While merkd mice and RCS rats share the same Mertk mutation and an upregulation of Fgf2 in response to subretinal electrical stimulation (SES), they have different neuroprotective responses to SES. This may be due to faster degeneration of the inner retina in the merkd mice.
PURPOSE:
Iontophoresis has been used for drug delivery across the cornea for many years. We sought to test whether small charged dyes and DNA can be transferred across human sclera by an electric field.
METHODS:
Full-thickness human scleral fragments were embedded vertically in an agarose gel and positioned to completely span individual gel lanes. The scleral fragments were located approximately 1 cm downstream from the gel wells. DNA or dyes were loaded into the wells and electrophoresis was carried out at about 3.3 V/cm for approximately 2 h per run. Movement of DNA and dyes through the agarose and sclera was measured with either digital time-lapse photography or through DNA extraction and purification from the gel. SYBR green stain was used as a sensitive method to detect DNA.
RESULTS:
Digital time-lapse photography of agarose gel electrophoresis revealed that two dyes, xylene cyanol and bromphenol blue, passed through the sclera in the presence of an electric field. Xylene cyanol was driven through the sclera virtually unimpeded except for some spreading of the dye. Bromphenol blue was slowed markedly by the sclera, but it too eventually passed through the tissue. Small DNAs, including a single stranded 51-mer and a double hairpin 68-mer oligonucleotide, passed through the sclera as detected by SYBR green staining. Linear double stranded DNAs ranging from 50 bp to 12,000 bp passed through the sclera. The larger the DNA, the slower the rate of passage through the sclera, and the greater the band spreading. pEGFP-1 (a 3 kb plasmid) passed through the sclera but was accompanied by a great amount of band spreading. Following completion of the initial electrophoresis run, the plasmid DNA was extracted from the smeared bands in the agarose distal to the sclera and re-run on a second gel without sclera. The initially smeared plasmid bands resolved into 2 distinct bands after extraction and purification and matched well with control plasmid bands.
CONCLUSIONS:
Charged molecules such as xylene cyanol, bromphenol blue, and DNAs ranging from 51 bp oligonucleotides to a 3 kb plasmid can be driven across human sclera by an electric field and directly detected. Passage of plasmids was efficient, but the plasmid bands were diffuse after transit. This technique offers promise as a noninvasive DNA delivery tool, where gene therapy can be accomplished by small RNA or DNA synthetic oligonucleotides, larger double stranded fragments, or even plasmids.
Electrical deep brain stimulation (DBS) of the subthalamic nucleus (STN) is effective for ameliorating the motor symptoms of Parkinson’s disease (PD) including bradykinesia. The STN receives its main excitatory input from cortex; however, the contribution of cortico-subthalamic projection neurons to the effects of DBS remains unclear. To isolate the consequences of stimulating layer 5 primary motor cortex (M1) projections to the STN, we used a dual virus transfection technique to selectively express opsins in these neurons in mice made parkinsonian by unilateral nigrostriatal 6-OHDA lesioning. AAVs containing WGA-Cre constructs were injected in the STN to retrogradely place Cre in STN afferents, while AAVs containing Cre-dependent ultrafast hChR2(E123T/T159C)-EYFP opsin constructs were injected in M1 layer 5, producing specific opsin expression in M1-STN projections. Under unstimulated conditions, lesioned mice showed bradykinesia and hypokinesia (decreased movement), along with electrophysiological changes similar to those observed in PD patients. Specifically, low frequency power (theta, alpha, low beta) was increased and gamma power was decreased, while M1/STN coherence and STN phase-amplitude-coupling (PAC) were increased. Optogenetic stimulation (100–130 Hz) of STN afferents in these mice ameliorated bradykinesia and hypokinesia and brought the neural dynamics closer to the non-parkinsonian state by reducing theta and alpha and increasing gamma power in M1, decreasing STN PAC, and reducing theta band coherence. Histological examination of the EYFP expression revealed that, in addition to orthodromic and antidromic effects, stimulation of cortico-subthalamic neurons may cause wide-spread increased glutamatergic activity due to collaterals that project to areas of the thalamus and other brain regions.
by
Charlotte Andrieu-Soler;
Mounia Halhal;
Jeffrey Boatright;
Staci A. Padove;
John Nickerson;
Eva Stodulkova;
Rachael E. Stewart;
Vincent Ciavatta;
Marc Doat;
Jean-Claude Jeanny;
Therese de Bizemont;
Florian Sennlaub;
Yves Courtois;
Francine Behar-Cohen
Purpose: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6brd1 (rd1) mouse retinas in vivo. Methods: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the β-subunit of rod photoreceptor cGMP-phosphodiesterase (β-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/ henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of β-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. Results: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. β-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of β-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. Conclusions: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable β-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.
by
Charlotte Andrieu-Soler;
Mounia Halhal;
Jeffrey Boatright;
Staci A. Padove;
John Nickerson;
Eva Stodulkova;
Rachael E. Stewart;
Vincent Thomas Ciavatta;
Marc Doat;
Jean-Claude Jeanny;
Therese de Bizemont;
Florian Sennlaub;
Yves Courtois;
Francine Behar-Cohen
Purpose
The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6brd1 (rd1) mouse retinas in vivo.
Methods
Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the β-subunit of rod photoreceptor cGMP-phosphodiesterase (β-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of β-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site.
Results
Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. β-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of β-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33.
Conclusions
Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable β-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.
Purpose
Test the hypothesis that subretinal electrical stimulation from a microphotodiode array (MPA) exerts a neuroprotective effect in RCS rats through the induction of growth factors.
Methods
RCS rats were divided into four groups at P21 in which one eye per rat received treatment: (A) active MPA, (M) minimally-active MPA, (S) sham surgery, or (C) no surgery and the opposite eye was unoperated. Dark- and light-adapted ERGs were recorded one week after surgery. A second set of A-, M-, and C-treated RCS rats had weekly ERG recordings for 4 weeks. Real-time RT-PCR was used to measure relative expression of mRNAs (Bdnf, Fgf2, Fgf1, Cntf, Gdnf, and Igf1) in retina samples collected 2 days after the final ERG.
Results
One week after surgery, there was a slight difference in dark-adapted ERG b-wave at the brightest flash intensity. Mean retinal Fgf2 expression in the treated eye relative to the opposite eye was greatest for the A group (4.67 +/−0.72) compared to the M group (2.80 +/−0.45, p=0.0501), S group (2.03 +/−0.45, p<0.01), and C group (1.30 +/−0.22, p<0.001). No significant change was detected for Bdnf, Cntf, Fgf1, Gdnf, and Igf1. Four weeks after surgery, the A group had significantly larger dark- and light-adapted ERG b-waves compared to the M and C groups (p<0.01). Simultaneously, mean relative Fgf2 expression was again greatest for the A group (3.28 +/−0.61) compared to the M (1.28 +/− 0.32, p<0.05) and C groups (1.05 +/−0.04, p<0.05).
Conclusion
The results show subretinal implantation of an MPA induces selective expression of Fgf2 above that expected from a retina-piercing injury. Preservation of ERG b-wave amplitude 4 weeks after implantation is accompanied by elevated Fgf2 expression. These results suggest that Fgf2 may play a role in the neuroprotection provided by subretinal electrical stimulation.
by
Jeffrey Boatright;
Anisha G. Moring;
Clinton McElroy;
Michael J. Phillips;
Vi T. Do;
Bo Chang ;
Norm L. Hawes;
Amber P. Boyd;
Sheree S. Sidney;
Rachael E. Stewart;
Steven C. Minear;
Rajashree Chaudhury;
Vincent Thomas Ciavatta;
Cecilia M.P. Rodrigues;
Clifford J. Steer;
John Nickerson;
Machelle Pardue
PURPOSE:
Bear bile has been used in Asia for over 3,000 years to treat visual disorders, yet its therapeutic potential remains unexplored in Western vision research. The purpose of this study was to test whether treatment of mice undergoing retinal degeneration with tauroursodeoxycholic acid (TUDCA), a primary constituent of bear bile, alters the course of degeneration.
METHODS:
Two retinal degeneration models were tested: the rd10 mouse, which has a point mutation in the gene encoding the beta subunit of rod phosphodiesterase, and light induced retinal damage (LIRD). For LIRD studies, albino Balb/C adult mice were subcutaneously injected with TUDCA (500 mg/kg body weight) or vehicle (0.15 M NaHCO(3)). Sixteen h later, each mouse received repeat injections. Half of each treatment group was then placed in bright light (10,000 lux) or dim light (200 lux) for seven h. At the end of exposure, animals were transferred to their regular housing. Electroretinograms (ERGs) were assessed 24 h later, mice sacrificed, eyes embedded in paraffin and sectioned, and retina sections assayed for morphology and apoptosis by TUNEL and anti-active caspase-3 immunoreactivity via fluorescent confocal microscopy. A subset of mice were sacrificed 8 and 15 days after exposure and retina sections analyzed for morphology and apoptosis. For rd10 studies, mice were injected subcutaneously with TUDCA or vehicle at postnatal (P) days 6, 9, 12, and 15. At p18, ERGs were recorded, mice were euthanized and eyes were harvested, fixed, and processed. Retinal sections were stained (toluidine blue), and retinal cell layers morphometrically analyzed by light microscopy. Consecutive sections were analyzed for apopotosis as above.
RESULTS:
By every measure, TUDCA greatly slowed retinal degeneration in LIRD and rd10 mice. ERG a-wave and b-wave amplitudes were greater in mice treated with TUDCA compared to those treated with vehicle. Retinas of TUDCA-treated mice had thicker outer nuclear layers, more photoreceptor cells, and more fully-developed photoreceptor outer segments. Finally, TUDCA treatments dramatically suppressed signs of apoptosis in both models.
CONCLUSIONS:
Systemic injection of TUDCA, a primary constituent of bear bile, profoundly suppressed apoptosis and preserved function and morphology of photoreceptor cells in two disparate mouse models of retinal degeneration. It may be that bear bile has endured so long in Asian pharmacopeias due to efficacy resulting from this anti-apoptotic and neuroprotective activity of TUDCA. These results also indicate that a systematic, clinical assessment of TUDCA may be warranted.
While merkd mice and RCS rats share the same Mertk mutation and an upregulation of Fgf2 in response to subretinal electrical stimulation (SES), they have different neuroprotective responses to SES. This may be due to faster degeneration of the inner retina in the merkd mice.
Although the brain relies on auditory information to calibrate vocal behavior, the neural substrates of vocal learning remain unclear. Here we demonstrate that lesions of the dopaminergic inputs to a basal ganglia nucleus in a songbird species (Bengalese finches, Lonchura striata var. domestica) greatly reduced the magnitude of vocal learning driven by disruptive auditory feedback in a negative reinforcement task. These lesions produced no measureable effects on the quality of vocal performance or the amount of song produced. Our results suggest that dopaminergic inputs to the basal ganglia selectively mediate reinforcement-driven vocal plasticity. In contrast, dopaminergic lesions produced no measurable effects on the birds' ability to restore song acoustics to baseline following the cessation of reinforcement training, suggesting that different forms of vocal plasticity may use different neural mechanisms.
SIGNIFICANCE STATEMENT During skill learning, the brain relies on sensory feedback to improve motor performance. However, the neural basis of sensorimotor learning is poorly understood. Here, we investigate the role of the neurotransmitter dopamine in regulating vocal learning in the Bengalese finch, a songbird with an extremely precise singing behavior that can nevertheless be reshaped dramatically by auditory feedback. Our findings show that reduction of dopamine inputs to a region of the songbird basal ganglia greatly impairs vocal learning but has no detectable effect on vocal performance. These results suggest a specific role for dopamine in regulating vocal plasticity.
Purpose
The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.
Methods
We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.
Results
Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.
Conclusions
The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.