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Role of cyclic nucleotide-gated channels in the modulation of mouse hippocampal neurogenesis.

Podda MV, Piacentini R, Barbati SA, Mastrodonato A, Puzzo D, D'Ascenzo M, Leone L, Grassi C - PLoS ONE (2013)

Bottom Line: Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype.The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade.The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Physiology, Medical School, Università Cattolica, Rome, Italy.

ABSTRACT
Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

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CNG channel-like conductance in differentiating hippocampal NSCs.Representative whole-cell patch-clamp recordings from NSCs grown for three days in differentiation medium and exhibiting morphological features of neurons. Application of 8-Br-cGMP (1 mM) elicited inward currents at holding potential of -80 mV (A) which were inhibited by the CNG channel blockers, 50 µM LCD (B) and 3 mM Cd2+ (C). (D) Voltage ramp protocol used to evoke leakage currents. (E) Typical current traces under control conditions and in the presence of 1 mM 8-Br-cGMP. 8-Br-cGMP-activated current (net current) was obtained by subtraction. (F) I–V plot of mean 8-Br-cGMP net currents (n=10).
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pone-0073246-g004: CNG channel-like conductance in differentiating hippocampal NSCs.Representative whole-cell patch-clamp recordings from NSCs grown for three days in differentiation medium and exhibiting morphological features of neurons. Application of 8-Br-cGMP (1 mM) elicited inward currents at holding potential of -80 mV (A) which were inhibited by the CNG channel blockers, 50 µM LCD (B) and 3 mM Cd2+ (C). (D) Voltage ramp protocol used to evoke leakage currents. (E) Typical current traces under control conditions and in the presence of 1 mM 8-Br-cGMP. 8-Br-cGMP-activated current (net current) was obtained by subtraction. (F) I–V plot of mean 8-Br-cGMP net currents (n=10).

Mentions: Since CNG channels are voltage-independent non-selective cation channels, we expected that application of the cGMP membrane permeant analogue, 8-Br-cGMP, would elicit non-inactivating inward currents in cells voltage-clamped at negative membrane potentials. Accordingly, cells locally perfused with 8-Br-cGMP (1 mM, for 1-2 min) displayed tonic currents of -22.8±0.9 pA at holding potential of -80 mV (n=22 out of 27 cells tested; Figure 4A). The responses were washed out when the cGMP analogue perfusion was replaced by control Tyrode’s solution. These responses were independent on PKG activation because they were observed in the presence of the selective PKG inhibitor, KT5823 (1 µM), which was dialyzed into the recorded cells via the patch pipette solution. No significant changes in membrane currents were, instead, observed when 8-Br-cGMP was applied to cells pre-treated for 3-5 min with the CNG channel blocker LCD (50 µM; -1.2±0.3 pA, n=12). Furthermore, when LCD was applied once the response to 8-Br-cGMP had fully developed it caused a marked reduction of current amplitudes (-98.4±5.2%; n=6; P<0.05; Figure 4B). 8-Br-cGMP-induced currents were also virtually abolished by the divalent cation, cadmium (3 mM; -99.3±0.4%; n=6; P<0.05; Figure 4C), which is a typical feature of CNG-channel mediated responses [42,43,53].


Role of cyclic nucleotide-gated channels in the modulation of mouse hippocampal neurogenesis.

Podda MV, Piacentini R, Barbati SA, Mastrodonato A, Puzzo D, D'Ascenzo M, Leone L, Grassi C - PLoS ONE (2013)

CNG channel-like conductance in differentiating hippocampal NSCs.Representative whole-cell patch-clamp recordings from NSCs grown for three days in differentiation medium and exhibiting morphological features of neurons. Application of 8-Br-cGMP (1 mM) elicited inward currents at holding potential of -80 mV (A) which were inhibited by the CNG channel blockers, 50 µM LCD (B) and 3 mM Cd2+ (C). (D) Voltage ramp protocol used to evoke leakage currents. (E) Typical current traces under control conditions and in the presence of 1 mM 8-Br-cGMP. 8-Br-cGMP-activated current (net current) was obtained by subtraction. (F) I–V plot of mean 8-Br-cGMP net currents (n=10).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750014&req=5

pone-0073246-g004: CNG channel-like conductance in differentiating hippocampal NSCs.Representative whole-cell patch-clamp recordings from NSCs grown for three days in differentiation medium and exhibiting morphological features of neurons. Application of 8-Br-cGMP (1 mM) elicited inward currents at holding potential of -80 mV (A) which were inhibited by the CNG channel blockers, 50 µM LCD (B) and 3 mM Cd2+ (C). (D) Voltage ramp protocol used to evoke leakage currents. (E) Typical current traces under control conditions and in the presence of 1 mM 8-Br-cGMP. 8-Br-cGMP-activated current (net current) was obtained by subtraction. (F) I–V plot of mean 8-Br-cGMP net currents (n=10).
Mentions: Since CNG channels are voltage-independent non-selective cation channels, we expected that application of the cGMP membrane permeant analogue, 8-Br-cGMP, would elicit non-inactivating inward currents in cells voltage-clamped at negative membrane potentials. Accordingly, cells locally perfused with 8-Br-cGMP (1 mM, for 1-2 min) displayed tonic currents of -22.8±0.9 pA at holding potential of -80 mV (n=22 out of 27 cells tested; Figure 4A). The responses were washed out when the cGMP analogue perfusion was replaced by control Tyrode’s solution. These responses were independent on PKG activation because they were observed in the presence of the selective PKG inhibitor, KT5823 (1 µM), which was dialyzed into the recorded cells via the patch pipette solution. No significant changes in membrane currents were, instead, observed when 8-Br-cGMP was applied to cells pre-treated for 3-5 min with the CNG channel blocker LCD (50 µM; -1.2±0.3 pA, n=12). Furthermore, when LCD was applied once the response to 8-Br-cGMP had fully developed it caused a marked reduction of current amplitudes (-98.4±5.2%; n=6; P<0.05; Figure 4B). 8-Br-cGMP-induced currents were also virtually abolished by the divalent cation, cadmium (3 mM; -99.3±0.4%; n=6; P<0.05; Figure 4C), which is a typical feature of CNG-channel mediated responses [42,43,53].

Bottom Line: Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype.The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade.The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Physiology, Medical School, Università Cattolica, Rome, Italy.

ABSTRACT
Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

Show MeSH
Related in: MedlinePlus