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Somatic integration of single ion channel responses of α7 nicotinic acetylcholine receptors enhanced by PNU-120596.

Uteshev VV - PLoS ONE (2012)

Bottom Line: The same concentration of a given nicotinic agent can be neuroprotective, ineffective or neurotoxic due to differences in the expression of α7 receptors and susceptibility to Ca(2+) influx among various subtypes of neurons.The results demonstrate that the membrane electrotonic properties do not impede somatic signaling, allowing reliable estimates of somatic ionic and Ca(2+) influx through α7 channels, while the somatic space-clamp error is minimal (~0.01 mV/µm).These research efforts could benefit optimization of potential α7-PAM-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America. Victor.Uteshev@unthsc.edu

ABSTRACT
Positive allosteric modulators of highly Ca(2+)-permeable α7 nicotinic acetylcholine receptors, such as PNU-120596, may become useful therapeutic tools supporting neuronal survival and function. However, despite promising results, the initial optimism has been tempered by the concerns for cytotoxicity. The same concentration of a given nicotinic agent can be neuroprotective, ineffective or neurotoxic due to differences in the expression of α7 receptors and susceptibility to Ca(2+) influx among various subtypes of neurons. Resolution of these concerns may require an ability to reliably detect, evaluate and optimize the extent of α7 somatic ionic influx, a key determinant of the likelihood of neuronal survival and function. In the presence of PNU-120596 and physiological choline (~10 µM), the activity of individual α7 channels can be detected in whole-cell recordings as step-like current/voltage deviations. However, the extent of α7 somatic influx remains elusive because the activity of individual α7 channels may not be integrated across the entire soma, instead affecting only specific subdomains located in the channel vicinity. Such a compartmentalization may obstruct detection and integration of α7 currents, causing an underestimation of α7 activity. By contrast, if step-like α7 currents are integrated across the soma, then a reliable quantification of α7 influx in whole-cell recordings is possible and could provide a rational basis for optimization of conditions that support survival of α7-expressing neurons. This approach can be used to directly correlate α7 single-channel activity to neuronal function. In this study, somatic dual-patch recordings were conducted using large hypothalamic and hippocampal neurons in acute coronal rat brain slices. The results demonstrate that the membrane electrotonic properties do not impede somatic signaling, allowing reliable estimates of somatic ionic and Ca(2+) influx through α7 channels, while the somatic space-clamp error is minimal (~0.01 mV/µm). These research efforts could benefit optimization of potential α7-PAM-based therapies.

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Somatic integration of individual α7 channel activity in hypothalamic TM neurons.A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 24.1 µm (A). Examples of patch-clamp recordings obtained from the neuron shown in (A) when both patch electrodes are in current-clamp (B) or voltage-clamp at −70 mV (C). The bottom traces in B–C) are results of subtraction of trace #2 from trace #1 indicating identical patterns of TM α7 single ion channel activity recorded by the two electrodes (see text). Horizontal bars in (B) indicate the membrane voltage of −75 mV. A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing. The baselines are indicated by arrowheads. Step-like responses (D) were completely (E) and reversibly (F) blocked by 20 nM MLA, a selective α7 nAChR antagonist, added to ACSF.
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pone-0032951-g002: Somatic integration of individual α7 channel activity in hypothalamic TM neurons.A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 24.1 µm (A). Examples of patch-clamp recordings obtained from the neuron shown in (A) when both patch electrodes are in current-clamp (B) or voltage-clamp at −70 mV (C). The bottom traces in B–C) are results of subtraction of trace #2 from trace #1 indicating identical patterns of TM α7 single ion channel activity recorded by the two electrodes (see text). Horizontal bars in (B) indicate the membrane voltage of −75 mV. A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing. The baselines are indicated by arrowheads. Step-like responses (D) were completely (E) and reversibly (F) blocked by 20 nM MLA, a selective α7 nAChR antagonist, added to ACSF.

Mentions: Large histaminergic TM neurons were identified in coronal hypothalamic slices on the basis of their size and morphology, location within the slice, and the expression of high densities of functional α7 nAChRs [12] as well as strong Ih and IA currents [13]. To activate α7 nAChRs, hypothalamic slices were perfused with ACSF containing 5 µM choline and 1 µM PNU-120596. In two experiments, 10 µM choline was used. To detect α7 single-channel events in whole-cell current-clamp experiments, spontaneous firing was inhibited by keeping the membrane voltage near −70 mV by injecting continuous hyperpolarizing currents (50–120 pA). The inter-patch distances were made as large as possible and images of the recorded TM neurons were taken during each experiment. The inter-patch distances were then measured off-line (Figures 1A and 2A, open circles). The average inter-patch distance was 21.0±2.9 µm (n = 16).


Somatic integration of single ion channel responses of α7 nicotinic acetylcholine receptors enhanced by PNU-120596.

Uteshev VV - PLoS ONE (2012)

Somatic integration of individual α7 channel activity in hypothalamic TM neurons.A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 24.1 µm (A). Examples of patch-clamp recordings obtained from the neuron shown in (A) when both patch electrodes are in current-clamp (B) or voltage-clamp at −70 mV (C). The bottom traces in B–C) are results of subtraction of trace #2 from trace #1 indicating identical patterns of TM α7 single ion channel activity recorded by the two electrodes (see text). Horizontal bars in (B) indicate the membrane voltage of −75 mV. A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing. The baselines are indicated by arrowheads. Step-like responses (D) were completely (E) and reversibly (F) blocked by 20 nM MLA, a selective α7 nAChR antagonist, added to ACSF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316542&req=5

pone-0032951-g002: Somatic integration of individual α7 channel activity in hypothalamic TM neurons.A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 24.1 µm (A). Examples of patch-clamp recordings obtained from the neuron shown in (A) when both patch electrodes are in current-clamp (B) or voltage-clamp at −70 mV (C). The bottom traces in B–C) are results of subtraction of trace #2 from trace #1 indicating identical patterns of TM α7 single ion channel activity recorded by the two electrodes (see text). Horizontal bars in (B) indicate the membrane voltage of −75 mV. A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing. The baselines are indicated by arrowheads. Step-like responses (D) were completely (E) and reversibly (F) blocked by 20 nM MLA, a selective α7 nAChR antagonist, added to ACSF.
Mentions: Large histaminergic TM neurons were identified in coronal hypothalamic slices on the basis of their size and morphology, location within the slice, and the expression of high densities of functional α7 nAChRs [12] as well as strong Ih and IA currents [13]. To activate α7 nAChRs, hypothalamic slices were perfused with ACSF containing 5 µM choline and 1 µM PNU-120596. In two experiments, 10 µM choline was used. To detect α7 single-channel events in whole-cell current-clamp experiments, spontaneous firing was inhibited by keeping the membrane voltage near −70 mV by injecting continuous hyperpolarizing currents (50–120 pA). The inter-patch distances were made as large as possible and images of the recorded TM neurons were taken during each experiment. The inter-patch distances were then measured off-line (Figures 1A and 2A, open circles). The average inter-patch distance was 21.0±2.9 µm (n = 16).

Bottom Line: The same concentration of a given nicotinic agent can be neuroprotective, ineffective or neurotoxic due to differences in the expression of α7 receptors and susceptibility to Ca(2+) influx among various subtypes of neurons.The results demonstrate that the membrane electrotonic properties do not impede somatic signaling, allowing reliable estimates of somatic ionic and Ca(2+) influx through α7 channels, while the somatic space-clamp error is minimal (~0.01 mV/µm).These research efforts could benefit optimization of potential α7-PAM-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America. Victor.Uteshev@unthsc.edu

ABSTRACT
Positive allosteric modulators of highly Ca(2+)-permeable α7 nicotinic acetylcholine receptors, such as PNU-120596, may become useful therapeutic tools supporting neuronal survival and function. However, despite promising results, the initial optimism has been tempered by the concerns for cytotoxicity. The same concentration of a given nicotinic agent can be neuroprotective, ineffective or neurotoxic due to differences in the expression of α7 receptors and susceptibility to Ca(2+) influx among various subtypes of neurons. Resolution of these concerns may require an ability to reliably detect, evaluate and optimize the extent of α7 somatic ionic influx, a key determinant of the likelihood of neuronal survival and function. In the presence of PNU-120596 and physiological choline (~10 µM), the activity of individual α7 channels can be detected in whole-cell recordings as step-like current/voltage deviations. However, the extent of α7 somatic influx remains elusive because the activity of individual α7 channels may not be integrated across the entire soma, instead affecting only specific subdomains located in the channel vicinity. Such a compartmentalization may obstruct detection and integration of α7 currents, causing an underestimation of α7 activity. By contrast, if step-like α7 currents are integrated across the soma, then a reliable quantification of α7 influx in whole-cell recordings is possible and could provide a rational basis for optimization of conditions that support survival of α7-expressing neurons. This approach can be used to directly correlate α7 single-channel activity to neuronal function. In this study, somatic dual-patch recordings were conducted using large hypothalamic and hippocampal neurons in acute coronal rat brain slices. The results demonstrate that the membrane electrotonic properties do not impede somatic signaling, allowing reliable estimates of somatic ionic and Ca(2+) influx through α7 channels, while the somatic space-clamp error is minimal (~0.01 mV/µm). These research efforts could benefit optimization of potential α7-PAM-based therapies.

Show MeSH
Related in: MedlinePlus