Off Starburst Amacrine Cells in the Retina Trigger Looming-Evoked Fear Responses in Mice.

A rapidly approaching dark object evokes an evolutionarily conserved fear response in both vertebrates and invertebrates, young to old. A looming visual stimulus mimics an approaching object and triggers a similarly robust fear response in mice, resulting in freeze and flight. However, the retinal neural pathway responsible for this innate response has not been fully understood. We first explored a variety of visual stimuli that reliably induced these innate responses, and found that a looming stimulus with 2-d acclimation consistently evoked fear responses. Because the fear responses were triggered by the looming stimulus with moving edges, but not by a screen flipping from light to dark, we targeted the starburst amacrine cells (SACs), crucial neurons for retinal motion detection. We used intraocular injection of diphtheria toxin (DT) in mutant mice expressing diphtheria toxin receptors (DTR) in SACs. The looming-evoked fear responses disappeared in half of the DT-injected mice, and the other mice still exhibited the fear responses. The optomotor responses (OMRs) were reduced or eliminated, which occurred independent of the disappearance of the fear responses. A histologic examination revealed that ON SACs were reduced in both mouse groups preserved or absent fear responses. In contrast, the number of OFF SACs was different among two groups. The OFF SACs were relatively preserved in mice exhibiting continued fear responses, whereas they were ablated in mice lacking fear response to looming stimulation. These results indicate that OFF SACs and the direction-selective pathway in the retina play a role in looming-induced fear behaviors.

Presynaptic depolarization differentially regulates dual neurotransmitter release from starburst amacrine cells in the mouse retina.

The retina is comprised of diverse neural networks, signaling from photoreceptors to ganglion cells to encode images. The synaptic connections between these retinal neurons are crucial points for information transfer; however, the input-output relations of many synapses are understudied. Starburst amacrine cells in the retina are known to contribute to retinal motion detection circuits, providing a unique window for understanding neural computations. We examined the dual transmitter release of GABA and acetylcholine from starburst amacrine cells by optogenetic activation of these cells, and conducted patch clamp recordings from postsynaptic ganglion cells to record excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). As starburst amacrine cells exhibit distinct kinetics in response to objects moving in a preferred or null direction, we mimicked their depolarization kinetics using optogenetic stimuli by varying slopes of the rising phase. The amplitudes of EPSCs and IPSCs in postsynaptic ganglion cells were reduced as the stimulus rising speed was prolonged. However, the sensitivity of postsynaptic currents to the stimulus slope differed. EPSC amplitudes were consistently reduced as the steepness of the rising phase fell. By contrast, IPSCs were less sensitive to the slope of the stimulus rise phase and maintained their amplitudes until the slope became shallow. These results indicate that distinct synaptic release mechanisms contribute to acetylcholine and GABA release from starburst amacrine cells, which could contribute to the ganglion cells' direction selectivity.

Patch clamp recording from bipolar cells in the wholemount mouse retina.

Bipolar cells are the second-order neurons in the retina that are less accessible for investigating their synaptic responses. Here, we present a protocol to conduct patch clamp recordings from bipolar cells in the wholemount retina from Ai32 mutant mice. We detail whole-cell patch-clamp recording from bipolar cells to examine their light-evoked responses to optogenetic stimulation, followed by imaging terminals of recorded cells to determine bipolar cell type. We describe light stimulus information to activate channelrhodopsin-2 (ChR2). For complete details on the use and execution of this protocol, please refer to Hellmer et al. (2021).

Cholinergic feedback to bipolar cells contributes to motion detection in the mouse retina.

Retinal bipolar cells are second-order neurons that transmit basic features of the visual scene to postsynaptic partners. However, their contribution to motion detection has not been fully appreciated. Here, we demonstrate that cholinergic feedback from starburst amacrine cells (SACs) to certain presynaptic bipolar cells via alpha-7 nicotinic acetylcholine receptors (α7-nAChRs) promotes direction-selective signaling. Patch clamp recordings reveal that distinct bipolar cell types making synapses at proximal SAC dendrites also express α7-nAChRs, producing directionally skewed excitatory inputs. Asymmetric SAC excitation contributes to motion detection in On-Off direction-selective ganglion cells (On-Off DSGCs), predicted by computational modeling of SAC dendrites and supported by patch clamp recordings from On-Off DSGCs when bipolar cell α7-nAChRs is eliminated pharmacologically or by conditional knockout. Altogether, these results show that cholinergic feedback to bipolar cells enhances direction-selective signaling in postsynaptic SACs and DSGCs, illustrating how bipolar cells provide a scaffold for postsynaptic microcircuits to cooperatively enhance retinal motion detection.

Dopaminergic Modulation of Signal Processing in a Subset of Retinal Bipolar Cells.

The retina and the olfactory bulb are the gateways to the visual and olfactory systems, respectively, similarly using neural networks to initiate sensory signal processing. Sensory receptors receive signals that are transmitted to neural networks before projecting to primary cortices. These networks filter sensory signals based on their unique features and adjust their sensitivities by gain control systems. Interestingly, dopamine modulates sensory signal transduction in both systems. In the retina, dopamine adjusts the retinal network for daylight conditions ("light adaptation"). In the olfactory system, dopamine mediates lateral inhibition between the glomeruli, resulting in odorant signal decorrelation and discrimination. While dopamine is essential for signal discrimination in the olfactory system, it is not understood whether dopamine has similar roles in visual signal processing in the retina. To elucidate dopaminergic effects on visual processing, we conducted patch-clamp recording from second-order retinal bipolar cells, which exhibit multiple types that can convey different temporal features of light. We recorded excitatory postsynaptic potentials (EPSPs) evoked by various frequencies of sinusoidal light in the absence and presence of a dopamine receptor 1 (D1R) agonist or antagonist. Application of a D1R agonist, SKF-38393, shifted the peak temporal responses toward higher frequencies in a subset of bipolar cells. In contrast, a D1R antagonist, SCH-23390, reversed the effects of SKF on these types of bipolar cells. To examine the mechanism of dopaminergic modulation, we recorded voltage-gated currents, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and low-voltage activated (LVA) Ca2+ channels. SKF modulated HCN and LVA currents, suggesting that these channels are the target of D1R signaling to modulate visual signaling in these bipolar cells. Taken together, we found that dopamine modulates the temporal tuning of a subset of retinal bipolar cells. Consequently, we determined that dopamine plays a role in visual signal processing, which is similar to its role in signal decorrelation in the olfactory bulb.

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 through the lateral geniculate nucleus to the visual cortex, the system is complex. Cellular and molecular studies have been conducted to elucidate the mechanisms underpinning visual processing and, by extension, disease mechanisms. These studies may contribute to the development of artificial visual systems. To validate the results of these studies, behavioral vision testing is necessary. Here, we show that the looming stimulation experiment is a reliable mouse vision test that requires a relatively simple setup. The looming experiment was conducted in a large enclosure with a shelter in one corner and a computer monitor located on the ceiling. A CCD camera positioned next to the computer monitor served to observe mouse behavior. A mouse was placed in the enclosure for 10 minutes and allowed to acclimate to and explore the surroundings. Then, the monitor projected a program-derived looming stimulus 10 times. The mouse responded to the stimuli either by freezing or by fleeing to the hiding place. The mouse's behavior before and after the looming stimuli was recorded, and the video was analyzed using motion tracking software. The velocity of the mouse movement significantly changed after the looming stimuli. In contrast, no reaction was observed in blind mice. Our results demonstrate that the simple looming experiment is a reliable test of mouse vision.

Bipolar Cell Type-Specific Expression and Conductance of Alpha-7 Nicotinic Acetylcholine Receptors in the Mouse Retina.

Purpose: Motion detection is performed by a unique neural network in the mouse retina. Starburst amacrine cells (SACs), which release acetylcholine and gamma-aminobutyric acid (GABA) into the network, are key neurons in the motion detection pathway. Although GABA contributions to the network have been extensively studied, the role of acetylcholine is minimally understood. Acetylcholine receptors are present in a subset of bipolar, amacrine, and ganglion cells. We focused on α7-nicotinic acetylcholine receptor (α7-nAChR) expression in bipolar cells, and investigated which types of bipolar cells possess α7-nAChRs. Methods: Retinal slice sections were prepared from C57BL/6J and Gus8.4-GFP mice. Specific expression of α7-nAChRs in bipolar cells was examined using α-bungarotoxin (αBgTx)-conjugated Alexa dyes co-labeled with specific bipolar cell markers. Whole-cell recordings were conducted from bipolar cells in retinal slice sections. A selective α7-nAChR agonist, PNU282987, was applied by a puff and responses were recorded. Results: αBgTx fluorescence was observed primarily in bipolar cell somas. We found that α7-nAChRs were expressed by the majority of type 1, 2, 4, and 7 bipolar cells. Whole-cell recordings revealed that type 2 and 7 bipolar cells depolarized by PNU application. In contrast, α7-nAChRs were not detected in most of type 3, 5, 6, and rod bipolar cells. Conclusions: We found that α7-nAChRs are present in bipolar cells in a type-specific manner. Because these bipolar cells provide synaptic inputs to SACs and direction selective ganglion cells, α7-nAChRs may play a role in direction selectivity by modulating these bipolar cells' outputs.

A group I metabotropic glutamate receptor controls synaptic gain between rods and rod bipolar cells in the mouse retina.

The canonical mGluR6-Trpm1 pathway that generates the sign-inverting signal between photoreceptors and ON bipolar cells has been well described. However, one type of ON bipolar cell, the rod bipolar cell (RBC), additionally is thought to express the group I mGluRs whose function is unknown. We examined the role of group I mGluRs in mouse RBCs and here provide evidence that it controls synaptic gain between rods and RBCs. In dark-adapted conditions, the mGluR1 antagonists LY367385 and (RS)-1-Aminoindan-1,5-dicarboxylic acid, but not the mGluR5 antagonist 2-Methyl-6-(phenylethynyl)pyridine hydrochloride reduced the light-evoked responses in RBCs indicating that mGluR1, but not mGluR5, serves to potentiate RBC responses. Perturbing the downstream phospholipase C (PLC)-protein kinase C (PKC) pathway by inhibiting PLC, tightly buffering intracellular Ca2+ , or preventing its release from intracellular stores reduced the synaptic potentiation by mGluR1. The effect of mGluR1 activation was dependent upon adaptation state, strongly increasing the synaptic gain in dark-, but not in light-adapted retinas, or in the presence of a moderate background light, consistent with the idea that mGluR1 activation requires light-dependent glutamate release from rods. Moreover, immunostaining revealed that protein kinase Cα (PKCα) is more strongly expressed in RBC dendrites in dark-adapted conditions, revealing an additional mechanism behind the loss of mGluR1 potentiation. In light-adapted conditions, exogenous activation of mGluR1 with the agonist 3,5-Dihydroxyphenylglycine increased the mGluR6 currents in some RBCs and decreased it in others, suggesting an additional action of mGluR1 that is unmasked in the light-adapted state. Elevating intracellular free Ca2+ , consistently resulted in a decrease in synaptic gain. Our results provide evidence that mGluR1 controls the synaptic gain in RBCs.

Functional and Morphological Analysis of OFF Bipolar Cells.

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.

Differential signalling and glutamate receptor compositions in the OFF bipolar cell types in the mouse retina.

KEY POINTS: Using whole-cell clamp methods, we characterized the temporal coding in each type of OFF bipolar cell. We found that type 2 and 3a cells are transient, type 1 and 4 cells are sustained, and type 3b cells are intermediate. The light-evoked excitatory postsynaptic potentials in some types were rectified, suggesting that they provide inputs to the non-linear ganglion cells. Visual signalling from the photoreceptors was mediated exclusively through the kainate receptors in the transient OFF bipolar cells, whereas both kainate and AMPA receptors contributed in the other cells. This study demonstrates, for the first time, that parallel visual encoding starts at the OFF bipolar cells in a type-specific manner. ABSTRACT: The retina is the entrance to the visual system, which receives various kinds of image signals and forms multiple encoding pathways. The second-order retinal neurons, the bipolar cells, are thought to initiate multiple neural streams by encoding various visual signals in different types of cells. However, the functions of each bipolar cell type have not been fully understood. We investigated whether OFF bipolar cells encode visual signals in a type-dependent manner. We recorded the changes in the bipolar cell voltage in response to two input functions: step and sinusoidal light stimuli. Type 1 and 4 OFF bipolar cells were sustained cells and responded to sinusoidal stimuli over a broad range of frequencies. Type 2 and 3a cells were transient and exhibited band-pass filtering. Type 3b cells were in the middle of these two groups. The distinct temporal responses might be attributed to different types of glutamate receptors. We examined the AMPA and kainate glutamate receptor composition in each bipolar cell type. The light responses in the transient OFF bipolar cells were exclusively mediated by kainate receptors. Although the kainate receptors mediated the light responses in the sustained cells, the AMPA receptors also mediated a portion of the responses in sustained cells. Furthermore, we found that some types of cells were rectified more than other types. Taken together, we found that the OFF bipolar cells encode diverse temporal image signals in a type-dependent manner, confirming that each type of OFF bipolar cell initiates diverse temporal visual processing in parallel.

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. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

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