Somatic Integration of Single Ion Channel Responses of α7 Nicotinic Acetylcholine Receptors Enhanced by PNU-120596

Positive allosteric modulators of highly Ca2+-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 Ca2+ 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 Ca2+ 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

Analysis of cross-correlations and space-clamp in hippocampal CA1 interneurons.

<p>An example of traces recorded by two patch-clamp electrodes in current-clamp (<b>A</b>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s3" target="_blank">Results</a> of cross-correlation analysis (<b>B</b>). The correlation coefficient at lag = 0 ms (R = 0.9991) was used to estimate the significance of cross-correlation between the two traces (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s2" target="_blank">Methods</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s3" target="_blank">Results</a> obtained in the same experiment when electrode #2 was switched to voltage-clamp at −80 mV (<b>C</b>). Electrode #1 remained in current-clamp and detected small (∼0.12 mV) space-clamp errors (C, top trace) in the quality of voltage-clamp provided by electrode #2. Horizontal bars indicate the membrane voltage of −79 mV (<b>A</b>) and −79.4 mV (<b>C</b>). A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing (<b>A</b>). The baselines are indicated by arrowheads.</p

Somatic integration of individual α7 channel activity in hypothalamic TM neurons.

<p>A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 24.1 µm (<b>A</b>). Examples of patch-clamp recordings obtained from the neuron shown in (<b>A</b>) when both patch electrodes are in current-clamp (<b>B</b>) or voltage-clamp at −70 mV (<b>C</b>). 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>B</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 (<b>D</b>) were completely (<b>E</b>) and reversibly (<b>F</b>) blocked by 20 nM MLA, a selective α7 nAChR antagonist, added to ACSF.</p

Analysis of cross-correlations and space-clamp in hypothalamic TM neurons.

<p>An example of traces recorded by two patch-clamp electrodes in current-clamp (<b>A</b>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s3" target="_blank">Results</a> of cross-correlation analysis (<b>B</b>). The correlation coefficient at lag = 0 ms (R = 0.9993) was used to estimate the significance of cross-correlation between the two traces (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s2" target="_blank">Methods</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032951#s3" target="_blank">Results</a> obtained in the same experiment when electrode #2 was switched to voltage-clamp at −70 mV (<b>C</b>). Electrode #1 remained in current-clamp and detected small (∼0.22 mV) space-clamp errors (C, top trace) in the quality of voltage-clamp provided by electrode #2. Horizontal bars indicate the membrane voltage of −70 mV (<b>A</b>) and −70.2 mV (<b>C</b>). A continuous hyperpolarizing current (50–120 pA) was injected into cells to cease spontaneous firing (<b>A</b>). The baselines are indicated by arrowheads.</p

Reliability of physiological responses.

<p>A large hypothalamic TM neuron with two attached patch-clamp electrodes and an inter-patch distance of 25.3 µm (<b>A</b>). Examples of patch-clamp recordings obtained from the neuron shown in (<b>A</b>) under two experimental conditions: (<b>B</b>) Electrode #1 is in current-clamp (B, top trace), while Electrode #2 is cell-attached (B, bottom trace); and (<b>C</b>) Electrode #1 is in voltage-clamp at −70 mV (C, top trace), while Electrode #2 is cell-attached (C, bottom trace). Open arrows in (C) point at spontaneous synaptic currents. Closed arrow points at a step-like deviation corresponding to an individual α7 nAChR-mediated ion channel opening. ACSF always contained 5 µM choline plus 1 µM PNU-120596. A horizontal bar in (<b>B</b>) top trace indicates the membrane voltage of −50 mV. Currents were not injected in current-clamp.</p

Expression and Function of Transient Receptor Potential Ankyrin 1 Ion Channels in the Caudal Nucleus of the Solitary Tract

The nucleus of the solitary tract (NTS) receives visceral information via the solitary tract (ST) that comprises the sensory components of the cranial nerves VII, IX and X. The Transient Receptor Potential Ankyrin 1 (TRPA1) ion channels are non-selective cation channels that are expressed primarily in pain-related sensory neurons and nerve fibers. Thus, TRPA1 expressed in the primary sensory afferents may modulate the function of second order NTS neurons. This hypothesis was tested and confirmed in the present study using acute brainstem slices and caudal NTS neurons by RT-PCR, immunostaining and patch-clamp electrophysiology. The expression of TRPA1 was detected in presynaptic locations, but not the somata of caudal NTS neurons that did not express TRPA1 mRNA or proteins. Moreover, caudal NTS neurons did not show somatodendritic responsiveness to TRPA1 agonists, while TRPA1 immunostaining was detected only in the afferent fibers. Electrophysiological recordings detected activation of presynaptic TRPA1 in glutamatergic terminals synapsing on caudal NTS neurons evidenced by the enhanced glutamatergic synaptic neurotransmission in the presence of TRPA1 agonists. The requirement of TRPA1 for modulation of spontaneous synaptic activity was confirmed using TRPA1 knockout mice where TRPA1 agonists failed to alter synaptic efficacy. Thus, this study provides the first evidence of the TRPA1-dependent modulation of the primary afferent inputs to the caudal NTS. These results suggest that the second order caudal NTS neurons act as a TRPA1-dependent interface for visceral noxious-innocuous integration at the level of the caudal brainstem