ERR2 and ERR3 promote the development of gamma motor neuron functional properties required for proprioceptive movement control

The ability of terrestrial vertebrates to effectively move on land is integrally linked to the diversification of motor neurons into types that generate muscle force (alpha motor neurons) and types that modulate muscle proprioception, a task that in mammals is chiefly mediated by gamma motor neurons. The diversification of motor neurons into alpha and gamma types and their respective contributions to movement control have been firmly established in the past 7 decades, while recent studies identified gene expression signatures linked to both motor neuron types. However, the mechanisms that promote the specification of gamma motor neurons and/or their unique properties remained unaddressed. Here, we found that upon selective loss of the orphan nuclear receptors ERR2 and ERR3 (also known as ERR beta, ERR gamma or NR3B2, NR3B3, respectively) in motor neurons in mice, morphologically distinguishable gamma motor neurons are generated but do not acquire characteristic functional properties necessary for regulating muscle proprioception, thus disrupting gait and precision movements. Complementary gain-of-function experiments in chick suggest that ERR2 and ERR3 could operate via transcriptional activation of neural activity modulators to promote a gamma motor neuron biophysical signature of low firing thresholds and high firing rates. Our work identifies a mechanism specifying gamma motor neuron functional properties essential for the regulation of proprioceptive movement control

Intravitreal Injection of Fluorochrome-Conjugated Peanut Agglutinin Results in Specific and Reversible Labeling of Mammalian Cones In Vivo

PURPOSE. To investigate whether intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo and to evaluate its clinical potential. METHODS. Fluorescein-or rhodamine-conjugated PNA (0.005%-0.5%) was intravitreally injected into anesthetized mouse, guinea pig, or monkey and retinas were removed at various intervals for fluorescence microscopy. Immunofluorescence and TUNEL assay were carried out to investigate whether PNA injection adversely affected other retinal neurons. Gross visual function was studied in a visual cliff test. The retina of an N-methyl, N-nitrosourea (MNU)-induced mouse model of retinal degeneration was stained with PNA to evaluate how spatiotemporal pattern of the staining would reflect the progression of degeneration. RESULTS. Intravitreally injected PNA resulted in specific labeling of cone outer and inner segments and cone pedicles within 30 minutes over the entire retina and in all tested species. The labeling was reversible; cones did not show any labeling 3 weeks after the injection but could be restained with PNA. TUNEL signal and expression pattern of several retinal proteins in PNA-injected mouse retina were indistinguishable from normal. Similarly, visual behavior of mouse 10 hours after the injection was normal. The pattern of PNA labeling in mice with MNU-induced retinal degeneration showed progressive disappearance of cones from the center to the periphery. CONCLUSIONS. Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo without causing any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina. (Invest Ophthalmol Vis Sci

Loss of photoreceptors results in upregulation of synaptic proteins in bipolar cells and amacrine cells.

Deafferentation is known to cause significant changes in the postsynaptic neurons in the central nervous system. Loss of photoreceptors, for instance, results in remarkable morphological and physiological changes in bipolar cells and horizontal cells. Retinal ganglion cells (RGCs), which send visual information to the brain, are relatively preserved, but show aberrant firing patterns, including spontaneous bursts of spikes in the absence of photoreceptors. To understand how loss of photoreceptors affects the circuitry presynaptic to the ganglion cells, we measured specific synaptic proteins in two mouse models of retinal degeneration. We found that despite the nearly total loss of photoreceptors, the synaptophysin protein and mRNA levels in retina were largely unaltered. Interestingly, the levels of synaptophysin in the inner plexiform layer (IPL) were higher, implying that photoreceptor loss results in increased synaptophysin in bipolar and/or amacrine cells. The levels of SV2B, a synaptic protein expressed by photoreceptors and bipolar cells, were reduced in whole retina, but increased in the IPL of rd1 mouse. Similarly, the levels of syntaxin-I and synapsin-I, synaptic proteins expressed selectively by amacrine cells, were higher after loss of photoreceptors. The upregulation of syntaxin-I was evident as early as one day after the onset of photoreceptor loss, suggesting that it did not require any massive or structural remodeling, and therefore is possibly reversible. Together, these data show that loss of photoreceptors results in increased synaptic protein levels in bipolar and amacrine cells. Combined with previous reports of increased excitatory and inhibitory synaptic currents in RGCs, these results provide clues to understand the mechanism underlying the aberrant spiking in RGCs

Retinal levels of synaptophysin protein and mRNA were largely unaltered following photoreceptor loss.

<p>A) Representative blots of synaptophysin and β-tubulin in whole retinas of wild-type and rd1 mice at different developmental stages (“A” is for “Adult”). B) Ratio of synaptophysin to β-tubulin for several animals (Mean ± SE). The ratio in rd1 mouse was not statistically different from that in wild-type mouse at any stage (n = 6, 8, 9, 9 and 7 for 7, 14, 21, 28 days old and adult animals respectively). C) Synaptophysin mRNA levels normalized to 18S rRNA (Mean ± SE) were also similar in adult rd1 and wild-type mice (n = 6). D) Representative blots of synaptophysin and β-tubulin in whole retinas of sham-injected control and at various days after MNU injection. E) Ratio of synaptophysin to β-tubulin for several animals (Mean ± SE). Similar to rd1 mouse, the levels of synaptophysin in MNU-injected animals were not significantly different from the control for up to at least 28 days after the injection, except at 7 days (p>0.1, except for PID-7 where p = 0.044; n = 6 for all stages). *p<0.05</p

Ribbon-specific protein SV2B was downregulated in retina, but upregulated in IPL of rd1 mouse.

<p>A) Representative blots of SV2B and β-tubulin in adult wild-type and rd1 mouse retinas. Each band is from a different animal. B) Ratio of SV2B to β-tubulin (mean ± SE) in adult rd1 mouse retina was significantly lower than in wild-type mouse (p = 0.01; n = 5). C) Relative mRNA levels (mean ± SE) of SV2B in adult rd1 mouse were approximately 40% lower than in wild-type, although the difference was not statistically significant (p = 0.062; n = 6). D, E) Representative images of retinal sections of adult wild-type (D) and rd1 (E) mouse retinas immunostained for SV2B. Scale bar: 50 µm. A) Intensity profile of SV2B through retinal depth of the images shown in D and E. Similar to synaptophysin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090250#pone-0090250-g002" target="_blank">Figs. 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090250#pone-0090250-g002" target="_blank">2B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090250#pone-0090250-g002" target="_blank">2C</a>), SV2B is nearly absent in OPL of rd1 mouse retina, whereas that in IPL is higher than in wild-type, particularly in the Off sublamina. B) Levels of SV2B (mean ± SE), measured with quantitative immunohistochemistry were more than twofold higher in IPL of adult rd1 mouse retina than in wild-type (p = 0.014; n = 5). The SV2B levels were higher in both On and Off sublaminas of IPL, although the increase was more pronounced in the Off sublamina (On: p = 0.031; Off: p = 0.005). *<i>p</i><0.05; **p<0.01.</p

List of antibodies used.

<p>List of antibodies used.</p