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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 amplitude and propagation of spontaneous network bursts in one-dimensional cultures observed by imaging of [Ca2+]i. Bicuculline was administered at 40 μM. a, Example of time course for the fluorescence signal for a diploid network, depicting the intensity measured in each of the three ROIs drawn in d. Fluorescence signals were normalized by the baseline fluorescence f0. To improve visualization, traces are shifted relative to one another by ± 0.03 . Inset: Zoom in on the initiation of the network bursts, from which arrival times of the activity at each ROI was obtained. b, Similar example of fluorescence intensity signal for Ts65Dn network. Inset as in a. c, Similar example of fluorescence intensity signal for Tc1 network. Inset as in a. d, Schematic sketch of the pattern used for growing the one dimensional culture. e, Imaging of [Ca2+]i of background fluorescence in a linear neuronal culture. Scale bar is 1 mm. Timing of the activity in the ROIs (marked black in d and red in e) is used to measure the velocity. f, Average fluorescence intensity () of cultures diploid (N = 12 cultures) vs. Ts65Dn cultures (N = 5 cultures), p = 0.021. g, Average propagation velocity of diploid (N = 12 cultures) cultures vs. Ts65Dn (N = 5 cultures) cultures, p < 0.001. h, Average fluorescence intensity () of diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.05. i, Average propagation velocity of activity in diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.01. For panels f–i data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
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f0005: Analysis of amplitude and propagation of spontaneous network bursts in one-dimensional cultures observed by imaging of [Ca2+]i. Bicuculline was administered at 40 μM. a, Example of time course for the fluorescence signal for a diploid network, depicting the intensity measured in each of the three ROIs drawn in d. Fluorescence signals were normalized by the baseline fluorescence f0. To improve visualization, traces are shifted relative to one another by ± 0.03 . Inset: Zoom in on the initiation of the network bursts, from which arrival times of the activity at each ROI was obtained. b, Similar example of fluorescence intensity signal for Ts65Dn network. Inset as in a. c, Similar example of fluorescence intensity signal for Tc1 network. Inset as in a. d, Schematic sketch of the pattern used for growing the one dimensional culture. e, Imaging of [Ca2+]i of background fluorescence in a linear neuronal culture. Scale bar is 1 mm. Timing of the activity in the ROIs (marked black in d and red in e) is used to measure the velocity. f, Average fluorescence intensity () of cultures diploid (N = 12 cultures) vs. Ts65Dn cultures (N = 5 cultures), p = 0.021. g, Average propagation velocity of diploid (N = 12 cultures) cultures vs. Ts65Dn (N = 5 cultures) cultures, p < 0.001. h, Average fluorescence intensity () of diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.05. i, Average propagation velocity of activity in diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.01. For panels f–i data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.

Mentions: Primary cultures were prepared from hippocampi of E17 embryos following (Feinerman et al., 2005; Papa et al., 1995). Briefly, the hippocampus was dissected in chilled Leibovitz L15 medium enriched with 0.6% glucose and in the presence of 15 μg/ml gentamicin. The medium was oxygenated and the dissection carried out at 4 °C. The tissue was mechanically and enzymatically dissociated (papain) in small volumes of L15 using a fire-polished Pasteur pipette. Tissue was suspended in plating medium, consisting of 5% fetal calf serum and 5% heat-inactivated horse serum prepared in Eagle's minimal essential medium enriched with 0.6% glucose, 2 mM glutamine, and 15 μg/ml gentamicin. Neurons were plated on poly-l-lysine-coated (15 μg/ml) 13 mm round glass coverslips, (# 1, Menzel-Glazer), in 24-well culture plates at a density of 650,000 cells per well (one-dimensional), or 750,000 cells per well (two-dimensional). Four days after plating, the medium was changed to one containing 10% heat-inactivated horse serum. In addition, the first change of medium also contained a mixture of 50 μg/ml uridine, and 20 μg/ml 5′-fluoro-2 deoxyuridine, to block the proliferation of glial cells. After additional four days, medium was changed again to one containing 10% heat-inactivated horse serum alone. Thereafter one third of the medium was replaced daily. Midbrain and cortex tissue from each pup were harvested and stored at − 70 °C for further genotyping. Each hippocampus is plated onto several separate coverslips immediately after dissection, so that neurons from different pups never mix. They grow in a standard incubator at 37 °C, 5% CO2 until use (1–3 weeks in culture). Since the seeding of the cells is time sensitive, genotyping is delayed and performed separately, within the next day or two. One-dimensional cultures were grown on a “C” shape, patterned as described by Feinerman et al. (2005). The pattern was designed to allow a long propagation length concurrent with three observation points that fit in the microscope view and cover the two edges of the line and its center, see Fig. 1d and e.


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 amplitude and propagation of spontaneous network bursts in one-dimensional cultures observed by imaging of [Ca2+]i. Bicuculline was administered at 40 μM. a, Example of time course for the fluorescence signal for a diploid network, depicting the intensity measured in each of the three ROIs drawn in d. Fluorescence signals were normalized by the baseline fluorescence f0. To improve visualization, traces are shifted relative to one another by ± 0.03 . Inset: Zoom in on the initiation of the network bursts, from which arrival times of the activity at each ROI was obtained. b, Similar example of fluorescence intensity signal for Ts65Dn network. Inset as in a. c, Similar example of fluorescence intensity signal for Tc1 network. Inset as in a. d, Schematic sketch of the pattern used for growing the one dimensional culture. e, Imaging of [Ca2+]i of background fluorescence in a linear neuronal culture. Scale bar is 1 mm. Timing of the activity in the ROIs (marked black in d and red in e) is used to measure the velocity. f, Average fluorescence intensity () of cultures diploid (N = 12 cultures) vs. Ts65Dn cultures (N = 5 cultures), p = 0.021. g, Average propagation velocity of diploid (N = 12 cultures) cultures vs. Ts65Dn (N = 5 cultures) cultures, p < 0.001. h, Average fluorescence intensity () of diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.05. i, Average propagation velocity of activity in diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.01. For panels f–i data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
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Related In: Results  -  Collection

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Show All Figures
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f0005: Analysis of amplitude and propagation of spontaneous network bursts in one-dimensional cultures observed by imaging of [Ca2+]i. Bicuculline was administered at 40 μM. a, Example of time course for the fluorescence signal for a diploid network, depicting the intensity measured in each of the three ROIs drawn in d. Fluorescence signals were normalized by the baseline fluorescence f0. To improve visualization, traces are shifted relative to one another by ± 0.03 . Inset: Zoom in on the initiation of the network bursts, from which arrival times of the activity at each ROI was obtained. b, Similar example of fluorescence intensity signal for Ts65Dn network. Inset as in a. c, Similar example of fluorescence intensity signal for Tc1 network. Inset as in a. d, Schematic sketch of the pattern used for growing the one dimensional culture. e, Imaging of [Ca2+]i of background fluorescence in a linear neuronal culture. Scale bar is 1 mm. Timing of the activity in the ROIs (marked black in d and red in e) is used to measure the velocity. f, Average fluorescence intensity () of cultures diploid (N = 12 cultures) vs. Ts65Dn cultures (N = 5 cultures), p = 0.021. g, Average propagation velocity of diploid (N = 12 cultures) cultures vs. Ts65Dn (N = 5 cultures) cultures, p < 0.001. h, Average fluorescence intensity () of diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.05. i, Average propagation velocity of activity in diploid cultures (N = 10 cultures) vs. Tc1 cultures (N = 13 cultures), p = 0.01. For panels f–i data are presented as mean ± SEM, * indicates p-value < 0.05, ** is p < 0.01, and *** indicates p < 0.001.
Mentions: Primary cultures were prepared from hippocampi of E17 embryos following (Feinerman et al., 2005; Papa et al., 1995). Briefly, the hippocampus was dissected in chilled Leibovitz L15 medium enriched with 0.6% glucose and in the presence of 15 μg/ml gentamicin. The medium was oxygenated and the dissection carried out at 4 °C. The tissue was mechanically and enzymatically dissociated (papain) in small volumes of L15 using a fire-polished Pasteur pipette. Tissue was suspended in plating medium, consisting of 5% fetal calf serum and 5% heat-inactivated horse serum prepared in Eagle's minimal essential medium enriched with 0.6% glucose, 2 mM glutamine, and 15 μg/ml gentamicin. Neurons were plated on poly-l-lysine-coated (15 μg/ml) 13 mm round glass coverslips, (# 1, Menzel-Glazer), in 24-well culture plates at a density of 650,000 cells per well (one-dimensional), or 750,000 cells per well (two-dimensional). Four days after plating, the medium was changed to one containing 10% heat-inactivated horse serum. In addition, the first change of medium also contained a mixture of 50 μg/ml uridine, and 20 μg/ml 5′-fluoro-2 deoxyuridine, to block the proliferation of glial cells. After additional four days, medium was changed again to one containing 10% heat-inactivated horse serum alone. Thereafter one third of the medium was replaced daily. Midbrain and cortex tissue from each pup were harvested and stored at − 70 °C for further genotyping. Each hippocampus is plated onto several separate coverslips immediately after dissection, so that neurons from different pups never mix. They grow in a standard incubator at 37 °C, 5% CO2 until use (1–3 weeks in culture). Since the seeding of the cells is time sensitive, genotyping is delayed and performed separately, within the next day or two. One-dimensional cultures were grown on a “C” shape, patterned as described by Feinerman et al. (2005). The pattern was designed to allow a long propagation length concurrent with three observation points that fit in the microscope view and cover the two edges of the line and its center, see Fig. 1d and e.

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