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The role Ttyh1 protein in regulation of dendritic tree and spine formation

100%

Gorniak-Walas M., Kaliszewska A., Lukasiuk K.

Acta Neurobiologiae Experimentalis

2015

tom 75

nr Supl.

BACKGROUND AND AIMS: Tweety homolog1 (Ttyh1) is a presumed volume-regulated Cl- channel, regulated by cell swelling. Ttyh1 overexpression in dissociated cultures of hippocampal neurons leads to increase in neuritognesis implicating its role in regulation of neuronal morphology. However, the role of Ttyh1 protein in regulation of dendritic tree and spine formation is not well understood. The aim of the study was to examine the influence of Ttyh1 protein in regulation of dendritic tree complexity and formation of dendritic spines in organotypic hippocampal cultures with preserved three dimensional structure. METHODS: Organotypic hippocampal slices were performed from 6 days old rats and transfected with plasmids coding RFP under β-actin promoter and Ttyh1-GFP under synapsin promoter or respective controls, using the Gene Gun. Pyramidal neurons in two areas of the hippocampus: CA1 and CA3 were analyzed using Neuromantic and Sholl software to quantify dendritic tree complexity and dendritic length. SpineMagick software was used to examine dendritic spine morphology and spine density. Proximal and distal parts of apical secondary branches in the stratum radiatum and basal dendrites were analyzed. Dendritic parameters from different groups were compared using a Student’s t test. RESULTS: Sholl analyses did not reveal significant differences in arborization of CA1 and CA3 neurons overexpressing Ttyh1 in comparison to control neurons. Dendritic spine analysis revealed considerable reduction in number and size of dendritic spines. Ttyh1 transfected neurons had varicose swelling of dendrites and more stubby and mushroom spines. CONCLUSIONS: On the basis of our results we conclude that Ttyh1 may participate in regulation of dendritic spine formation. Ttyh1 protein does not affect on dendritic complexity. Formation of dendritic beading may be dependent on Na2+ and Cl- intracellular movement, followed by water movement to maintain osmolarity.

Morphological changes of CA1 and CA3 pyramidal neurons in rat hippocampal slice cultures following TTYH1 overexpression

100%

Gorniak-Walas M., Kaliszewska A., Leski S., Lukasiuk K.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: Tweety homolog1 (Ttyh1) is a presumed volume-regulated Cl-channel. It has been proposed to participate in the regulation of neuronal morphology. AIM(S): We aimed to examine dendritic arborization and spine morphology of pyramidal neurons following TTYH1 overexpression in organotypic hippocampal cultures. METHOD(S): Rat organotypic cultures were co-transfected with TTYH1‑GFP‑Synapsin and RFP‑β‑actin constructs, using biolistic transfection (Gene-Gun, BioRad). Neuronal reconstructions of CA1 and CA3 pyramidal cells were obtained with confocal microscopy and Neuromantic software. Morphometric assessments of individual neurons were performed with Sholl method. L-measure software was used to extract more complex quantitative measurements from neuronal reconstructions. Changes in spine morphology and density on CA1 and CA3 neurons were studied with SpineMagick software. RESULTS: Sholl method did not reveal signifficant differences in dendritic arborization of neurons overexpressing TTYH1 compared to control neurons. L-measure revealed that CA3 neurons overexpressing TTYH1 showed increased average branch length in the seventh branch order of apical dendrites (P<0.05) and increased number of branches in the third branch order of basal dendrites (P<0.01). CA1 pyramidal neurons overexpressing TTYH1 showed reduced average branch length in the third (P<0.05) and fourth (P<0.001) branch orders of basal dendrites. TTYH1 overexpression led to increased number of stubby spines on CA1 neurons (apical proximal and distal dendrites: P<0.01; basal dendrites: P<0.05) and CA3 neurons (apical proximal dendrites: P<0.01). Decrease in the number of long spines on CA1 neurons (apical proximal and distal dendrites: P<0.01) and CA3 neurons was confirmed (apical proximal dendrites: P<0.05). CONCLUSIONS: The influence of TTYH1 overexpression on dendritic complexity and spines morphology suggests that TTYH1 protein may be involved in neuronal plasticity. FINANCIAL SUPPORT: This research was supported by Polish National Science Centre Grant 2011/03/B/ NZ4/00302.

Ograniczanie wyników

2 Acta Neurobiologiae Experimentalis

2 Gorniak-Walas M.

2 Kaliszewska A.

2 Lukasiuk K.

1 Leski S.

1 2017

1 2015

Wyniki wyszukiwania

The role Ttyh1 protein in regulation of dendritic tree and spine formation

Morphological changes of CA1 and CA3 pyramidal neurons in rat hippocampal slice cultures following TTYH1 overexpression