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Comparative Study
. 2012 Apr 4;32(14):4959-71.
doi: 10.1523/JNEUROSCI.5835-11.2012.

Synaptic activity unmasks dopamine D2 receptor modulation of a specific class of layer V pyramidal neurons in prefrontal cortex

Steven Gee  1 , Ian Ellwood, Tosha Patel, Francisco Luongo, Karl Deisseroth, Vikaas S Sohal
Affiliations
Comparative Study

Synaptic activity unmasks dopamine D2 receptor modulation of a specific class of layer V pyramidal neurons in prefrontal cortex

Steven Gee et al. J Neurosci. .

Abstract

Dopamine D2 receptors (D2Rs) play a major role in the function of the prefrontal cortex (PFC), and may contribute to prefrontal dysfunction in conditions such as schizophrenia. Here we report that in mouse PFC, D2Rs are selectively expressed by a subtype of layer V pyramidal neurons that have thick apical tufts, prominent h-current, and subcortical projections. Within this subpopulation, the D2R agonist quinpirole elicits a novel afterdepolarization that generates voltage fluctuations and spiking for hundreds of milliseconds. Surprisingly, this afterdepolarization is masked in quiescent brain slices, but is readily unmasked by physiologic levels of synaptic input which activate NMDA receptors, possibly explaining why this phenomenon has not been reported previously. Notably, we could still elicit this afterdepolarization for some time after the cessation of synaptic stimulation. In addition to NMDA receptors, the quinpirole-induced afterdepolarization also depended on L-type Ca(2+) channels and was blocked by the selective L-type antagonist nimodipine. To confirm that D2Rs can elicit this afterdepolarization by enhancing Ca(2+) (and Ca(2+)-dependent) currents, we measured whole-cell Ca(2+) potentials that occur after blocking Na(+) and K(+) channels, and found quinpirole enhanced these potentials, while the selective D2R antagonist sulpiride had the opposite effect. Thus, D2Rs can elicit a Ca(2+)-channel-dependent afterdepolarization that powerfully modulates activity in specific prefrontal neurons. Through this mechanism, D2Rs might enhance outputs to subcortical structures, contribute to reward-related persistent firing, or increase the level of noise in prefrontal circuits.

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Figures

Figure 1.
Figure 1.
H-current distinguishes two populations of layer V pyramidal neurons that differ in their projection targets. A, High-power confocal image of layer V of mPFC showing the distribution of fluorescently labeled retrogradely transported microspheres within individual neurons. Microspheres were injected into MD thalamus (red) or the contralateral PFC (green). Scale bar, 10 μm. B, Sample recordings from corticothalamic (CT) or corticocortical (CC) pyramidal neurons identified using retogradely transported microspheres showing responses to hyperpolarizing or depolarizing current injection. Note the voltage sag and rebound afterdepolarization in response to hyperpolarizing current injection that is visible in the CT neuron but not the CC neuron (arrows). C, The amount of h-current, measured as the sum of the voltage sag and rebound afterdepolarization in response to hyperpolarizing current pulses, in CT (n = 18) and CC neurons (n = 8). Thick horizontal bars indicate the means of each distribution, and the dotted line indicates a threshold that unambiguously separates the two nonoverlapping distributions. D, Input resistances (Rin) and resting membrane potentials (Vrest) for CT and CC neurons. ***p < 0.001.
Figure 2.
Figure 2.
Type A and B pyramidal neurons have different morphologies. A, Confocal images of representative neurons in which the amount of h-current falls either above (“type A,” left) or below (“type B,” right) the threshold in Fig. 1 C. B, Type A and B neurons differ in the widths of the shafts of their apical dendrites (left) and in the number of primary branches of their apical dendrites (right) (n = 4 neurons in each group).
Figure 3.
Figure 3.
D2Rs are selectively expressed in type A pyramidal neurons which can be distinguished using the h-current. A, Low-power confocal images of infralimbic cortex showing the pattern of fluorescence in Drd1-Cre (D1-Cre) and Drd2-Cre (D2-Cre) transgenic mice injected with virus to drive Cre-dependent expression of ChR2-YFP. The corpus callosum and midline lie below and above both images, respectively. ml, midline. Scale bar, 0.1 mm (both images are to the same scale). B, The amount of h-current (measured as above) in identified corticothalamic (CT, n = 18), corticocortical (CC, n = 8), D2R-expressing (D2, n = 14), or D1R-expressing (D1, n = 10) pyramidal neurons in layer V of mPFC. The dotted line indicates the threshold that separates the distributions of h-current from CT and CC neurons. *p < 0.05, **p < 0.01.
Figure 4.
Figure 4.
Synaptic stimulation unmasks a novel D2R-mediated afterdepolarization in specific layer V pyramidal neurons. A, Experimental design. We recorded from ChR2-negative layer V neurons while stimulating ChR2-positive axons from the contralateral mPFC with trains of light flashes (470 nm, 2.5 ms, ∼2 mW). B, Responses of a type A layer V pyramidal neuron to current injection before (left) and immediately following (middle and right) optogenetic stimulation of synaptic inputs. Blue bars indicate the times of light flashes. C, Before synaptic stimulation, no quinpirole-induced afterdepolarization is observed; however, the same current injection elicits a marked afterdepolarization (along with spike height rundown) following weak synaptic stimulation. D, The quinpirole-induced afterdepolarization is eliminated by the addition of AP5. E, Lower doses of quinpirole (5 μm) also induce an afterdepolarization following synaptic stimulation, which can be blocked by nimodipine (1 μm). F, Recording from a type A neuron showing a prolonged quinpirole-induced afterdepolarization following synaptic stimulation. G, Average time constants for the membrane potential to return to baseline following a depolarizing current pulse (300–400 pA, 250–500 ms) delivered immediately following the pattern of synaptic stimulation shown above. Data are shown for control conditions (black; n = 12), quinpirole (purple; 5 μm, n = 7; 20 μm, n = 6), quinpirole in the absence of synaptic stimulation (hollow purple bar; 5 μm, n = 6), nimodipine (gray; 1 μm, n = 3), sulpiride (green; 5 μm, n = 4), or AP5 (green; 50 μm, n = 3). *p < 0.05, **p < 0.01.
Figure 5.
Figure 5.
NMDA can unmask the quinpirole-induced afterdepolarization in type A neurons. A, Responses of a type A neuron to depolarizing current pulses in various pharmacologic conditions showing that bath application of quinpirole and NMDA, but not NMDA alone, induces an afterdepolarization (arrow) that is reversed by sulpiride. B, Top, Summary data showing the effect of quinpirole, NMDA, and sulpiride on the time constant for the membrane potential to return to baseline following depolarizing current pulses (350 pA, 250 ms) in type A neurons (n = 4 for each condition): control (black), NMDA (blue; 4 μm), (±)quinpirole (purple; 20 μm) + NMDA), or sulpiride (green; 5 μm) + quinpirole + NMDA). B, Bottom, Quinpirole does not elicit a similar afterdepolarization in type B neurons. The time constant for the membrane potential to return to baseline following depolarization current pulses (350 pA, 250 ms) in type B neurons is shown for various conditions: control (black; n = 3), (±)quinpirole (20 μm) following optogenetic synaptic stimulation (purple; n = 3), (±)quinpirole (20 μm) plus NMDA (4 μm) (purple; n = 4). *p < 0.05, **p < 0.01.
Figure 6.
Figure 6.
Quinpirole also induces an afterdepolarization during perforated-patch recordings from type A neurons. A, Recordings from a type A neuron in perforated-patch configuration (top three recordings) showing the quinpirole-induced afterdepolarization that occurs in the presence of NMDA, and is reversed by sulpiride. Bottom, shows a recording from the same neuron after breaking in and shifting to a whole-cell recording. B, Fluorescent dye in the recording pipette was excluded from the neuron while in the perforated-patch configuration (top), but entered the neuron after breaking in and shifting to a whole-cell configuration (bottom). C, Summary data showing that (±)quinpirole (20 μm) plus NMDA (6 μm) prolongs the time constant for the membrane potential to return to baseline following depolarizing current pulses (350 pA, 250 ms), and that this is reversed by the addition of sulpiride (5 μm) (n = 5). *p < 0.05, **p < 0.01.
Figure 7.
Figure 7.
Quinpirole reversibly prolongs calcium-dependent plateau potentials. A, A short current pulse elicits a brief Ca2+ spike that is followed by a prolonged plateau potential in a type A neurons after application of TTX and TEA. Each experiment recorded plateau potentials in control conditions, then while applying (±)quinpirole (20 μm), and finally while applying quinpirole + sulpiride (5 μm). B, We quantified the size of plateau potentials by measuring the area under the voltage trace. The average size of the plateau potentials are shown as a function of time (n = 4 cells in each condition). For the magenta trace, t = 0 represents the beginning of quinpirole application, and quinpirole and sulpiride were both applied after t = 20 min. The dark blue trace represents recordings in control ACSF. Shaded regions represent ± 1 SEM. C, Summary data for the size of plateau potentials in each condition. Each bar represents data collected from 5 min before until 5 min after the end of drug application, or corresponding time points during recordings in control ACSF. We measured plateau potentials every 5 min during this period and used repeated-measures ANOVA and corrections for multiple-comparisons to assess statistical significance. *p < 0.05, ***p < 0.001.
Figure 8.
Figure 8.
The Ca2+ chelator BAPTA eliminates the quinpirole-induced afterdepolarization. A, Perforated patch recording from a type A pyramidal neuron in control conditions (top) and after application of quinpirole + NMDA elicits an afterdepolarization (middle). Bottom, In the same neuron, after breaking in and switching to a whole-cell recording configuration, the quinpirole-induced afterdepolarization is abolished. BAPTA (5 mm) is present in the pipette solution. B, Summary data showing time constants for the membrane potential to return to baseline following depolarizing current pulses (50–150 pA, 250 ms) under various conditions. “Control,” perforated patch recordings in control ACSF (black; n = 3); “qpl + NMDA, perf patch,” the same perforated patch recordings after applying quinpirole and NMDA (orange; n = 3); “qpl + NMDA, postbreak in w/BAPTA,” whole-cell recordings from the same cells that were initially recorded in perforated patch configuration (in quinpirole and NMDA) (purple; n = 3); “qpl + NMDA, whole-cell w/BAPTA,” recordings from cells that broke in and switched to whole-cell configuration during the application of quinpirole + NMDA (red; n = 5). **p < 0.01.
Figure 9.
Figure 9.
Blocking SK channels and applying quinpirole produces an afterdepolarization that requires L-type Ca2+ channels. A, Responses of a type A neuron to depolarizing current pulses in various pharmacologic conditions showing that bath application of quinpirole, bicuculline, and AP5 induces an afterdepolarization (middle) that is reversed by nimodipine (bottom). B, Responses of another type A neuron showing that application of quinpirole and apamin induces a similar afterdepolarization (middle). C, Summary data showing the effect of various conditions on the time constant for the membrane potential to return to baseline following depolarizing current pulses (350 pA, 250 ms) in type A neurons: control (black; n = 10), quinpirole + bicuculline + AP5 (red; n = 3), quinpirole + bicuculline + AP5 + nimodipine (gray; n = 3), apamin + quinpirole (magenta; n = 3), quinpirole + gabazine (orange; 10 μm, n = 4). **p < 0.01, ***p < 0.001.
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