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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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Related in: MedlinePlus

Testing the effects of SKF81297 on membrane potential based on an analysis of K+ channels. To get a surrogate measure of transmembrane voltage in cell-attached mode, the reversal potential for delayed rectifier K+ channel openings was used. For these experiments, the patch solutions were altered by removing K+ channel blockers and matching the [K+] in the patch pipette to the intracellular concentration, yielding a reversal potential near 0 mV. Voltage ramps started at the resting membrane potential and moved to +120 mV depolarized from rest (bottom schematic). The resting membrane potential for the presented cell was −80 mV. Multiple continuous openings were evoked. These openings started as outward but flipped to inward as the patch was depolarized. The reversal occurred at a transmembrane potential of −3.45 mV (top). Following the bath application of SKF81297 (3–5 µM), the reversal occurred at a transmembrane potential of −3.43 mV (bottom). Black and red lines are single sweeps. Sweeps with channel openings across a wide range of voltages were chosen. The background current was subtracted.
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Figure 4: Testing the effects of SKF81297 on membrane potential based on an analysis of K+ channels. To get a surrogate measure of transmembrane voltage in cell-attached mode, the reversal potential for delayed rectifier K+ channel openings was used. For these experiments, the patch solutions were altered by removing K+ channel blockers and matching the [K+] in the patch pipette to the intracellular concentration, yielding a reversal potential near 0 mV. Voltage ramps started at the resting membrane potential and moved to +120 mV depolarized from rest (bottom schematic). The resting membrane potential for the presented cell was −80 mV. Multiple continuous openings were evoked. These openings started as outward but flipped to inward as the patch was depolarized. The reversal occurred at a transmembrane potential of −3.45 mV (top). Following the bath application of SKF81297 (3–5 µM), the reversal occurred at a transmembrane potential of −3.43 mV (bottom). Black and red lines are single sweeps. Sweeps with channel openings across a wide range of voltages were chosen. The background current was subtracted.

Mentions: Prior to analyzing the effects of the D1/D5 receptor agonist SKF81297 on Na+ channel gating, it was important to determine whether the drug affected the membrane potential, since a change in voltage would alter all voltage-dependent measurements. To test this the K+ reversal potential was analyzed under baseline conditions and following the administration of SKF81297 (3–5 µM) in the bath. The delayed rectifier K+ current was chosen because it is very prominent in cell-attached recordings from mPFC neurons in the absence of TEA. To measure changes in K+ reversal potential, we recorded the delayed rectifier K+ channel using a patch solution with a potassium concentration of 150 mM. This was close to the internal potassium concentration, thereby bringing the K+ reversal potential in the patch close to 0. We used the following ramping voltage protocol: from the resting membrane potential, the voltage was slowly increased to 120 mV more positive than the resting membrane potential at a rate of 0.2 mV/ms. By delivering such ramping protocols it allowed us directly record the reversal potential of the current with an accuracy of ±0.5 mV. In 5 patches tested, the K+ reversal potential changed by less than 1 mV (range −0.8 mV + 0.6 mV) following D1/D5 receptor agonist administration (Figure 4). This indicated that any impact of SKF81297 on membrane potential was negligible and should not contaminate our analysis of its effects on INap.


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)

Testing the effects of SKF81297 on membrane potential based on an analysis of K+ channels. To get a surrogate measure of transmembrane voltage in cell-attached mode, the reversal potential for delayed rectifier K+ channel openings was used. For these experiments, the patch solutions were altered by removing K+ channel blockers and matching the [K+] in the patch pipette to the intracellular concentration, yielding a reversal potential near 0 mV. Voltage ramps started at the resting membrane potential and moved to +120 mV depolarized from rest (bottom schematic). The resting membrane potential for the presented cell was −80 mV. Multiple continuous openings were evoked. These openings started as outward but flipped to inward as the patch was depolarized. The reversal occurred at a transmembrane potential of −3.45 mV (top). Following the bath application of SKF81297 (3–5 µM), the reversal occurred at a transmembrane potential of −3.43 mV (bottom). Black and red lines are single sweeps. Sweeps with channel openings across a wide range of voltages were chosen. The background current was subtracted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325928&req=5

Figure 4: Testing the effects of SKF81297 on membrane potential based on an analysis of K+ channels. To get a surrogate measure of transmembrane voltage in cell-attached mode, the reversal potential for delayed rectifier K+ channel openings was used. For these experiments, the patch solutions were altered by removing K+ channel blockers and matching the [K+] in the patch pipette to the intracellular concentration, yielding a reversal potential near 0 mV. Voltage ramps started at the resting membrane potential and moved to +120 mV depolarized from rest (bottom schematic). The resting membrane potential for the presented cell was −80 mV. Multiple continuous openings were evoked. These openings started as outward but flipped to inward as the patch was depolarized. The reversal occurred at a transmembrane potential of −3.45 mV (top). Following the bath application of SKF81297 (3–5 µM), the reversal occurred at a transmembrane potential of −3.43 mV (bottom). Black and red lines are single sweeps. Sweeps with channel openings across a wide range of voltages were chosen. The background current was subtracted.
Mentions: Prior to analyzing the effects of the D1/D5 receptor agonist SKF81297 on Na+ channel gating, it was important to determine whether the drug affected the membrane potential, since a change in voltage would alter all voltage-dependent measurements. To test this the K+ reversal potential was analyzed under baseline conditions and following the administration of SKF81297 (3–5 µM) in the bath. The delayed rectifier K+ current was chosen because it is very prominent in cell-attached recordings from mPFC neurons in the absence of TEA. To measure changes in K+ reversal potential, we recorded the delayed rectifier K+ channel using a patch solution with a potassium concentration of 150 mM. This was close to the internal potassium concentration, thereby bringing the K+ reversal potential in the patch close to 0. We used the following ramping voltage protocol: from the resting membrane potential, the voltage was slowly increased to 120 mV more positive than the resting membrane potential at a rate of 0.2 mV/ms. By delivering such ramping protocols it allowed us directly record the reversal potential of the current with an accuracy of ±0.5 mV. In 5 patches tested, the K+ reversal potential changed by less than 1 mV (range −0.8 mV + 0.6 mV) following D1/D5 receptor agonist administration (Figure 4). This indicated that any impact of SKF81297 on membrane potential was negligible and should not contaminate our analysis of its effects on INap.

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