Light-Induced Fos Expression in Intrinsically Photosensitive Retinal Ganglion Cells in Melanopsin Knockout (Opn4−/−) Mice

Retinal ganglion cells that express the photopigment melanopsin are intrinsically photosensitive (ipRGCs) and exhibit robust synaptically driven ON-responses to light, yet they will continue to depolarize in response to light when all synaptic input from rod and cone photoreceptors is removed. The light-evoked increase in firing of classical ganglion cells is determined by synaptic input from ON-bipolar cells in the proximal sublamina of the inner plexiform layer. OFF-bipolar cells synapse with ganglion cell dendrites in the distal sublamina of the inner plexiform layer. Of the several types of ipRGC that have been described, M1 ipRGCs send dendrites exclusively into the OFF region of the inner plexiform layer where they stratify near the border of the inner nuclear layer. We tested whether M1 ipRGCs with dendrites restricted to the OFF sublamina of the inner plexiform layer receive synaptic ON-bipolar input by examining light-induced gene expression in vivo using melanopsin knockout mice. Mice in which both copies of the melanopsin gene (opn4) have been replaced with the tau-lacZ gene (homozygous tau-lacZ+/+ knockin mice) are melanopsin knockouts (opn4−/−) but M1 ipRGCs are specifically identified by their expression of β-galactosidase. Approximately 60% of M1 ipRGCs in Opn4−/− mice exposed to 3 hrs of light expressed c-Fos; no β-galactosidase-positive RGCs expressed c-Fos in the dark. Intraocular application of L-AP4, a compound which blocks transmission of visual signals between photoreceptors and ON-bipolar cells significantly reduced light-evoked c-Fos expression in M1 ipRGCs compared to saline injected eyes (66% saline vs 27% L-AP4). The results are the first description of a light-evoked response in an ipRGC lacking melanopsin and provide in vivo confirmation of previous in vitro observations illustrating an unusual circuit in the retina in which ganglion cells sending dendrites to the OFF sublamina of the inner plexiform layer receive excitatory synaptic input from ON-bipolar cells

A Herpesvirus Encoded Deubiquitinase Is a Novel Neuroinvasive Determinant

The neuroinvasive property of several alpha-herpesviruses underlies an uncommon infectious process that includes the establishment of life-long latent infections in sensory neurons of the peripheral nervous system. Several herpesvirus proteins are required for replication and dissemination within the nervous system, indicating that exploiting the nervous system as a niche for productive infection requires a specialized set of functions encoded by the virus. Whether initial entry into the nervous system from peripheral tissues also requires specialized viral functions is not known. Here we show that a conserved deubiquitinase domain embedded within a pseudorabies virus structural protein, pUL36, is essential for initial neural invasion, but is subsequently dispensable for transmission within and between neurons of the mammalian nervous system. These findings indicate that the deubiquitinase contributes to neurovirulence by participating in a previously unrecognized initial step in neuroinvasion

Intraocular application of L-AP4 inhibits light-induced Fos expression in M1 ipRGCs lacking melanopsin.

<p>The number of Fos-positive β-galactosidase cells is presented as a percentage of the number of β-galactosidase cells observed for each eye of each animal (#1–#3) after intravitreal injection of 1 µl saline (sal) or 1 µl L-AP4 (1 mM) followed by 3 h of light stimulation or 3 h of darkness.</p

Light-induced c-Fos expression in an ipRGC lacking melanopsin.

<p>(upper panel) An M1 ipRGC in the retina of a <i>tau-lacZ^+/+</i> mouse identified by β-galactosidase immunostaining. Note the dendrite emanating from this ipRGC extends to the outer aspect of the IPL, bifurcates and stratifies along the border of the INL, contributing to the definition of this ganglion cell as an M1 ipRGC. (middle panel) Same section as in upper panel illustrating light-induced Fos expression in the nuclei of neurons in the ganglion cell layer and INL. (bottom panel) Merged image illustrating light-induced Fos expression in an M1 ipRGC lacking melanopsin.</p

Retrograde transmission following CNS injection.

<p>Stereotactic injection route resulting in exposure of viral inoculum to SCN neurons. RGC ipsilateral projection to the SCN indicates route of viral transmission to the eye. (A) Representative image of virus fluorescence in the SCN of a coronal brain slice (region imaged is indicated by the doted box in right panel). 3 V, third ventricle. Scale bar = 40 µm. (B) Virus detected in the eye following retrograde transmission from the SCN is seen as punctate fluorescence in the RGCs of the ganglion cell layer (GCL) and in bipolar/amacrine cells of the inner nuclear layer (INL) of the retina. The bright fluorescent band near the top of the image is autofluorescence emitted from the retinal pigmented epithelium (RPE) at the back of the retina, and is not of viral origin. Scale bar = 10 µm.</p

Propagation of PRV with a mutated amino terminus in cultured cells.

<p>(A) Illustration of the PRV-Becker genome with region encoding the UL36 gene (which encodes the pUL36 protein) and neighboring UL37 gene expanded. The position of the codon change resulting in the C26A point mutation is indicated. Promoters are represented as black triangles. IR, internal repeat. TR, terminal repeat. (B) Purified extracellular virions encoding mRFP1-capsids and either a wild-type UL36 allele (WT; PRV-GS847) or UL36 allele encoding the amino-terminal point mutation (C26A; PRV-GS1652) were examined by Western blot analysis for the incorporation of the UL37 protein. The major capsid protein, VP5, was used as a loading control. (C) Comparison of plaque diameters resulting from infection of Vero cells with the virus encoding wild-type UL36 (WT) or mutated UL36 (C26A) allele. Each virus also encodes the mRFP1-VP26 fusion (red capsids). Error bars are standard error of the means (SEM). (D) Propagation kinetics of viruses encoding wild-type UL36 (WT), mutated UL36 (C26A), or a wild-type revertant of the C26A allele (Rev). All viruses also encode the mRFP1-VP26 fusion (red capsids). Infectious virions were detected as plaque-forming units harvested from either the cells (cells) or tissue culture supernatant (sups).</p

Retrograde transmission defect following anterior chamber injection.

<p>Anterior chamber injection route resulting in exposure of viral inoculum to the iris. The route of viral encephalitic spread is indicated: autonomic oculomotor nerve innervation of the iris from the ciliary ganglion (CG), which in turn receives innervation from parasympathetic neurons of the Edinger-Westphal nucleus (EW) of the midbrain. (A) Representative coronal images of EW (shown as a dashed box in coronal illustration) following anterior chamber injection or either wild-type or C26A virus. (B) Co-infection with PRV-152 and the C26A virus. Diffused GFP fluorescence and punctate RFP capsid signals are emitted from PRV-152 and the C26A viruses, respectively. Scale bars = 10 µm.</p

Viral infection in peripheral tissue of the eye.

<p>(A) Images of the ciliary body following co-infection with PRV-152 (diffuse GFP signal) and the C26A virus (punctate RFP signal) in the anterior chamber. Cells infected with both viruses are evident in the merged image. (B) Illustration of the peripheral tissues in the eye (iris and ciliary body) exposed to viral inoculum and imaged in these studies. (C) C26A virus fluorescence from nuclei of cells in the iris following anterior chamber injection. Scale bars = 10 µm.</p

Summary of PRV neuroinvasion by examination of trans-synaptically labeled neurons.

a<p>site imaged.</p>b<p>site injected.</p>c<p>one animal showed fluorescence signal in the oculomotor nucleus.</p>d<p>1∶1 mixture of PRV-152 (Bartha; green fluorescence) and PRV-GS1652 (C26A; red fluorescence).</p>e<p>all 3 animals emitted red and green fluorescence in the EW indicative of PRV-152 and PRV-GS1652 co-infection.</p><p>SC superior colliculus.</p><p>EW Edinger-Westphal nucleus.</p><p>RGC retinal ganglion cells.</p><p>CNS central nervous system.</p><p>WT virus encoding wild-type pUL36.</p><p>C26A virus encoding C26A mutant isoform of pUL36.</p><p>Rev revertant of C26A virus (encodes wild-type pUL36).</p><p>n/d no data.</p

Viral transport dynamics in axons of cultured primary neurons.

<p>(A) Representative example of retrograde transport of an individual capsid in a dorsal root sensory axon after infection with the C26A virus, shown as a time-lapse montage. All frames are 1.68×10.8 µm. (B) Retrograde transport efficiency measured as frame-by-frame velocities of individual capsid particles in axons (as documented in panel A). (C) Anterograde transport efficiency measured by accumulation of newly replicated capsids in axons.</p