Impairment of Pupillary Responses and Optokinetic Nystagmus in the mGluR6-deficient Mouse

doi:10.1016/S0028-3908(96)00167-0

Neuropharmacology

Volume 36, Issue 2, February 1997, Pages 135-143

https://doi.org/10.1016/S0028-3908(96)00167-0 Get rights and content

Abstract

Retinal bipolar cells receive glutamatergic transmission from photoreceptors and mediate a key process in segregating visual signals into ON-center and OFF-center pathways. The segregation of ON responses involves a G protein-coupled metabotropic glutamate receptor (mGluR). The mGluR6 subtype is expressed restrictedly at the postsynaptic site of retinal ON-bipolar cells. Ablation of mGluR6 in the ON-bipolar cells by gene targeting results in a loss of ON responses but unchanged OFF responses in visual transmission. Thus, mGluR6 is essential for inducing ON responses. The aims of this study are analyses of visual responsiveness and possible visual dysfunction in mGluR6-deficient mice. We report here that mGluR6-deficient mice have unaltered locomotor activity in a daily light-dark cycle and exhibit light-stimulated induction of Fos immunoreactivity in the suprachiasmatic nucleus. These findings indicate that mGluR6-deficient mice are capable of responding to light stimulation. The mGluR6 deficiency, however, markedly reduces the sensitivity of pupillary responses to light stimulus and severely impairs the ability to drive optokinetic nystagmus in response to visual contrasts. This study thus demonstrates that mGluR6 contributes to discrimination of visual contrasts. © 1997 Elsevier Science Ltd. All rights reserved.

Section snippets

Daily locomotor activity

Male littermates (9–11 weeks old) were housed individually with a light-dark cycle of 12 hr light/12 hr dark for more than 9 days (light on 7 a.m. with 70–140 lux at the level of the cage floor, light off 7 p.m.). The locomotor activity was monitored continuously by counting the animal movements with an activity monitoring system (sensor unit 0603, scanner 1099 and printer 0130, Panlab, Stockholm, Sweden) which measures the changes in capacitance of a resonance circuit installed beneath a cage.

Daily locomotor activity

In mammals, visual responses to light and dark stimuli serve as the primary cues responsible for generation and entrainment of circadian rhythms in daily locomotor activities (Rusak and Zucker, 1979). We first examined whether the light-dark cycle of daily locomotor activities is maintained in mGluR6-deficient mice. Adult male littermates of wild-type and mGluR6-deficient mutant mice were housed individually and adapted to a light-dark cycle of 12 hr light/12 hr dark for more than 9 days. The

DISCUSSION

In a previous study, we showed that targeted disruption of the mGluR6 gene completely abolishes ON responses to light stimulus without causing any change in OFF responses (Masu et al., 1995). This investigation is concerned with the analysis of the visual responsiveness of mGluR6-deficient mice to light stimulation and characterization of possible visual dysfunction that results from the deficit of ON responses. The analyses of both daily locomotor activity and Fos immunostaining indicate that

Acknowledgements

We thank Dr Y. Fukuda for helpful advice, Dr T. Hirano for advice and technical assistance of OKN and Drs S.R. Nash and M. Yokoi for reading the manuscript. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan and the Ministry of Health and Welfare of Japan, the Uehara Memorial Foundation and the Sankyo Foundation.

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Remarkably, animals with a functional OFF pathway free run (lacking any circadian photoentrainment) even under light/dark conditions, similar to animals maintained under constant conditions. It has been shown previously, using mGluR6 KO mice, that the OFF pathway drives the PLR, a non-image-forming visual behavior, (Iwakabel et al., 1997). However, we showed in this study that the OFF pathway cannot drive the PLR and that the PLR is dependent on the outer retinal ON pathways for normal function.
Image- and non-image-forming vision are essential for animal behavior. Here we use genetically modified mouse lines to examine retinal circuits driving image- and non-image-functions. We describe the outer retinal circuits underlying the pupillary light response (PLR) and circadian photoentrainment, two non-image-forming behaviors. Rods and cones signal light increments and decrements through the ON and OFF pathways, respectively. We find that the OFF pathway drives image-forming vision but cannot drive circadian photoentrainment or the PLR. Cone light responses drive image formation but fail to drive the PLR. At photopic levels, rods use the primary and secondary rod pathways to drive the PLR, whereas at the scotopic and mesopic levels, rods use the primary pathway to drive the PLR, and the secondary pathway is insufficient. Circuit dynamics allow rod ON pathways to drive two non-image-forming behaviors across a wide range of light intensities, whereas the OFF pathway is potentially restricted to image formation.
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Since the rod ON BC dependent primary ON (rod BC → AIIAC → cone ON BC) and OFF (rod BC → AIIAC → cone OFF BC) pathways are absent in Bhlhb4−/− mice (Bramblett et al., 2004) and rod-cone, AIIAC-AIIAC and AIIAC-cone ON BC electrical coupling is disrupted in Cx36−/− mice (Deans et al., 2002), our results suggest that rod-driven OKRs persist in the absence of the primary and secondary rod pathways. In particular the known ON BC dependence of OKRs (Iwakabe et al., 1997) implies that the tertiary rod pathway is involved in mediating these scotopic OKRs. Taken together with the high-sensitivity RGC ON responses observed in the Cx36−/− and Bhlhb4−/− mice (Fig. 2Aiii and iv), our scotopic OKR data indicate that the tertiary rod pathways play a significant role in mediating rod inputs to RGCs and scotopic vision in awake animals.
Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα^−/− mice, but indistinguishable from controls in Cx36^−/− and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα^−/− mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36^−/− mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night vision’s ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways.
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It has been shown that mGlu6 receptor mutations leading to the suppression of receptor trafficking are associated with congenital stationary night blindness in humans (Nomura et al., 1994; Hashimoto et al., 1997; Snellman & Nawy, 2002; Zeitz et al., 2007; Flor & Acher, 2012). Mice with mGlu6 receptor gene deletion demonstrate impaired pupillary responses, optokinetic nystagmus and behavioral suppression by light (Iwakabe et al., 1997; Takao et al., 2000). In pharmacological studies, l-APB, an mGlu6 receptor agonist, suppressed normal short-latency ON responses what leads to the disclosure of long-latency ON responses, followed by disrupting the accurate detection of light stimulus timing (Renteria et al., 2006).
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OKR testing showed that, in the absence of Fz4, rod and cone signals are not transmitted effectively, and this block arises from a primary defect in the vasculature (Figures 2D and 2E). Interestingly, the OKR defect in Fz4−/− and Fz4CKOAP/−;Tie2-Cre mice is more severe than the OKR defect associated with genetic silencing of the ON pathway in mGluR6−/− mice (Iwakabe et al., 1997), consistent with a model in which the OKR is driven by both the ON and OFF pathways and indicating that both pathways are compromised in Fz4−/− and Fz4CKOAP/−;Tie2-Cre mice. In contrast to the OKR, light-dependent pupil constriction is minimally impaired by the loss of Fz4 (Figure 2F), indicating that the melanopsin-expressing intrinsically photosensitive RGCs, which control pupil constriction (Hattar et al., 2003), remain active in the Fz4−/− retina.
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

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Impairment of Pupillary Responses and Optokinetic Nystagmus in the mGluR6-deficient Mouse

Abstract

Section snippets

Daily locomotor activity

Daily locomotor activity

DISCUSSION

Acknowledgements

Neurosci. Lett.

Vision Res

Cell

Trends Neurosci.

J. Biol. Chem.

Neuron

Trends Neurosci.

Neuron

Cell

Neuropharmacology

Brain Res. Bull.

Trends Neurosci.

Curr. Biol.

Physiol. Behav.

Brain Res.

Luminance and darkness detectors in the olivary and posterior pretectal nuclei and their relationship to the pupillary light reflex in the rat.I. Studies with steady luminance levels

Exp. Brain Res.