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Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter.

Bedore J, Martyn AC, Li AK, Dolinar EA, McDonald IS, Coupland SG, Prado VF, Prado MA, Hill KA - PLoS ONE (2015)

Bottom Line: One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits.Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Western Ontario, London, Ontario, Canada N6A 5B7.

ABSTRACT

Background: Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina.

Methods & results: A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5) deletion of VAChT (VAChTSix3-Cre-flox/flox) and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.

Significance: This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.

No MeSH data available.


Related in: MedlinePlus

Assessment of gene and protein expression of cholinergic markers.(A, D & G) Quantitative real-time PCR (qRT-PCR) assessed expression levels of VAChT, ChAT and CHT1 mRNA from retinal tissue of VAChTSix3-Cre-flox/flox (n = 5) and littermate control mice (VAChTflox/flox, n = 6). (B, E & H) Immunoblotting followed by (C, F & I) densitometry assessed protein levels of cholinergic proteins (A) Expression of VAChT mRNA in the retina of VAChTSix3-Cre-flox/flox mice is significantly reduced relative to control. Expression of VAChT protein ChAT increased. (B) Immunoblotting. (B, E, H) Expression levels of VAChT, ChAT and CHT1 protein in the retina of VAChTSix3-Cre-flox/flox and littermate control mice were quantified using densitometry. Expression of VAChT protein in the retina of VAChTSix3-Cre-flox/flox mice (n = 5) is reduced relative to littermate control (n = 6) (C). Synaptophysin protein was used as a loading control for VAChT, while actin protein was used for ChAT and CHT1. Values in (A) and (C) represent mean ± SEM, γ, P < 0.01; β, P < 0.001 versus control mice.
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pone.0133989.g002: Assessment of gene and protein expression of cholinergic markers.(A, D & G) Quantitative real-time PCR (qRT-PCR) assessed expression levels of VAChT, ChAT and CHT1 mRNA from retinal tissue of VAChTSix3-Cre-flox/flox (n = 5) and littermate control mice (VAChTflox/flox, n = 6). (B, E & H) Immunoblotting followed by (C, F & I) densitometry assessed protein levels of cholinergic proteins (A) Expression of VAChT mRNA in the retina of VAChTSix3-Cre-flox/flox mice is significantly reduced relative to control. Expression of VAChT protein ChAT increased. (B) Immunoblotting. (B, E, H) Expression levels of VAChT, ChAT and CHT1 protein in the retina of VAChTSix3-Cre-flox/flox and littermate control mice were quantified using densitometry. Expression of VAChT protein in the retina of VAChTSix3-Cre-flox/flox mice (n = 5) is reduced relative to littermate control (n = 6) (C). Synaptophysin protein was used as a loading control for VAChT, while actin protein was used for ChAT and CHT1. Values in (A) and (C) represent mean ± SEM, γ, P < 0.01; β, P < 0.001 versus control mice.

Mentions: Real-time PCR (qRT-PCR) and western blotting were used to confirm the deletion of VAChT from the retina of VAChTSix3-Cre-flox/flox mice. qRT-PCR showed that VAChT mRNA is reduced significantly in VAChTSix3-Cre-flox/flox retinas (P<0.01; Fig 2A). There is a significant increase in ChAT gene expression (P<0.01; Fig 2D), while expression of the high affinity choline transporter CHT1 mRNA is not changed (Fig 2G). Immunoblotting followed by densitometry demonstrated that VAChT protein is significantly reduced in the retinas of VAChTSix3-Cre-flox/flox mice (P<0.001; Fig 2B and 2C), while ChAT (Fig 2E and 2F) and CHT1 (Fig 2H and 2I) protein expression are not altered.


Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter.

Bedore J, Martyn AC, Li AK, Dolinar EA, McDonald IS, Coupland SG, Prado VF, Prado MA, Hill KA - PLoS ONE (2015)

Assessment of gene and protein expression of cholinergic markers.(A, D & G) Quantitative real-time PCR (qRT-PCR) assessed expression levels of VAChT, ChAT and CHT1 mRNA from retinal tissue of VAChTSix3-Cre-flox/flox (n = 5) and littermate control mice (VAChTflox/flox, n = 6). (B, E & H) Immunoblotting followed by (C, F & I) densitometry assessed protein levels of cholinergic proteins (A) Expression of VAChT mRNA in the retina of VAChTSix3-Cre-flox/flox mice is significantly reduced relative to control. Expression of VAChT protein ChAT increased. (B) Immunoblotting. (B, E, H) Expression levels of VAChT, ChAT and CHT1 protein in the retina of VAChTSix3-Cre-flox/flox and littermate control mice were quantified using densitometry. Expression of VAChT protein in the retina of VAChTSix3-Cre-flox/flox mice (n = 5) is reduced relative to littermate control (n = 6) (C). Synaptophysin protein was used as a loading control for VAChT, while actin protein was used for ChAT and CHT1. Values in (A) and (C) represent mean ± SEM, γ, P < 0.01; β, P < 0.001 versus control mice.
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pone.0133989.g002: Assessment of gene and protein expression of cholinergic markers.(A, D & G) Quantitative real-time PCR (qRT-PCR) assessed expression levels of VAChT, ChAT and CHT1 mRNA from retinal tissue of VAChTSix3-Cre-flox/flox (n = 5) and littermate control mice (VAChTflox/flox, n = 6). (B, E & H) Immunoblotting followed by (C, F & I) densitometry assessed protein levels of cholinergic proteins (A) Expression of VAChT mRNA in the retina of VAChTSix3-Cre-flox/flox mice is significantly reduced relative to control. Expression of VAChT protein ChAT increased. (B) Immunoblotting. (B, E, H) Expression levels of VAChT, ChAT and CHT1 protein in the retina of VAChTSix3-Cre-flox/flox and littermate control mice were quantified using densitometry. Expression of VAChT protein in the retina of VAChTSix3-Cre-flox/flox mice (n = 5) is reduced relative to littermate control (n = 6) (C). Synaptophysin protein was used as a loading control for VAChT, while actin protein was used for ChAT and CHT1. Values in (A) and (C) represent mean ± SEM, γ, P < 0.01; β, P < 0.001 versus control mice.
Mentions: Real-time PCR (qRT-PCR) and western blotting were used to confirm the deletion of VAChT from the retina of VAChTSix3-Cre-flox/flox mice. qRT-PCR showed that VAChT mRNA is reduced significantly in VAChTSix3-Cre-flox/flox retinas (P<0.01; Fig 2A). There is a significant increase in ChAT gene expression (P<0.01; Fig 2D), while expression of the high affinity choline transporter CHT1 mRNA is not changed (Fig 2G). Immunoblotting followed by densitometry demonstrated that VAChT protein is significantly reduced in the retinas of VAChTSix3-Cre-flox/flox mice (P<0.001; Fig 2B and 2C), while ChAT (Fig 2E and 2F) and CHT1 (Fig 2H and 2I) protein expression are not altered.

Bottom Line: One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits.Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Western Ontario, London, Ontario, Canada N6A 5B7.

ABSTRACT

Background: Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina.

Methods & results: A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5) deletion of VAChT (VAChTSix3-Cre-flox/flox) and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.

Significance: This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.

No MeSH data available.


Related in: MedlinePlus