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Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice.

Stern S, Segal M, Moses E - EBioMedicine (2015)

Bottom Line: We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents.Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity.Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics of Complex Systems, Weizmann Institute of Science, P.O. Box 26, Rehovot 76100 Israel.

ABSTRACT
Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca(2+)]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype.

No MeSH data available.


Related in: MedlinePlus

Analysis of spontaneous network bursts in two-dimensional networks observed by imaging of [Ca2+]i. a, Example of time course of the fluorescence intensity () recording of a spontaneous network burst of a two-dimensional diploid vs. Ts65Dn network. b, Amplitude () of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.014. c, Mean duration of a burst (see Methods for a definition of the duration) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.0019. d, Mean normalized fluorescence () intensity of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with inhibitory activity blocked by 40 μM bicuculline, p = 0.019. e, Mean FWHM duration of a burst (see Methods) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with 40 μM bicuculline, p = 0.03. f, Example of time course for the fluorescence signal of a diploid culture. g, Example of time course for the fluorescence signal of a Ts65Dn culture. h, Same diploid culture as in f, but with 30 μM of GABA applied. i, Same Ts65Dn culture as in f, but with 30 μM of GABA applied. j, Mean baclofen concentration required to stop network activity in diploid networks (N = 5 cultures) vs. the concentration needed to stop activity in Ts65Dn networks (N = 6 cultures), p = 0.0135. k–n, Inhibition plots for baclofen. k, Inhibition plot showing the duration of time that the network was active bursting divided by the total recording time for diploid and Ts65Dn cultures as a function of the baclofen concentration. l, Inhibition plot showing the duration of time that the network was active normalized by the duration of time that the network was active at 0 baclofen for diploid and Ts65Dn cultures as a function of the baclofen concentration. m, Inhibition plot showing the duration that the network was active divided by the total recording time for diploid and Ts65Dn cultures with 80 μM saclofen in the recording medium, as a function of the baclofen concentration. Inset graph shows duration of activity divided by the total recording time as a function of the concentration of saclofen applied. n, Inhibition plot showing the duration that the network was active normalized by the duration that the network was active at 0 baclofen for diploid and Ts65Dn cultures at 80 μM saclofen, as a function of the baclofen concentration. o, Fura-2 ratio of the baseline fluorescent signal at 340 nm to 380 nm in diploid networks (N = 7 cultures) vs. that in Ts65Dn networks (N = 7 cultures), p = 0.185. For panels b–e and j–o data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
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f0010: Analysis of spontaneous network bursts in two-dimensional networks observed by imaging of [Ca2+]i. a, Example of time course of the fluorescence intensity () recording of a spontaneous network burst of a two-dimensional diploid vs. Ts65Dn network. b, Amplitude () of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.014. c, Mean duration of a burst (see Methods for a definition of the duration) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.0019. d, Mean normalized fluorescence () intensity of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with inhibitory activity blocked by 40 μM bicuculline, p = 0.019. e, Mean FWHM duration of a burst (see Methods) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with 40 μM bicuculline, p = 0.03. f, Example of time course for the fluorescence signal of a diploid culture. g, Example of time course for the fluorescence signal of a Ts65Dn culture. h, Same diploid culture as in f, but with 30 μM of GABA applied. i, Same Ts65Dn culture as in f, but with 30 μM of GABA applied. j, Mean baclofen concentration required to stop network activity in diploid networks (N = 5 cultures) vs. the concentration needed to stop activity in Ts65Dn networks (N = 6 cultures), p = 0.0135. k–n, Inhibition plots for baclofen. k, Inhibition plot showing the duration of time that the network was active bursting divided by the total recording time for diploid and Ts65Dn cultures as a function of the baclofen concentration. l, Inhibition plot showing the duration of time that the network was active normalized by the duration of time that the network was active at 0 baclofen for diploid and Ts65Dn cultures as a function of the baclofen concentration. m, Inhibition plot showing the duration that the network was active divided by the total recording time for diploid and Ts65Dn cultures with 80 μM saclofen in the recording medium, as a function of the baclofen concentration. Inset graph shows duration of activity divided by the total recording time as a function of the concentration of saclofen applied. n, Inhibition plot showing the duration that the network was active normalized by the duration that the network was active at 0 baclofen for diploid and Ts65Dn cultures at 80 μM saclofen, as a function of the baclofen concentration. o, Fura-2 ratio of the baseline fluorescent signal at 340 nm to 380 nm in diploid networks (N = 7 cultures) vs. that in Ts65Dn networks (N = 7 cultures), p = 0.185. For panels b–e and j–o data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.

Mentions: Fig. 2a shows examples of recorded network bursts of diploid and Ts65Dn networks. The amplitude of the fluorescence signal during activity was smaller in Ts65Dn cultures by about a factor of 2, going from 6.5 ± 1% (N = 12 cultures, n = 266 network bursts) in diploid cultures to 3.3 ± 0.4% in Ts65Dn cultures (N = 10 cultures, n = 253 network bursts, p = 0.014) (Fig. 2b). The average burst duration (see Methods) was also significantly lower (Fig. 2c). We measured 7.1 ± 0.8 s (N = 12 cultures, n = 266 network bursts) in diploid cultures vs. 3.5 ± 0.6 s (N = 10 cultures, n = 253 network bursts) in Ts65Dn cultures (p = 0.0019). To see the contribution of excitatory cells only, we added bicuculline to block GABAA inhibition, and measured the amplitude of the fluorescence signal (Fig. 2d) giving again a ratio of about 2, with 22 ± 5% (N = 9 cultures, n = 64 network bursts) in diploid cultures versus 9 ± 1.3% (N = 9 cultures, n = 42 network bursts) in Ts65Dn cultures (p = 0.019). Similarly, for the duration of network bursts we measured an average of 2.5 ± 0.3 s (N = 9 cultures, n = 64 network bursts) for the diploid cultures versus 1.8 ± 0.2 s for the Ts65Dn (N = 9 cultures, n = 42 network bursts, p = 0.03) (Fig. 2e). Gradual application of GABA had the effect of reducing network activity until it completely stops. The minimal concentration of GABA needed to stop activity was typically 10 μM to 50 μM depending on how active the culture was. An example is shown in Fig. 2f–i. Fig. 2f shows a recording of the fluorescence signal in a diploid culture with no GABA. Similarly, Fig. 2g shows an example recording for a Ts65Dn culture. Fig. 2h is a recording of the same culture used in Fig. 2f, but with 30 μM GABA application. Fig. 2i similarly shows the Ts65Dn network recording with 30 μM GABA for the same culture used in Fig. 2g. We conclude that network bursts in 2D cultures of the Ts65Dn mouse model were reduced in both amplitude and duration. The ratio of fluorescence amplitudes before and after blocking inhibition is somewhat larger in Ts65Dn, though not statistically significant. This increase may indicate that possible differences in the ratio of excitatory to inhibitory contributions might also play a role.


Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice.

Stern S, Segal M, Moses E - EBioMedicine (2015)

Analysis of spontaneous network bursts in two-dimensional networks observed by imaging of [Ca2+]i. a, Example of time course of the fluorescence intensity () recording of a spontaneous network burst of a two-dimensional diploid vs. Ts65Dn network. b, Amplitude () of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.014. c, Mean duration of a burst (see Methods for a definition of the duration) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.0019. d, Mean normalized fluorescence () intensity of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with inhibitory activity blocked by 40 μM bicuculline, p = 0.019. e, Mean FWHM duration of a burst (see Methods) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with 40 μM bicuculline, p = 0.03. f, Example of time course for the fluorescence signal of a diploid culture. g, Example of time course for the fluorescence signal of a Ts65Dn culture. h, Same diploid culture as in f, but with 30 μM of GABA applied. i, Same Ts65Dn culture as in f, but with 30 μM of GABA applied. j, Mean baclofen concentration required to stop network activity in diploid networks (N = 5 cultures) vs. the concentration needed to stop activity in Ts65Dn networks (N = 6 cultures), p = 0.0135. k–n, Inhibition plots for baclofen. k, Inhibition plot showing the duration of time that the network was active bursting divided by the total recording time for diploid and Ts65Dn cultures as a function of the baclofen concentration. l, Inhibition plot showing the duration of time that the network was active normalized by the duration of time that the network was active at 0 baclofen for diploid and Ts65Dn cultures as a function of the baclofen concentration. m, Inhibition plot showing the duration that the network was active divided by the total recording time for diploid and Ts65Dn cultures with 80 μM saclofen in the recording medium, as a function of the baclofen concentration. Inset graph shows duration of activity divided by the total recording time as a function of the concentration of saclofen applied. n, Inhibition plot showing the duration that the network was active normalized by the duration that the network was active at 0 baclofen for diploid and Ts65Dn cultures at 80 μM saclofen, as a function of the baclofen concentration. o, Fura-2 ratio of the baseline fluorescent signal at 340 nm to 380 nm in diploid networks (N = 7 cultures) vs. that in Ts65Dn networks (N = 7 cultures), p = 0.185. For panels b–e and j–o data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
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f0010: Analysis of spontaneous network bursts in two-dimensional networks observed by imaging of [Ca2+]i. a, Example of time course of the fluorescence intensity () recording of a spontaneous network burst of a two-dimensional diploid vs. Ts65Dn network. b, Amplitude () of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.014. c, Mean duration of a burst (see Methods for a definition of the duration) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures), p = 0.0019. d, Mean normalized fluorescence () intensity of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with inhibitory activity blocked by 40 μM bicuculline, p = 0.019. e, Mean FWHM duration of a burst (see Methods) of diploid networks (N = 8 cultures) vs. Ts65Dn networks (N = 6 cultures) with 40 μM bicuculline, p = 0.03. f, Example of time course for the fluorescence signal of a diploid culture. g, Example of time course for the fluorescence signal of a Ts65Dn culture. h, Same diploid culture as in f, but with 30 μM of GABA applied. i, Same Ts65Dn culture as in f, but with 30 μM of GABA applied. j, Mean baclofen concentration required to stop network activity in diploid networks (N = 5 cultures) vs. the concentration needed to stop activity in Ts65Dn networks (N = 6 cultures), p = 0.0135. k–n, Inhibition plots for baclofen. k, Inhibition plot showing the duration of time that the network was active bursting divided by the total recording time for diploid and Ts65Dn cultures as a function of the baclofen concentration. l, Inhibition plot showing the duration of time that the network was active normalized by the duration of time that the network was active at 0 baclofen for diploid and Ts65Dn cultures as a function of the baclofen concentration. m, Inhibition plot showing the duration that the network was active divided by the total recording time for diploid and Ts65Dn cultures with 80 μM saclofen in the recording medium, as a function of the baclofen concentration. Inset graph shows duration of activity divided by the total recording time as a function of the concentration of saclofen applied. n, Inhibition plot showing the duration that the network was active normalized by the duration that the network was active at 0 baclofen for diploid and Ts65Dn cultures at 80 μM saclofen, as a function of the baclofen concentration. o, Fura-2 ratio of the baseline fluorescent signal at 340 nm to 380 nm in diploid networks (N = 7 cultures) vs. that in Ts65Dn networks (N = 7 cultures), p = 0.185. For panels b–e and j–o data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
Mentions: Fig. 2a shows examples of recorded network bursts of diploid and Ts65Dn networks. The amplitude of the fluorescence signal during activity was smaller in Ts65Dn cultures by about a factor of 2, going from 6.5 ± 1% (N = 12 cultures, n = 266 network bursts) in diploid cultures to 3.3 ± 0.4% in Ts65Dn cultures (N = 10 cultures, n = 253 network bursts, p = 0.014) (Fig. 2b). The average burst duration (see Methods) was also significantly lower (Fig. 2c). We measured 7.1 ± 0.8 s (N = 12 cultures, n = 266 network bursts) in diploid cultures vs. 3.5 ± 0.6 s (N = 10 cultures, n = 253 network bursts) in Ts65Dn cultures (p = 0.0019). To see the contribution of excitatory cells only, we added bicuculline to block GABAA inhibition, and measured the amplitude of the fluorescence signal (Fig. 2d) giving again a ratio of about 2, with 22 ± 5% (N = 9 cultures, n = 64 network bursts) in diploid cultures versus 9 ± 1.3% (N = 9 cultures, n = 42 network bursts) in Ts65Dn cultures (p = 0.019). Similarly, for the duration of network bursts we measured an average of 2.5 ± 0.3 s (N = 9 cultures, n = 64 network bursts) for the diploid cultures versus 1.8 ± 0.2 s for the Ts65Dn (N = 9 cultures, n = 42 network bursts, p = 0.03) (Fig. 2e). Gradual application of GABA had the effect of reducing network activity until it completely stops. The minimal concentration of GABA needed to stop activity was typically 10 μM to 50 μM depending on how active the culture was. An example is shown in Fig. 2f–i. Fig. 2f shows a recording of the fluorescence signal in a diploid culture with no GABA. Similarly, Fig. 2g shows an example recording for a Ts65Dn culture. Fig. 2h is a recording of the same culture used in Fig. 2f, but with 30 μM GABA application. Fig. 2i similarly shows the Ts65Dn network recording with 30 μM GABA for the same culture used in Fig. 2g. We conclude that network bursts in 2D cultures of the Ts65Dn mouse model were reduced in both amplitude and duration. The ratio of fluorescence amplitudes before and after blocking inhibition is somewhat larger in Ts65Dn, though not statistically significant. This increase may indicate that possible differences in the ratio of excitatory to inhibitory contributions might also play a role.

Bottom Line: We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents.Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity.Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics of Complex Systems, Weizmann Institute of Science, P.O. Box 26, Rehovot 76100 Israel.

ABSTRACT
Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca(2+)]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype.

No MeSH data available.


Related in: MedlinePlus