All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation.

Neuro-prosthetic devices aim to restore impaired function through artificial stimulation of the nervous system. A lingering technological bottleneck in this field is the realization of soft, micron sized electrodes capable of injecting enough charge to evoke localized neuronal activity without causing neither electrode nor tissue damage. Direct stimulation with micro electrodes will offer the high efficacy needed in applications such as cochlear and retinal implants. Here we present a new flexible neuronal micro electrode device, based entirely on carbon nanotube technology, where both the conducting traces and the stimulating electrodes consist of conducting carbon nanotube films embedded in a polymeric support. The use of carbon nanotubes bestows the electrodes flexibility and excellent electrochemical properties. As opposed to contemporary flexible neuronal electrodes, the technology presented here is both robust and the resulting stimulating electrodes are nearly purely capacitive. Recording and stimulation tests with chick retinas were used to validate the advantageous properties of the electrodes and demonstrate their suitability for high-efficacy neuronal stimulation applications.

Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration.

Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.

A role for vascular deficiency in retinal pathology in a mouse model of ataxia-telangiectasia.

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm(-/-)). At 2 months of age, angiography revealed faint retinal vasculature in Atm(-/-) animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm(-/-) retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm(-/-) retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm(-/-) retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm(-/-) mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte-endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration.

Sema-3A indirectly disrupts the regeneration process of goldfish optic nerve after controlled injury.

BACKGROUND: Neurons of adult mammalian CNS are prevented from regenerating injured axons due to formation of a non-permissive environment. The retinal ganglion cells (RGC), which are part of the CNS, share this characteristic. In sharp contrast, the RGC of lower vertebrates, such as fish, are capable of re-growing injured optic nerve axons, and achieve, through a complex multi-factorial process, functional vision after injury. Semaphorin-3A (sema-3A), a member of the class 3 semaphorins known for its repellent and apoptotic activities, has previously been shown to play a key role in the formation of a non-permissive environment after CNS injury in mammalians. METHODS: The expression of sema-3A and its effect on regenerative processes in injured gold fish retina and optic nerve were investigated in this study. Unilateral optic nerve axotomy or crush was induced in goldfish. 2 microl sema-3A was injected intraviterally 48 hours post injury. Neuronal viability was measured using the lipophilic neurotracer dye 4-Di-10-Asp. Axonal regeneration was initiated using the anterograde dye dextran. Retinas and optic nerves were collected at intervals of 2, 3, 7, 14 and 28 days after the procedure. Using Western blot and immunohistochemical analysis, the expression levels of semaphorin-3A, axonal regeneration, the removal of myelin debris and macrophage invasion were studied. RESULTS: We found a decrease in sema-3A levels in the retina at an early stage after optic nerve injury, but no change in sema-3A levels in the injured optic nerve. Intravitreal injection of sema-3A to goldfish eye, shortly after optic nerve injury, led to destructive effects on several pathways of the regenerative processes, including the survival of retinal ganglion cells, axonal growth, and clearance of myelin debris from the lesion site by macrophages. CONCLUSIONS: Exogenous administration of sema-3A in fish indirectly interferes with the regeneration process of the optic nerve. The findings corroborate our previous findings in mammals, and further validate sema-3A as a key factor in the generation of a non-permissive environment after transection of the optic nerve.

Photopic ERG negative response from amacrine cell signaling in RCS rat retinal degeneration.

PURPOSE: The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. METHODS: Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. RESULTS: As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. CONCLUSIONS: The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat.

Electrical stimulation of different retinal components and the effect of asymmetric pulses.

BACKGROUND: High resolution electrical stimulation of neural tissue is a fundamental challenge in applications such as deep brain stimulation and artificial vision. In artificial vision, achieving and validating local selective epi-retinal stimulation of different layers in the retina is particularly challenging owing to plurality of retinal cell types and delocalized wiring. RESULTS: Strong selectivity and non-localized responses to epi-retinal stimulation, over a wide range of realistic stimulation parameters, was achieved and validated using asymmetric pulses. NEW METHOD: The reported method consists of multi electrode array (MEA) stimulation and recording from a developing chick retina combined with calcium imaging. Data show direct and indirect neuronal activation in the chick retina model. In particular, axonal activation, orientation and conduction velocity are derived, and the non-local nature of the responses to direct axonal stimulation is demonstrated. COMPARISON WITH EXISTING METHODS: Some of the previous research with mammalian retinas demonstrated local responses around the stimulating electrode, revealing little as to axonal activation. Recent studies showed activation along the nerve fibers and studied the effect of pulse duration to improve stimulation localization (Twyford and Fried, 2016; Weitz et al., 2015). The chick retina offers a straight forward mapping of axonal activation. Here we demonstrate that the chick retina, combined with MEA recording and stimulation along with calcium imaging is a powerful tool to study retinal activation and in particular the effect of asymmetry on axonal activation. CONCLUSIONS: MEA recording and stimulation from the chick retina is exceptionally powerful in distinguishing between direct and indirect responses. This method facilitates comparison between different stimulation strategies. We show that asymmetric electrical stimulations allow control over the intensity of direct activation.

Haploinsufficiency is not the key mechanism of pathogenesis in a heterozygous Elovl4 knockout mouse model of STGD3 disease.

PURPOSE: Autosomal dominant Stargardt-like (STGD3) disease results from mutations in the ELOVL4 gene (elongation of very-long-chain fatty acids). This study was undertaken to characterize a mouse model with a targeted deletion of Elovl4 and to explore the role of this gene in retinal/macular degeneration. METHODS: A construct targeted to exon 2 of the Elovl4 gene was used to suppress expression of the gene. Elovl4 homozygous pups were nonviable and were not available for study. Hence, the analysis was performed on heterozygous Elovl4(+/-) mice 16 to 22 month of age and littermate wild-type (WT) control mice of the same age. Characterization included examining gene message and protein levels, electroretinogram (ERG), retinal morphology and ultrastructure, and plasma and retinal fatty acid composition. RESULTS: Although the level of Elovl4 mRNA was reduced in Elovl4(+/-) retinas, only minimal morphologic abnormalities were found, and the retinal (ERG) function was essentially normal in Elovl4(+/-) retinas compared with the WT control retinas. Systemic fatty acid profiles of Elovl4(+/-) mice were unremarkable, although the concentration of several fatty acids was significantly lower in Elovl4(+/-) retinas, particularly the monounsaturated fatty acids. CONCLUSIONS: The detailed characterization of this animal model provides the first in vivo evidence that Elovl4 haploinsufficiency is not the underlying key disease mechanism in STGD3. The results are consistent with a dominant negative mechanism for the deletion mutation. The Elovl4 knockout mouse is one of three complementary animal models that will help elucidate the disease mechanism.

Elovl4 5-bp-deletion knock-in mice develop progressive photoreceptor degeneration.

PURPOSE: To develop and characterize a heterozygous knock-in mouse model carrying the 5-bp deletion in Elovl4 (E_mut+/-) and to study the pathology underlying Stargardt-like macular degeneration (STGD3). METHODS: E_mut+/- mice were generated by targeting a 5-bp deletion (AACTT) in the Elovl4 gene by homologous recombination. E_mut+/- mice of age 2 to 18 months and age-matched wild-type (Wt) littermate control animals were analyzed for the expression of Elovl4 transcript, ELOVL4 protein, photoreceptor-specific genes, and retinal fatty acid composition. Functional retinal changes were evaluated by electroretinography (ERG) and by morphologic and ultrastructural criteria. RESULTS: E_mut+/- mice retinas showed the presence of both Wt and mutant Elovl4 transcripts and proteins. Morphologic evaluation revealed cone photoreceptor ultrastructural abnormalities as early as 2 months of age, accumulation of lipofuscin in retinal pigment epithelium (RPE), and subretinal deposits at later ages. Shortening of rod outer segments (OS) was observed at approximately 10 months of age. Both cone and rod changes progressed with age. Unlike rod-specific genes, expression of selected cone specific genes was significantly reduced by 7 months of age. Mixed rod-cone and light-adapted b-waves were higher than normal at both 8 and 15 months. Levels of the fatty acids 20:5 (P = 0.027), 22:5 (P = 0.040) and 24:6 (P = 0.005) were found to be significantly lower in the retinas of E_mut+/- mice than in retinas of control subjects. CONCLUSIONS: E_mut+/- animals display characteristic features associated with Stargardt-like macular degeneration and serve as a model for the study of the mechanism underlying STGD3.

Photoreceptor protection by adeno-associated virus-mediated LEDGF expression in the RCS rat model of retinal degeneration: probing the mechanism.

PURPOSE: Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored. METHODS: Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control. Retinal function was evaluated 8 weeks later by the electroretinogram and compared with photoreceptor cell layer count. LEDGF mRNA and protein levels and mRNA levels of known stress-related factors were compared in treated and control retinas to explore the mechanism of LEDGF protection. Nine RCS rats were treated with adenovirus-heat shock protein 27 (Ad-HSP27) and examined for protection. RESULTS: Significant photoreceptor protection was evident functionally and morphologically in 65% to 100% of the RCS rats treated at early ages of up to 7 weeks. Cell protection was more prominent in the superior retinal hemisphere which has a slower natural degeneration rate in untreated eyes. Although many of the heat shock proteins and other stress-related genes showed significant elevation in the AAV-Ledgf-treated eyes, all increases were approximately twofold or less. Transduction of retinal cells with Ad-HSP27 also resulted in photoreceptor protection. AAV-Ledgf elicited no photoreceptor functional protection in P23H rhodopsin transgenic rat retina. CONCLUSIONS: Chronic LEDGF treatment via AAV-Ledgf administration gave successful protection of photoreceptors in the RCS rat retina and retarded cell death by about 2 weeks. Induction of heat shock proteins also gave photoreceptor protection. However, compelling evidence was not found that LEDGF protection was associated with upregulation of heat shock proteins.

Conditional inactivation of the NBS1 gene in the mouse central nervous system leads to neurodegeneration and disorganization of the visual system.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of the human NBS1, Nbs1, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. Here we report that conditional targeted disruption of the murine NBS1 gene in the CNS results in mal-development, degeneration, disorganization and dysfunction of the murine visual system, especially in the optic nerve. Nbs1 deletion resulted in reduced diameters of Nbs1-CNS-Delta eye and optic nerve. MRI analysis revealed defective white matter development and organization. Nbs1 inactivation altered the morphology and organization of the glial cells. Interestingly, at the age of two-month-old the levels of the axonal guidance molecule semaphorin-3A and its receptor neuropilin-1 were up-regulated in the retina of the mutant mice, a typical injury response. Electroretinogram analysis revealed marked reduction in a- and b-waves, indicative of decreased retinal function. Our study points to a novel role for Nbs1 in the development, organization and function of the visual system.