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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

Electroretinogram and OP component modelling.(A) Schematic representation of a trace ERG elicited by a control dark-adapted mouse retina in response to a light stimulus of 25 cd˖s/m2 showing major characteristic components of the scotopic ERG. The ERG has 3 components, the a-wave, b-wave, and the oscillatory potentials (OPs). The OPs are the ascending waves between the a- and b-wave. Signal conditioning of OPs from the ERG trace waveform. (B) A digital subtraction of photoreceptor contribution (dashed line) was performed by a mathematical fitting and subtraction of the a-wave from the initial ERG (solid line). (C) The ERG waveform following complete a-wave digital subtraction. (D) The final waveform was passed through a fifth order Butterworth transformation (65–300 Hz) to remove any low or high frequency noise resulting in the final “OP extracted waveform” (solid line). Following waveform extraction, measurements of OP implicit time (ms) and amplitude (μV) were taken for OP2-5. Amplitude of each OP was defined as the difference between the trough and the peak immediately preceding it. Initial implicit time was defined as the time from stimulus to the onset of the OP2 peak. Interpeak distance was the measurement of time (ms) between adjacent peaks (E) Measurement of individual OP features in the frequency-domain. A fast Fourier transform (FFT) was applied to the extracted OP waveform and gave a single-sided smoothed frequency power spectrum (dotted line). The single-sided smoothed frequency power spectrum was fitted to a two-term Gaussian envelope (solid line) and measurements of peak power (Ppeak), peak frequency (Fpeak) and total energy (waveform integration) were taken.
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pone.0133989.g001: Electroretinogram and OP component modelling.(A) Schematic representation of a trace ERG elicited by a control dark-adapted mouse retina in response to a light stimulus of 25 cd˖s/m2 showing major characteristic components of the scotopic ERG. The ERG has 3 components, the a-wave, b-wave, and the oscillatory potentials (OPs). The OPs are the ascending waves between the a- and b-wave. Signal conditioning of OPs from the ERG trace waveform. (B) A digital subtraction of photoreceptor contribution (dashed line) was performed by a mathematical fitting and subtraction of the a-wave from the initial ERG (solid line). (C) The ERG waveform following complete a-wave digital subtraction. (D) The final waveform was passed through a fifth order Butterworth transformation (65–300 Hz) to remove any low or high frequency noise resulting in the final “OP extracted waveform” (solid line). Following waveform extraction, measurements of OP implicit time (ms) and amplitude (μV) were taken for OP2-5. Amplitude of each OP was defined as the difference between the trough and the peak immediately preceding it. Initial implicit time was defined as the time from stimulus to the onset of the OP2 peak. Interpeak distance was the measurement of time (ms) between adjacent peaks (E) Measurement of individual OP features in the frequency-domain. A fast Fourier transform (FFT) was applied to the extracted OP waveform and gave a single-sided smoothed frequency power spectrum (dotted line). The single-sided smoothed frequency power spectrum was fitted to a two-term Gaussian envelope (solid line) and measurements of peak power (Ppeak), peak frequency (Fpeak) and total energy (waveform integration) were taken.

Mentions: The amplitude of the ERG a-wave was measured from the potential at stimulus onset to the trough of the descending arm and the implicit time was measured as the time from stimulus onset to the inflecting trough (Fig 1A). The ERG a-wave amplitude and implicit time were examined at the three highest luminances only. The amplitude of the b-wave was measured from the trough of the descending arm to the peak of the ascending arm. The implicit time of the b-wave was measured from the stimulus onset to the highest peak of the ascending limb of the ERG. Therefore, implicit times of a- and b- waves were measured from the onset of the light stimulus to the respective a-wave and b-wave negative and positive peaks. To compare the magnitude of a- and b-wave reduction in VAChTSix3-Cre-flox/flox mice, a- and b-wave amplitudes for each VAChTSix3-Cre-flox/flox mouse were divided by their respective age-matched littermate control mean amplitude to produce a ratio representing the proportion of the control amplitude. The a-wave proportion was then plotted against the b-wave proportion for each VAChTSix3-Cre-flox/flox mouse for each of the three highest luminances on a summary plot.


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)

Electroretinogram and OP component modelling.(A) Schematic representation of a trace ERG elicited by a control dark-adapted mouse retina in response to a light stimulus of 25 cd˖s/m2 showing major characteristic components of the scotopic ERG. The ERG has 3 components, the a-wave, b-wave, and the oscillatory potentials (OPs). The OPs are the ascending waves between the a- and b-wave. Signal conditioning of OPs from the ERG trace waveform. (B) A digital subtraction of photoreceptor contribution (dashed line) was performed by a mathematical fitting and subtraction of the a-wave from the initial ERG (solid line). (C) The ERG waveform following complete a-wave digital subtraction. (D) The final waveform was passed through a fifth order Butterworth transformation (65–300 Hz) to remove any low or high frequency noise resulting in the final “OP extracted waveform” (solid line). Following waveform extraction, measurements of OP implicit time (ms) and amplitude (μV) were taken for OP2-5. Amplitude of each OP was defined as the difference between the trough and the peak immediately preceding it. Initial implicit time was defined as the time from stimulus to the onset of the OP2 peak. Interpeak distance was the measurement of time (ms) between adjacent peaks (E) Measurement of individual OP features in the frequency-domain. A fast Fourier transform (FFT) was applied to the extracted OP waveform and gave a single-sided smoothed frequency power spectrum (dotted line). The single-sided smoothed frequency power spectrum was fitted to a two-term Gaussian envelope (solid line) and measurements of peak power (Ppeak), peak frequency (Fpeak) and total energy (waveform integration) were taken.
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getmorefigures.php?uid=PMC4520552&req=5

pone.0133989.g001: Electroretinogram and OP component modelling.(A) Schematic representation of a trace ERG elicited by a control dark-adapted mouse retina in response to a light stimulus of 25 cd˖s/m2 showing major characteristic components of the scotopic ERG. The ERG has 3 components, the a-wave, b-wave, and the oscillatory potentials (OPs). The OPs are the ascending waves between the a- and b-wave. Signal conditioning of OPs from the ERG trace waveform. (B) A digital subtraction of photoreceptor contribution (dashed line) was performed by a mathematical fitting and subtraction of the a-wave from the initial ERG (solid line). (C) The ERG waveform following complete a-wave digital subtraction. (D) The final waveform was passed through a fifth order Butterworth transformation (65–300 Hz) to remove any low or high frequency noise resulting in the final “OP extracted waveform” (solid line). Following waveform extraction, measurements of OP implicit time (ms) and amplitude (μV) were taken for OP2-5. Amplitude of each OP was defined as the difference between the trough and the peak immediately preceding it. Initial implicit time was defined as the time from stimulus to the onset of the OP2 peak. Interpeak distance was the measurement of time (ms) between adjacent peaks (E) Measurement of individual OP features in the frequency-domain. A fast Fourier transform (FFT) was applied to the extracted OP waveform and gave a single-sided smoothed frequency power spectrum (dotted line). The single-sided smoothed frequency power spectrum was fitted to a two-term Gaussian envelope (solid line) and measurements of peak power (Ppeak), peak frequency (Fpeak) and total energy (waveform integration) were taken.
Mentions: The amplitude of the ERG a-wave was measured from the potential at stimulus onset to the trough of the descending arm and the implicit time was measured as the time from stimulus onset to the inflecting trough (Fig 1A). The ERG a-wave amplitude and implicit time were examined at the three highest luminances only. The amplitude of the b-wave was measured from the trough of the descending arm to the peak of the ascending arm. The implicit time of the b-wave was measured from the stimulus onset to the highest peak of the ascending limb of the ERG. Therefore, implicit times of a- and b- waves were measured from the onset of the light stimulus to the respective a-wave and b-wave negative and positive peaks. To compare the magnitude of a- and b-wave reduction in VAChTSix3-Cre-flox/flox mice, a- and b-wave amplitudes for each VAChTSix3-Cre-flox/flox mouse were divided by their respective age-matched littermate control mean amplitude to produce a ratio representing the proportion of the control amplitude. The a-wave proportion was then plotted against the b-wave proportion for each VAChTSix3-Cre-flox/flox mouse for each of the three highest luminances on a summary plot.

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