GABA(A) receptors, comprised another surround pathway that was most effectively activated by bright illumination. Our results suggest that surround activation of lateral pathways in the IPL and OPL differently modulate the sensitivity of the ganglion cell to centre illumination.

Inner and outer retinal pathways both contribute to surround inhibition of salamander ganglion cells.

The retinal network increases its sensitivity in low-light conditions to detect small visual inputs and decreases its sensitivity in bright-light conditions to prevent saturation. However, the cellular mechanisms that adjust visual signaling in the retinal network are not known. Here, we show that voltage-gated sodium channels in bipolar cells dynamically control retinal light sensitivity. In dim conditions, sodium channels amplified light-evoked synaptic responses mediated by cone pathways. Conversely, in bright conditions, sodium channels were inactivated by dopamine released from amacrine cells, and they did not amplify synaptic inputs, minimizing signal saturation. Our findings demonstrate that bipolar cell sodium channels mediate light adaptation by controlling retinal signaling gain.

Ambient light regulates sodium channel activity to dynamically control retinal signaling.

Excitatory amino acid transporters (EAATs) terminate signaling in the CNS by clearing released glutamate. Glutamate also evokes an EAAT-mediated Cl(-) current, but its role in CNS signaling is poorly understood. We show in mouse retina that EAAT-mediated Cl(-) currents that were evoked by light inhibit rod pathway signaling. EAATs reside on rod bipolar cell axon terminals where GABA and glycine receptors also mediate light-evoked inhibition. We found that the mode of inhibition depended on light intensity. Dim light evoked GABAergic and glycinergic inhibition with rapid kinetics and a large spatial extent. Bright light evoked predominantly EAAT-mediated inhibition with slow kinetics and a small spatial extent. The switch to EAAT-mediated signaling in bright light supplements receptor-mediated signaling to expand the dynamic range of inhibition and contributes to the transition from rod to cone signaling by suppressing rod pathway signaling in bright light conditions.

The mode of retinal presynaptic inhibition switches with light intensity.

In the visual system, diverse image processing starts with bipolar cells, which are the second-order neurons of the retina. Thirteen subtypes of bipolar cells have been identified, which are thought to encode different features of image signaling and to initiate distinct signal-processing streams. Although morphologically identified, the functional roles of each bipolar cell subtype in visual signal encoding are not fully understood. Here, we investigated how ON cone bipolar cells of the mouse retina encode diverse temporal image signaling. We recorded bipolar cell voltage changes in response to two different input functions: sinusoidal light and step light stimuli. Temporal tuning in ON cone bipolar cells was diverse and occurred in a subtype-dependent manner. Subtypes 5s and 8 exhibited low-pass filtering property in response to a sinusoidal light stimulus, and responded with sustained fashion to step-light stimulation. Conversely, subtypes 5f, 6, 7, and XBC exhibited bandpass filtering property in response to sinusoidal light stimuli, and responded transiently to step-light stimuli. In particular, subtypes 7 and XBC were high-temporal tuning cells. We recorded responses in different ways to further examine the underlying mechanisms of temporal tuning. Current injection evoked low-pass filtering, whereas light responses in voltage-clamp mode produced bandpass filtering in all ON bipolar cells. These findings suggest that cone photoreceptor inputs shape bandpass filtering in bipolar cells, whereas intrinsic properties of bipolar cells shape low-pass filtering. Together, our results demonstrate that ON bipolar cells encode diverse temporal image signaling in a subtype-dependent manner to initiate temporal visual information-processing pathways.

Roles of ON cone bipolar cell subtypes in temporal coding in the mouse retina.

Green fluorescent protein (GFP) and its derivatives are broadly used in biomedical experiments for labeling particular cells or molecules. In the mouse retina, the light (~500 nm) used to excite GFP can also lead to photoreceptor bleaching (peak ~500 nm), which diminishes photoreceptor-mediated synaptic transmission in the retinal network. To overcome this problem, we investigated the use of infrared fluorescent protein (iRFP) as a marker since it is excited by light in the near-infrared range that would not damage the photoresponsiveness of the retina. Initially, we tested iRFP expression in human embryonic kidney 293 (HEK293) cells to confirm that conventional fluorescence microscopy can detect iRFP fluorescence. We next introduced the iRFP plasmid into adeno-associated virus 2 (AAV-2) and injected the resulting AAV-2 solution into the intraocular space. Retinal neurons were found to successfully express iRFP three weeks post-injection. Light-evoked responses in iRFP-marked cells were assessed using patch clamping, and light sensitivity was found to be similar in iRFP-expressing cells and non-iRFP-expressing cells, an indication that iRFP expression and detection do not affect retinal light responsiveness. Taken together, our results suggest iRFP can be a new tool for vision research, allowing for single-cell recordings from an iRFP marked neuron using conventional fluorescence microscopy.

Marking cells with infrared fluorescent proteins to preserve photoresponsiveness in the retina.

Retinal bipolar cells are second-order neurons in the visual system, which initiate multiple image feature-based neural streams. Among more than ten types of bipolar cells, type-5 cells are thought to play a role in motion detection pathways. Multiple subsets of type-5 cells have been reported; however, detailed characteristics of each subset have not yet been elucidated. Here, we found that they exhibit distinct morphological features as well as unique voltage-gated channel expression. We have conducted electrophysiological and immunohistochemical analysis of retinal bipolar cells. We defined type-5 cells by their axon terminal ramification in the inner plexiform layer between the border of ON/OFF sublaminae and the ON choline acetyltransferase (ChAT) band. We found three subsets of type-5 cells: XBCs had the widest axon terminals that stratified at a close approximation of the ON ChAT band as well as exhibiting large voltage-gated Na(+) channel activity, type-5-1 cells had compact terminals and no Na(+) channel activity, and type-5-2 cells contained umbrella-shaped terminals as well as large voltage-gated Na(+) channel activity. Hyperpolarization-activated cyclic nucleotide-gated (HCN) currents were also evoked in all type-5 bipolar cells. We found that XBCs and type-5-2 cells exhibited larger HCN currents than type-5-1 cells. Furthermore, the former two types showed stronger HCN1 expression than the latter. Our previous observations (Ichinose et al., 2014) match the current study: low temporal tuning cells that we named 5S corresponded to 5-1 in this study, while high temporal tuning 5f cells from the previous study corresponded to 5-2 cells. Taken together, we found three subsets of type-5 bipolar cells based on their morphologies and physiological features. 

Morphological and physiological analysis of type-5 and other bipolar cells in the Mouse Retina.

Retinal first-order neurons, photoreceptors, receive visual inputs and convert them to neural signals. The second-order neurons, bipolar cells then sort out the visual signals and encode them through multiple neural streams. Approximately 15 morphological types of bipolar cells have been identified, which are thought to encode different aspects of visual signals such as motion and color (Ichinose et al. J Neurosci 34(26):8761-8771, 2014; Euler et al. Nat Rev Neurosci 15(8):507-519, 2014). To investigate functional aspects of OFF bipolar cells, single cell recordings are preferred; however, bipolar cells in the mouse retina are small and hard to distinguish from other types of cells. Here, we describe our methodology and tips for immunohistochemistry and patch clamp recordings for analyzing light-evoked responses in each type of OFF bipolar cell.

Tomomi Ichinose

Anatomy and Cell Biology,
Ophthalmology,
Department of Ophthalmology,
Visual and Anatomical Sciences,
Department of Ophthalmology, Visual and Anatomical Sciences

Wayne State University School of Medicine

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

Using Looming Visual Stimuli to Evaluate Mouse Vision

Tomomi Ichinose

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Anatomy and Cell Biology,Ophthalmology,Department of Ophthalmology,Visual and Anatomical Sciences,Department of Ophthalmology, Visual and Anatomical Sciences

Wayne State University School of Medicine

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

Using Looming Visual Stimuli to Evaluate Mouse Vision

Anatomy and Cell Biology,
Ophthalmology,
Department of Ophthalmology,
Visual and Anatomical Sciences,
Department of Ophthalmology, Visual and Anatomical Sciences