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Cell-attached single-channel recordings in intact prefrontal cortex pyramidal neurons reveal compartmentalized D1/D5 receptor modulation of the persistent sodium current.

Gorelova N, Seamans JK - Front Neural Circuits (2015)

Bottom Line: While past studies have tested the effects of dopamine on I(Nap), the results have been contradictory largely because of difficulties in measuring I(Nap) using somatic whole-cell recordings.As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region.By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of I(Nap), we demonstrate conclusively that D1/D5 receptor activation can increase the I(Nap) generated proximally, however questions still remain as to how D1/D5 receptor modulates Na(+) currents in the more distal initial segment where most of the I Nap is normally generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Brain Research Centre, University of British Columbia Vancouver, BC, Canada.

ABSTRACT
The persistent Na(+) current (I(Nap)) is believed to be an important target of dopamine modulation in prefrontal cortex (PFC) neurons. While past studies have tested the effects of dopamine on I(Nap), the results have been contradictory largely because of difficulties in measuring I(Nap) using somatic whole-cell recordings. To circumvent these confounds we used the cell-attached patch-clamp technique to record single Na(+) channels from the soma, proximal dendrite (PD) or proximal axon (PA) of intact prefrontal layer V pyramidal neurons. Under baseline conditions, numerous well resolved Na(+) channel openings were recorded that exhibited an extrapolated reversal potential of 73 mV, a slope conductance of 14-19 pS and were blocked by tetrodotoxin (TTX). While similar in most respects, the propensity to exhibit prolonged bursts lasting >40 ms was many fold greater in the axon than the soma or dendrite. Bath application of the D1/D5 receptor agonist SKF81297 shifted the ensemble current activation curve leftward and increased the number of late events recorded from the PD but not the soma or PA. However, the greatest effect was on prolonged bursting where the D1/D5 receptor agonist increased their occurrence 3 fold in the PD and nearly 7 fold in the soma, but not at all in the PA. As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region. Therefore, D1/D5 receptor modulation appears to be targeted mainly to Na(+) channels in the PD/soma and not the PA. By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of I(Nap), we demonstrate conclusively that D1/D5 receptor activation can increase the I(Nap) generated proximally, however questions still remain as to how D1/D5 receptor modulates Na(+) currents in the more distal initial segment where most of the I Nap is normally generated.

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Characteristics of Na+ channel gating in mPFC neurons. (A) Schematic of the recording arrangement. Cell-attached patch-clamp recordings were made from the soma, the proximal dendrite (PD) or the proximal axon (PA), within 15 um of the soma. (B) Example traces from a cell-attached recording of Na+ channel openings in the PD evoked by a +60 mV voltage step from a holding potential −20 mV below the presumed resting membrane potential of −80 mV (illustrated in the bottom schematic). Openings varied widely in duration. In some cases prolonged bursts were recorded that lasted hundreds of ms. (C) Group plot of the slope conductances derived from all recordings from the PD (top), soma (middle) or PA (bottom). The x-axis gives the transmembrane potential to which the patch was stepped (starting from a holding potential −40 mV hyperpolarized from rest) and the y-axis gives the average amplitude of all single openings >2 ms in duration evoked by the step. Each blue dot is data from a single patch and the red line is the regression fit (with 95% confidence intervals) to the dots. The extrapolated slope conductance and reversal potentials are provided in the insets. (D) An example PD patch recording in which prolonged burst events were sufficiently frequent so as to allow for an investigation of the current throughout a voltage ramp. Channel openings began at an approximate transmembrane potential of −45 mV and decreased in amplitude as the driving force collapsed. The extrapolated slope conductance and reversal potential are given in the inset. The voltage ramp protocol is given in the bottom schematic and involved holding the patch −40 mV below rest and sweeping the voltage to 80 mV above rest. The resting transmembrane potential, obtained after break in, is given by the dotted gray line.
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Figure 1: Characteristics of Na+ channel gating in mPFC neurons. (A) Schematic of the recording arrangement. Cell-attached patch-clamp recordings were made from the soma, the proximal dendrite (PD) or the proximal axon (PA), within 15 um of the soma. (B) Example traces from a cell-attached recording of Na+ channel openings in the PD evoked by a +60 mV voltage step from a holding potential −20 mV below the presumed resting membrane potential of −80 mV (illustrated in the bottom schematic). Openings varied widely in duration. In some cases prolonged bursts were recorded that lasted hundreds of ms. (C) Group plot of the slope conductances derived from all recordings from the PD (top), soma (middle) or PA (bottom). The x-axis gives the transmembrane potential to which the patch was stepped (starting from a holding potential −40 mV hyperpolarized from rest) and the y-axis gives the average amplitude of all single openings >2 ms in duration evoked by the step. Each blue dot is data from a single patch and the red line is the regression fit (with 95% confidence intervals) to the dots. The extrapolated slope conductance and reversal potentials are provided in the insets. (D) An example PD patch recording in which prolonged burst events were sufficiently frequent so as to allow for an investigation of the current throughout a voltage ramp. Channel openings began at an approximate transmembrane potential of −45 mV and decreased in amplitude as the driving force collapsed. The extrapolated slope conductance and reversal potential are given in the inset. The voltage ramp protocol is given in the bottom schematic and involved holding the patch −40 mV below rest and sweeping the voltage to 80 mV above rest. The resting transmembrane potential, obtained after break in, is given by the dotted gray line.

Mentions: Layer V pyramidal cells were visualized in brain slices using infrared differential interference contrast optics (Axioskop Zeiss). Recordings were made from cell bodies, proximal apical dendrites (PD, 5–10 µm from soma) and proximal axons (PA, axon initial segment, 3–15 µm from soma) (Figure 1A). Pipettes were brought next to the neuron and very weak positive pressure was used to clean the surface before seal formation. Single channel recordings were made in cell-attached configuration. Patch pipettes were made from thick wall borosilicate glass capillaries with an outer diameter of 1.5 mm. The internal surface of the glass capillaries was treated with Sigmacote and allowed to dry at room temperature at least 3 days before being used for manufacturing patch pipettes. This treatment significantly reduced capacitance and improved the quality of the seal, which approached values >40 GΩ. To reduce the number of single channels in a patch we used pipettes with resistances of 15–25 MΩ when filled with patch solution. The pipette solution for recording Na+ channels contained the following (in mM): 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 0.1 CdCl2, 0.02 CNQX, 0.05 AP5, 10 D-glucose, 5 tetraethylammonium chloride, 1 4-AP and 10 HEPES with a pH of 7.3. The pipette solution for recording delayed rectifier K+ channels contained the following (in mM); 150 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, 10 D-glucose with a pH of 7.4.


Cell-attached single-channel recordings in intact prefrontal cortex pyramidal neurons reveal compartmentalized D1/D5 receptor modulation of the persistent sodium current.

Gorelova N, Seamans JK - Front Neural Circuits (2015)

Characteristics of Na+ channel gating in mPFC neurons. (A) Schematic of the recording arrangement. Cell-attached patch-clamp recordings were made from the soma, the proximal dendrite (PD) or the proximal axon (PA), within 15 um of the soma. (B) Example traces from a cell-attached recording of Na+ channel openings in the PD evoked by a +60 mV voltage step from a holding potential −20 mV below the presumed resting membrane potential of −80 mV (illustrated in the bottom schematic). Openings varied widely in duration. In some cases prolonged bursts were recorded that lasted hundreds of ms. (C) Group plot of the slope conductances derived from all recordings from the PD (top), soma (middle) or PA (bottom). The x-axis gives the transmembrane potential to which the patch was stepped (starting from a holding potential −40 mV hyperpolarized from rest) and the y-axis gives the average amplitude of all single openings >2 ms in duration evoked by the step. Each blue dot is data from a single patch and the red line is the regression fit (with 95% confidence intervals) to the dots. The extrapolated slope conductance and reversal potentials are provided in the insets. (D) An example PD patch recording in which prolonged burst events were sufficiently frequent so as to allow for an investigation of the current throughout a voltage ramp. Channel openings began at an approximate transmembrane potential of −45 mV and decreased in amplitude as the driving force collapsed. The extrapolated slope conductance and reversal potential are given in the inset. The voltage ramp protocol is given in the bottom schematic and involved holding the patch −40 mV below rest and sweeping the voltage to 80 mV above rest. The resting transmembrane potential, obtained after break in, is given by the dotted gray line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Characteristics of Na+ channel gating in mPFC neurons. (A) Schematic of the recording arrangement. Cell-attached patch-clamp recordings were made from the soma, the proximal dendrite (PD) or the proximal axon (PA), within 15 um of the soma. (B) Example traces from a cell-attached recording of Na+ channel openings in the PD evoked by a +60 mV voltage step from a holding potential −20 mV below the presumed resting membrane potential of −80 mV (illustrated in the bottom schematic). Openings varied widely in duration. In some cases prolonged bursts were recorded that lasted hundreds of ms. (C) Group plot of the slope conductances derived from all recordings from the PD (top), soma (middle) or PA (bottom). The x-axis gives the transmembrane potential to which the patch was stepped (starting from a holding potential −40 mV hyperpolarized from rest) and the y-axis gives the average amplitude of all single openings >2 ms in duration evoked by the step. Each blue dot is data from a single patch and the red line is the regression fit (with 95% confidence intervals) to the dots. The extrapolated slope conductance and reversal potentials are provided in the insets. (D) An example PD patch recording in which prolonged burst events were sufficiently frequent so as to allow for an investigation of the current throughout a voltage ramp. Channel openings began at an approximate transmembrane potential of −45 mV and decreased in amplitude as the driving force collapsed. The extrapolated slope conductance and reversal potential are given in the inset. The voltage ramp protocol is given in the bottom schematic and involved holding the patch −40 mV below rest and sweeping the voltage to 80 mV above rest. The resting transmembrane potential, obtained after break in, is given by the dotted gray line.
Mentions: Layer V pyramidal cells were visualized in brain slices using infrared differential interference contrast optics (Axioskop Zeiss). Recordings were made from cell bodies, proximal apical dendrites (PD, 5–10 µm from soma) and proximal axons (PA, axon initial segment, 3–15 µm from soma) (Figure 1A). Pipettes were brought next to the neuron and very weak positive pressure was used to clean the surface before seal formation. Single channel recordings were made in cell-attached configuration. Patch pipettes were made from thick wall borosilicate glass capillaries with an outer diameter of 1.5 mm. The internal surface of the glass capillaries was treated with Sigmacote and allowed to dry at room temperature at least 3 days before being used for manufacturing patch pipettes. This treatment significantly reduced capacitance and improved the quality of the seal, which approached values >40 GΩ. To reduce the number of single channels in a patch we used pipettes with resistances of 15–25 MΩ when filled with patch solution. The pipette solution for recording Na+ channels contained the following (in mM): 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 0.1 CdCl2, 0.02 CNQX, 0.05 AP5, 10 D-glucose, 5 tetraethylammonium chloride, 1 4-AP and 10 HEPES with a pH of 7.3. The pipette solution for recording delayed rectifier K+ channels contained the following (in mM); 150 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, 10 D-glucose with a pH of 7.4.

Bottom Line: While past studies have tested the effects of dopamine on I(Nap), the results have been contradictory largely because of difficulties in measuring I(Nap) using somatic whole-cell recordings.As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region.By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of I(Nap), we demonstrate conclusively that D1/D5 receptor activation can increase the I(Nap) generated proximally, however questions still remain as to how D1/D5 receptor modulates Na(+) currents in the more distal initial segment where most of the I Nap is normally generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Brain Research Centre, University of British Columbia Vancouver, BC, Canada.

ABSTRACT
The persistent Na(+) current (I(Nap)) is believed to be an important target of dopamine modulation in prefrontal cortex (PFC) neurons. While past studies have tested the effects of dopamine on I(Nap), the results have been contradictory largely because of difficulties in measuring I(Nap) using somatic whole-cell recordings. To circumvent these confounds we used the cell-attached patch-clamp technique to record single Na(+) channels from the soma, proximal dendrite (PD) or proximal axon (PA) of intact prefrontal layer V pyramidal neurons. Under baseline conditions, numerous well resolved Na(+) channel openings were recorded that exhibited an extrapolated reversal potential of 73 mV, a slope conductance of 14-19 pS and were blocked by tetrodotoxin (TTX). While similar in most respects, the propensity to exhibit prolonged bursts lasting >40 ms was many fold greater in the axon than the soma or dendrite. Bath application of the D1/D5 receptor agonist SKF81297 shifted the ensemble current activation curve leftward and increased the number of late events recorded from the PD but not the soma or PA. However, the greatest effect was on prolonged bursting where the D1/D5 receptor agonist increased their occurrence 3 fold in the PD and nearly 7 fold in the soma, but not at all in the PA. As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region. Therefore, D1/D5 receptor modulation appears to be targeted mainly to Na(+) channels in the PD/soma and not the PA. By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of I(Nap), we demonstrate conclusively that D1/D5 receptor activation can increase the I(Nap) generated proximally, however questions still remain as to how D1/D5 receptor modulates Na(+) currents in the more distal initial segment where most of the I Nap is normally generated.

Show MeSH
Related in: MedlinePlus