Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current

doi:10.1016/0006-8993(93)90353-O

Brain Research

Volume 604, Issues 1–2, 26 February 1993, Pages 69-78

https://doi.org/10.1016/0006-8993(93)90353-O Get rights and content

Abstract

Mouse trisomy 16 is an animal model for Down's syndrome (human trisomy 21). The whole-cell patch-clamp technique was used to compare passive and active electrical properties of trisomy 16 and diploid mouse 16 fetal hippocampal neurons maintained in culture for 2–5 weeks. There was no significant difference in any mean passive property, including resting potential, membrane resistance, capacitance and time constant. However, in trisomic neurons, the action potential had a 20% significantly slower rising phase and a 20% significantly smaller inward sodium current and inward sodium conductance than did control neurons. The outward conductance was not altered. The ratio of maximum inward conductance to maximum outward conductance was 30% less in the trisomy 16 cells. These results indicate that trisomy 16 hippocampal neurons have abnormal active electrical properties, most likely reflecting reduced sodium channel membrane density. Such subtle differences may influence elaboration of the hippocampus during development.

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Ts16 mice carry an extra copy of the whole of mouse chromosome 16 and are trisomic for a larger number of genes than Ts65Dn mice (Lana-Elola et al., 2011), but some of these trisomic genes are orthologous to genes on human chromosomes other than 21). The changes observed include faster and shorter APs in Ts16 mouse and trisomy 21 DRG cells (Ault et al., 1989; Caviedes et al., 1990) but slower and smaller APs in Ts16 mouse hippocampal neurons (Galdzicki et al., 1993), faster sodium currents with reduced inactivation in trisomy 21 DRG cells (Caviedes et al., 1990) but smaller sodium currents in Ts16 mouse hippocampal neurons (Galdzicki et al., 1993), and smaller and more slowly-activating calcium currents in Ts16 DRG cells (Caviedes et al., 2006) but increased calcium currents in Ts16 mouse hippocampal neurons (Galdzicki et al., 1998). Input resistance was usually unchanged but resting potential and input capacitance were affected in some studies but not in others (Ault et al., 1989; Best et al., 2011; Galdzicki et al., 1993, 1998).
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Likewise, the number of spikes per current step was similar between both genotypes (Supplementary Fig. 1B; two-way ANOVA, p = 0.98). These findings are in contrast to previous reports from full trisomy 16 mouse cultured hippocampal neurons (Galdzicki et al., 1993). Changes in the intrinsic properties of CA1 pyramidal cells in Ts65Dn mice could potentially compensate for alterations in GABAergic signaling.
GABAergic dysfunction is implicated in hippocampal deficits of the Ts65Dn mouse model of Down syndrome (DS). Since Ts65Dn mice overexpress G-protein coupled inward-rectifying potassium (GIRK2) containing channels, we sought to evaluate whether increased GABAergic function disrupts the functioning of hippocampal circuitry. After confirming that GABA_B/GIRK current density is significantly elevated in Ts65Dn CA1 pyramidal neurons, we compared monosynaptic inhibitory inputs in CA1 pyramidal neurons in response to proximal (stratum radiatum; SR) and distal (stratum lacunosum moleculare; SLM) stimulation of diploid and Ts65Dn acute hippocampal slices. Synaptic GABA_B and GABA_A mediated currents evoked by SR stimulation were generally unaffected in Ts65Dn CA1 neurons. However, the GABA_B/GABA_A ratios evoked by stimulation within the SLM of Ts65Dn hippocampus were significantly larger in magnitude, consistent with increased GABA_B/GIRK currents after SLM stimulation. These results indicate that GIRK overexpression in Ts65Dn has functional consequences which affect the balance between GABA_B and GABA_A inhibition of CA1 pyramidal neurons, most likely in a pathway specific manner, and may contribute to cognitive deficits reported in these mice.
Neuronal dysfunction in Down syndrome: Contribution of neuronal models in cell culture
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Down syndrome (DS) in humans, or trisomy of autosome 21, represents the hyperdiploidy that most frequently survives gestation, reaching an incidence of 1 in 700 live births. The condition is associated with multisystemic anomalies, including those affecting the central nervous system (CNS), determining a characteristic mental retardation. At a neuronal level, our group and others have shown that the condition determines marked alterations of action potential and ionic current kinetics, which may underlie abnormal processing of information by the CNS.
Since the use of human tissue presents both practical and ethical problems, animal models of the human condition have been sought. Murine trisomy 16 (Ts16) is a model of the human condition, due to the great homology between human autosome 21 and murine 16. Both conditions share the same alterations of electrical membrane properties. However, the murine Ts16 condition is unviable (animals die in utero), thus limiting the quantity of tissue procurable. To overcome this obstacle, we have established immortal cell lines from normal and Ts16 mice with a method developed by our group that allows the stable in vitro immortalization of mammalian tissue, yielding cell lines which retain the characteristics of the originating cells. Cell lines derived from cerebral cortex, hippocampus, spinal cord and dorsal root ganglion of Ts16 animals show alterations of intracellular Ca²⁺ signals in response to several neurotransmitters (glutamate, acetylcholine, and GABA). Gene overdose most likely underlies these alterations in cell function, and the identification of the relative contribution of DS associated genes on such specific neuronal dysfunction should be investigated. This could enlighten our understanding on the contribution of these genes in DS, and identify new therapeutic targets.
Cell lines derived from hippocampal neurons of the normal and trisomy 16 mouse fetus (a model for Down syndrome) exhibit neuronal markers, cholinergic function, and functional neurotransmitter receptors
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We haveestablished hippocampal cell lines from normal and trisomy 16 fetal mice, a model of human trisomy 21. Both cell lines, named H1b (derived from a normal animal) and HTk (trisomic) possess neuronal markers by immunohistochemistry (enolase, synaptophysin, microtubule associated protein-2, and choline acetyltransferase) and lack glial markers (glial fibrillary acidic protein and S-100). Also, we evaluated intracellular Ca²⁺ levels ([Ca²⁺]_i) in response to neurotransmitter agonists, in cells loaded with the fluorescent Ca²⁺ indicators Indo-1 and Fluo-3. Both cell lines responded to glutamatergic stimuli induced by glutamate, N-methyl-d-aspartate, I-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate. Glutamate responses were only partially prevented by addition of 5 mM EGTA and the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), increased [Ca²⁺]_i in both cell types. These results confirm the presence of glutamatergic metabotropic receptors. In glutamate-induced responses, HTk cells exhibited slower time-dependent decay kinetics than H1b cells. Cholinergic agonists (nicotine and muscarine) induced a rapid, transient increase in [Ca²⁺]_i in both cell types. Furthermore, some cells were sensitive to histamine and norepinephrine. All responses to the aforementioned agonists were prevented by addition of specific antagonists. We also studied incorporation and release of [³H]choline in the cells, and observed no differences in uptake parameters. However, release induced by K⁺ and nicotine depolarization was greatly reduced in HTk cells. The results show that H1b and HTk cells retain neuronal characteristics and respond to specific neurotransmitter stimuli. The HTk differences could be related to neuronal pathophysiology in Down syndrome.
Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides
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The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-d-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal γ-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[γ10K/γ14K] and con-R[γ11A/γ15A] displayed 5.6- and 8.4-fold decreases in τ_off, respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the τ_off was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 μM spermine enhanced the onset rate constant from 3.1×10³ M⁻¹ s⁻¹ to 12.6×10³ M⁻¹ s⁻¹. From analysis of equilibrium dose–inhibition curves using the Cheng–Prusoff equation, a K_i value of 1.1 μM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 μM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.
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2001, Brain Research Reviews
Down syndrome (DS, trisomy 21, Ts21) is the most common known cause of mental retardation. In vivo structural brain imaging in young DS adults, and post-mortem studies, indicate a normal brain size after correction for height, and the absence of neuropathology. Functional imaging with positron emission tomography (PET) shows normal brain glucose metabolism, but fewer significant correlations between metabolic rates in different brain regions than in controls, suggesting reduced functional connections between brain circuit elements. Cultured neurons from Ts21 fetuses and from fetuses of an animal model for DS, the trisomy 16 (Ts16) mouse, do not differ from controls with regard to passive electrical membrane properties, including resting potential and membrane resistance. On the other hand, the trisomic neurons demonstrate abnormal active electrical and biochemical properties (duration of action potential and its rates of depolarization and repolarization, altered kinetics of active Na⁺, Ca²⁺ and K⁺ currents, altered membrane densities of Na⁺ and Ca²⁺ channels). Another animal model, the adult segmental trisomy 16 mouse (Ts65Dn), demonstrates reduced long-term potentiation and increased long-term depression (models for learning and memory related to synaptic plasticity) in the CA1 region of the hippocampus. Evidence suggests that the abnormalities in the trisomy mouse models are related to defective signal transduction pathways involving the phosphoinositide cycle, protein kinase A and protein kinase C. The phenotypes of DS and its mouse models do not involve abnormal gene products due to mutations or deletions, but result from altered expression of genes on human chromosome 21 or mouse chromosome 16, respectively. To the extent that the defects in signal transduction and in active electrical properties, including synaptic plasticity, that are found in the Ts16 and Ts65Dn mouse models, are found in the brain of DS subjects, we postulate that mental retardation in DS results from such abnormalities. Changes in timing and synaptic interaction between neurons during development can lead to less than optimal functioning of neural circuitry and signaling then and in later life.

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Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current

Abstract

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Neurosci. Lett.

Trends Neurosci.

Neurosci. Lett.

Dev. Brain Res.

Trends Neurosci.

Brain Res.

Brain Res.

Dev. Brain Res.

Dev. Brain Res.

Brain Res. Bull.

Genomics

Progr. Brain Res.

Neuroscience

Rat hippocampal neurons in dispersed cell culture

Brain Res.

Genomic imprinting: normal complementation of murine chromosome 16

Genet. Res. Camb.

Electrophysiology of isolated hippocampal dendrites

J. Neurosci.

Inactivation of the sodium channel. I. Sodium current experiments

J. Gen. Physiol.

Passive electrical constants in three classes of hippocampal neurons

J. Neurophysiol.

Hebbian synapses: biophysical mechanisms and algorithms

Annu. Rev. Neurosci.

Nerve growth factor regulates the action potential duration of mature sensory neurons