scholarly journals Mechanical tension can specify axonal fate in hippocampal neurons

2002 ◽  
Vol 159 (3) ◽  
pp. 499-508 ◽  
Author(s):  
Phillip Lamoureux ◽  
Gordon Ruthel ◽  
Robert E. Buxbaum ◽  
Steven R. Heidemann
Keyword(s):  
Growth Cone ◽  
Hippocampal Neurons ◽  
De Novo ◽  
High Rate ◽  
Neurite Length ◽  
Mechanical Tension ◽  
Spontaneous Growth ◽  
Developmental Course ◽  
Cultured Hippocampal Neurons

Here we asked whether applied mechanical tension would stimulate undifferentiated minor processes of cultured hippocampal neurons to become axons and whether tension could induce a second axon in an already polarized neuron. Experimental tension applied to minor processes produced extensions that demonstrated axonal character, regardless of the presence of an existing axon. Towed neurites showed a high rate of spontaneous growth cone advance and could continue to grow out for 1–3 d after towing. The developmental course of experimental neurites was found to be similar to that of unmanipulated spontaneous axons. Furthermore, the experimentally elongated neurites showed compartmentation of the axonal markers dephospho-tau and L-1 in towed outgrowth after 24 h. Extension of a second axon from an already polarized neuron does not lead to the loss of the spontaneous axon either immediately or after longer term growth. In addition, we were able to initiate neurites de novo that subsequently acquired axonal character even though spontaneous growth cone advance began while the towed neurite was still no longer than its sibling processes. This suggests that tension rather than the achievement of a critical neurite length determined axonal specification.

scholarly journals GABA Withdrawal Modifies Network Activity in Cultured Hippocampal Neurons

2000 ◽  
Vol 7 (1-2) ◽  
pp. 31-42 ◽  
Author(s):  
H. Golan ◽  
K. Mikenberg ◽  
V. Greenberger ◽  
M. Segal
Keyword(s):  
Withdrawal Syndrome ◽  
Hippocampal Neurons ◽  
High Rate ◽  
Network Activity ◽  
Cultured Neurons ◽  
Synaptic Currents ◽  
Firing Rates ◽  
Cultured Hippocampal Neurons

Dissociated hippocampal neurons, grown in culture for 2 to 3 weeks, tended to fire bursts of synaptic currents at fairly regular intervals, representing network activity. A brief exposure of cultured neurons to GABA caused a total suppression of the spontaneous network activity. Following a washout of GABA, the activity was no longer clustered in bursts and instead, the cells fired at a high rate tonic manner. The effect of removing GABA could be seen as long as 1 to 2 days after GABA withdrawal and is expressed as an increase in the number of active cells in a network, as well as in their firing rates. Such striking effects of GABA removal may underlie part of the GABA withdrawal syndrome seen elsewhere.

scholarly journals Delayed Retraction of Filopodia in Gelsolin Null Mice

1997 ◽  
Vol 138 (6) ◽  
pp. 1279-1287 ◽  
Author(s):  
Mei Lu ◽  
Walter Witke ◽  
David J. Kwiatkowski ◽  
Kenneth S. Kosik
Keyword(s):  
Growth Cone ◽  
Hippocampal Neurons ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Wild Type ◽  
Dynamic Studies ◽  
Shape Changes ◽  
Time Lapse Video ◽  
Cultured Hippocampal Neurons

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.

scholarly journals Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides.

1992 ◽  
Vol 117 (3) ◽  
pp. 595-606 ◽  
Author(s):  
A Ferreira ◽  
J Niclas ◽  
R D Vale ◽  
G Banker ◽  
K S Kosik
Keyword(s):  
Neurite Outgrowth ◽  
Cell Body ◽  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Hippocampal Neurons ◽  
Full Recovery ◽  
Synapsin I ◽  
Neurite Length ◽  
Protein Levels ◽  
Cultured Hippocampal Neurons

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.

scholarly journals AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 324
Author(s):  
Matthias Deutsch ◽  
Anne Günther ◽  
Rodrigo Lerchundi ◽  
Christine R. Rose ◽  
Sabine Balfanz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Hippocampal Neurons ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Physiological Role ◽  
High Specificity ◽  
Hcn Channels ◽  
Proliferating Cells ◽  
Neuronal Tissues

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

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