The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

Abstract

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Fig. 1

(a) Diagram of Cacna1f, indicating the location of the intragenic insertion into exon 2. Exon 2 nucleotides are shown as capitals. The 6-bp duplication at the insertion site is underlined. (b) Immunofluorescent staining for α_1F is localized to the OPL and the IPL of the WT retina but is absent in the nob2 retina. (c) Comparison of retinal anatomy in adult WT and nob2 mice. In the nob2 retina, the OPL is disorganized but other retinal layers appear normal. GCL, ganglion cell layer; IPL, inner plexiform layer INL, inner nuclear layer; OPL, outer plexiform layer; and ONL, outer nuclear layer. Scale bar indicates 20 μm (b) or 40 μm (c).

Fig. 2

Dark-adapted electroretinograms. (a) Comparison of dark-adapted ERGs recorded from nob2 mice (red tracings) with those obtained from WT (black tracings) or nob (blue tracings) mice. Numbers indicate flash intensity in log cd s/m². (b) Amplitude of the dark-adapted a-wave (diamonds) or b-wave (circles) for nob2 (red), nob (blue), and WT (black) mice. Data points indicate the mean ± S.E.M. response for four mice tested at 6 weeks of age. (c) b-wave implicit times of nob2 (red circles) and WT (black circles) mice. Data points in (b & c) indicate the mean ± S.E.M. response for four nob2 or WT mice, tested at 6 weeks of age. (d) Relative amplitude of a-wave parameter Rm_P3 (open circles) or b-wave amplitude obtained to a 0.3 log cd s/m² stimulus (filled circles) plotted as a function of age for nob2 mice relative to the WT mean. Data points in (d) indicate the mean ± S.E.M. Response for 5–7 nob2 mice, which are expressed relative to the mean value obtained from 9–10 WT mice.

Fig. 3

Light-adapted electroretinograms. (a) Comparison of ERGs recorded from nob2 mice (red tracings) with those obtained from WT (black tracings) or nob (blue tracings) mice. Numbers indicate flash intensity in log cd s/m². Amplitude (b) and implicit time(c) of the cone ERG b-wave for nob2 (red circles), nob (blue circles) and WT (black circles) mice. Data points in (b &c) indicate the mean ± S.E.M. response for four mice tested at 6 weeks of age.

Fig. 4

Bipolar and horizontal cells extend dendritic processes into the ONL in the nob2 but not WT retina. Retinal sections from WT (a, c, & e) or nob2 (b, d, & f) retinas immunolabeled with antibodies against the rod bipolar cell marker, PKCα (a & b), the horizontal cell marker, calbindin (c & d), or the HBC marker NK3R (e &f). INL, inner nuclear layer; OPL, outer plexiform layer; and ONL, outer nuclear layer. DIC images were obtained from a WT retina. Scale bar indicates 20 μm. Mice were studied at 5 months of age.

Fig. 5

Synaptic abnormalities in nob2 retina. (a–f) micrographs of vertical sections through WT (a, c, & e) or nob2 (b, d, & f) retinas immunolabeled with PNA (red)/Ribeye (green; [a & b]), mGluR6 (red)/Ribeye (green; [c & d] or mGluR6 (red)/PNA (green; [e & f]. Scale bar indicates 10 μm (a, b) or 5 μm (c–f). Mice were studied at 5 months of age.

Fig. 6

Ganglion cell responses in nob2 mice. Representative raw traces from an ON-center RGC in a nob2 mouse are shown in (a) and were obtained by recording from the optic nerve in vivo. The cell’s response to the 30 cd/m2 adapting background is shown at the top and the responses to increasing intensity spot stimuli from 90 cd/m² to 1860 cd/m² are shown from top to bottom. A profile of the stimulus is shown at the bottom of the column. Intensity–response functions are shown in (b) for ON- and (c) for OFF-center RGCs for nob2 and WT animals. Responses are plotted as the mean spike rate (spikes/s) determined for the first 0.5 s of the stimulus, expressed as a percentage of the maximum response obtained for that cell. Responses are plotted against the log (normalized intensity) for ON-center cells and −log (normalized intensity) for OFF-center cells), as defined in the Methods. Data points indicate mean ± S.E.M. for 16 WT ON-center cells, 13 nob2 ON-center cells, 11 WT OFF-center cells, or 11 nob2 OFF-center cells. Curves represent the Hill equation fitted to the averaged data set, to derive the semisaturation constant and slope. Mice were studied at 5 months of age.