Dopamine D2-Like Receptors Modulate Intrinsic Properties and Synaptic Transmission of Parvalbumin Interneurons in the Mouse Primary Motor Cortex

Abstract

Dopamine (DA) plays a crucial role in the control of motor and higher cognitive functions such as learning, working memory, and decision making. The primary motor cortex (M1), which is essential for motor control and the acquisition of motor skills, receives dopaminergic inputs in its superficial and deep layers from the midbrain. However, the precise action of DA and DA receptor subtypes on the cortical microcircuits of M1 remains poorly understood. The aim of this work was to investigate in mice how DA, through the activation of D2-like receptors (D2Rs), modulates the cellular and synaptic activity of M1 parvalbumin-expressing interneurons (PVINs) which are crucial to regulate the spike output of pyramidal neurons (PNs). By combining immunofluorescence, ex vivo electrophysiology, pharmacology and optogenetics approaches, we show that D2R activation increases neuronal excitability of PVINs and GABAergic synaptic transmission between PVINs and PNs in Layer V of M1. Our data reveal how cortical DA modulates M1 microcircuitry, which could be important in the acquisition of motor skills.

Keywords: D2 receptors; electrophysiology; neuromodulation; parvalbumin interneuron; primary motor cortex.

Figure 1.

Distribution of D2R-expressing neurons in M1 in Drd2-Cre:Ribotag mice. A, Coronal section from Drd2-Cre:Ribotag mice stained with hemagglutinin (HA) showing the distribution of D2R-expressing neurons in the different layers of M1. Scale bars: 500 μm (left) and 50 μm (right). B, Histogram showing the distribution of HA-labeled neurons in Layer I, Layers II–III, and Layers V–VI of the M1 (17 hemispheres analyzed, 5 mice). The distribution is expressed as a percentage of HA-positive neurons in all layers. The number of HA-positive cells counted is indicated between parentheses. C, HA (cyan) and calbindin-D28k (CB), Calretinin (CR), parvalbumin (PV), neuropeptide Y (NPY), and nNOS (orange) immunofluorescence in M1 Layers V–VI of Drd2-Cre:Ribotag mice. Magenta arrowheads indicate HA/markers-positive neurons. Scale bars: 40 μm. D, Histograms showing the co-expression as a percentage of HA-positive cells in M1 Layers V–VI of Drd2-Cre:Ribotag mice (blue, left). The total numbers of HA- and marker-positive cells counted are indicated between parentheses. DS: dorsal striatum; cc: corpus callosum; Acb: nucleus accumbens; aca: anterior commissure.

Figure 2.

Electrophysiological characterization of D2R-expressing neurons in motor cortex M1 in Drd2-Cre:Ai9T mice. A, Firing behavior of the three types of D2R-expressing neurons in Layer V of M1. A depolarizing current injection (200 pA, 1 s) evoked a high-frequency spike firing pattern in FS (left) and a lower frequency of discharge in RSNP (middle) and PNs (right). Next to each trace, an expanded view of single spikes and AHP is presented for the three groups of neurons. B, Histogram showing the percentage of each type of D2R-expressing neurons in Layer V of M1 (n = 21). C, Summary of resting membrane potential (Vrest.), maximal firing frequency (Max. freq.), rheobase (Rh), and input resistance (Rin) in the three cell types.

Figure 3.

Quinpirole increases the excitability of M1 PVINs. A, left, Schematic of the experiment. PVINs were identified as tdTomato (tdTom in inset) positive neurons in slices from PV-Cre:Ai9T mouse brain. Representative voltage responses to +50-pA current injection in a PVIN in control bath solution (green, middle) and after 10 min of perfusion of the D2R agonist quinpirole (Quinp, 2 μm, red, right). B, Quinpirole enhanced the firing frequency (Firing freq.) of PVINs and significantly shifted the input-output curve to the left (p < 0.0001, n = 10, F_(1,96) = 42.64, two-way ANOVA). C, Quinpirole does not increase the firing frequency of PVINs in presence of sulpiride (p = 0.7645, n = 13, F_(1,144) = 0.09,011, two-way ANOVA). Each symbol represents mean ± SEM. D, Summary of the quinpirole effect on resting membrane potential (Vrest.), maximal firing frequency (Max. freq.), rheobase (Rh), and input resistance (Rin; WSR test). The thick bar and the color block represent the mean and the SEM, respectively. GABA_A, NMDA, and AMPA/kainate receptors were blocked throughout all of the recordings with PTX (50 μm), D-AP5 (50 μm), and DNQX (10 μm), respectively.*p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant.

Figure 4.

Effect of quinpirole on the electrical activity and sIPSCs (sIPSCs and mIPSCs) of PNs. A, Schematic of the experiment. PNs were identified by their morphology, the absence of tdTomato in their soma as well as their intrinsic properties when possible. Example of voltage responses to +50-pA current injection recorded in a representative PN in control (left, blue) and in quinpirole (right, red). B, Quinpirole did not change the firing frequency of PNs (p = 0.6453, n = 7, F_(1,72) = 0.21, two-way ANOVA), nor the resting potential or input resistance (p = 1.000 and p = 0.8982, respectively; n = 7, WSR test). C, Representative traces of sIPSCs recorded from a M1 PN (left) in control conditions (top trace, blue) and in the presence of quinpirole (bottom trace, red). D, Cumulative distribution (left) and mean (right) of sIPSC instantaneous frequency in the control (blue) and in the presence of quinpirole (red). No differences were observed (p = 0.9987, K-S test and p > 0.9999, WSR test). E, Cumulative distribution (left) and mean (middle) of sIPSC amplitude and value of τ (right). Note that cumulative distribution of the amplitude and decay time differed significantly between control and quinpirole conditions (p < 0.0001, K-S, and p = 0.0059, WSR test, n = 10). F–H, Same representations as in C–E for mIPSCs. Note that similarly to sIPSCs, only the cumulative distribution (p < 0.05, K-S test) and mean of mIPSC amplitude differed significantly between control and quinpirole conditions (p = 0.0098; n = 10, WSR test). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant.

Figure 5.

Quinpirole increases GABAergic synaptic transmission at the PVIN-PN synapse. A, Schematic of the experiment. An AAV.DIO.ChR2.EYFP virus was injected in M1 two weeks before ex vivo recordings. Representative slice showing the expression of ChR2-EYFP in M1. PVINs (tdTom-positive) were patched to verify our ability to manipulate their activity. B, Light reliably induced action potentials in a PVIN (left). Each flash of light (blue line) evoked one or two action potentials in PVINs as presented in an example (left) of an intracellularly recorded PVIN and in the raster plot of spiking in different trials (right). Each blue tick represents a flash of light (473 nm, 1 ms) and each green tick represents a spike. C, Schematic of the recording configuration from a postsynaptic PN during photoactivation of PVINs (left). Soma of PNs had a triangular shape, were tdTom-negative and PV-negative (middle). Representative firing pattern of recorded PNs to a 150-pA, 500-ms current step (blue, right). D, Sample traces and quantification of light-evoked IPSCs recorded in the same PN before (blue, control trace) and after 10 min of quinpirole perfusion (red). Mean of the amplitude of the evoked response (p = 0.0371, n = 10, WSR). Mean and SEM are represented. E, Sample traces and quantification of responses to repetitive photostimulation (10 Hz) recorded before (blue) and after bath application of quinpirole (red). Photoactivation of PVINs produced large initial IPSCs that depress rapidly. In the graph, the IPSC amplitudes were normalized to that of the first IPSC in the control condition for each neuron recorded (p < 0.0001, n = 10, F_(1,180) = 19.36). F, Short-term synaptic dynamics of the eIPSCs in PNs induced by the photoactivation of PVINs were not changed in presence of quinpirole. IPSC amplitudes were normalized to the first IPSC of the train in each condition (p = 0.1563, n = 10, F_(1,180) = 0.4749). *p < 0.05, ***p < 0.001; n.s., not significant.