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.

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).

Asymmetric Distributions of Achromatic Bipolar Cells in the Mouse Retina.

In the retina, evolutionary changes can be traced in the topography of photoreceptors. The shape of the visual streak depends on the height of the animal and its habitat, namely, woods, prairies, or mountains. Also, the distribution of distinct wavelength-sensitive cones is unique to each animal. For example, UV and green cones reside in the ventral and dorsal regions in the mouse retina, respectively, whereas in the rat retina these cones are homogeneously distributed. In contrast with the abundant investigation on the distribution of photoreceptors and the third-order neurons, the distribution of bipolar cells has not been well understood. We utilized two enhanced green fluorescent protein (EGFP) mouse lines, Lhx4-EGFP (Lhx4) and 6030405A18Rik-EGFP (Rik), to examine the topographic distributions of bipolar cells in the retina. First, we characterized their GFP-expressing cells using type-specific markers. We found that GFP was expressed by type 2, type 3a, and type 6 bipolar cells in the Rik mice and by type 3b, type 4, and type 5 bipolar cells in the Lhx4 mice. All these types are achromatic. Then, we examined the distributions of bipolar cells in the four cardinal directions and three different eccentricities of the retinal tissue. In the Rik mice, GFP-expressing bipolar cells were more highly observed in the nasal region than those in the temporal retina. The number of GFP cells was not different along with the ventral-dorsal axis. In contrast, in the Lhx4 mice, GFP-expressing cells occurred at a higher density in the ventral region than in the dorsal retina. However, no difference was observed along the nasal-temporal axis. Furthermore, we examined which type of bipolar cells contributed to the asymmetric distributions in the Rik mice. We found that type 3a bipolar cells occurred at a higher density in the temporal region, whereas type 6 bipolar cells were denser in the nasal region. The asymmetricity of these bipolar cells shaped the uneven distribution of the GFP cells in the Rik mice. In conclusion, we found that a subset of achromatic bipolar cells is asymmetrically distributed in the mouse retina, suggesting their unique roles in achromatic visual processing.