New Results

Cell-Type-Specific Decrease of the Intrinsic Excitability of Motor Cortical Pyramidal Neurons in Parkinsonian Mice

Liqiang Chen, Yerim Kim, Hong-Yuan Chu
doi: https://doi.org/10.1101/2020.10.20.347732

Abstract

The hypokinetic motor symptoms of Parkinson’s disease (PD) are closely linked with a decreased motor cortical output as a consequence of elevated basal ganglia inhibition. However, whether and how the loss of dopamine alters the cellular properties of motor cortical neurons in PD remains undefined. We induced experimental parkinsonism in adult C57BL6 mice of both sexes by injecting neurotoxin, 6-hydroxydopamine, into the medial forebrain bundle. By using ex vivo patch-clamp recording and retrograde tracing approach, we found that the intrinsic excitability of pyramidal tract neurons (PTNs) in the motor cortical layer 5b was greatly decreased following the degeneration of midbrain dopaminergic neurons; but the intratelencephalic neurons (ITNs) were not affected. The cell-type-specific intrinsic adaptations were associated with a significant broadening of the action potentials in PTNs but not in ITNs. Moreover, the loss of midbrain dopaminergic neurons impaired the capability of M1 PTNs to sustain high-frequency firing, which could underlie their abnormal pattern of activity in the parkinsonian state. We also showed that the decreased excitability and broadened action potentials were largely caused by a disrupted function of the large conductance, Ca2+-activated K+ channels. The restoration of dopaminergic neuromodulation failed to rescue the impaired intrinsic excitability of M1 PTNs in parkinsonian mice. Altogether, our data show cell-type-specific decreases of the excitability of M1 pyramidal neurons following the loss of midbrain dopaminergic neurons. Thus, intrinsic adaptations in the motor cortex, together with pathological basal ganglia inhibition, underlie the decreased motor cortical output in parkinsonian state and exacerbate parkinsonian motor deficits.

Significance statement The degeneration of midbrain dopaminergic neurons in Parkinson’s disease remodels the connectivity and function of cortico–basal ganglia–thalamocortical network. However, whether and how the loss of dopamine and aberrant basal ganglia activity alter motor cortical circuitry remain undefined. We found that pyramidal neurons in the layer 5b of the primary motor cortex (M1) exhibit distinct adaptations in response to the loss of midbrain dopaminergic neurons, depending on their long-range projections. Besides the decreased thalamocortical synaptic excitation as proposed by the classical model of Parkinson’s pathophysiology, these results, for the first time, show novel cellular and molecular mechanisms underlying the abnormal motor cortical output in parkinsonian state.

Introduction

The degeneration of dopamine (DA) neurons in the substantia nigra (SN) alters the connection and computation of cortico–basal ganglia–thalamocortical network, which underlies the devastating motor symptoms in Parkinson’s disease (PD), including akinesia, bradykinesia, and rigidity (Albin et al., 1989; Galvan and Wichmann, 2008; McGregor and Nelson, 2019). Specially, the loss of SN DA neurons increases and decreases the activities of indirect and direct pathways, respectively, which disrupts the balanced activity between striatal direct and indirect pathways, leading to the hypokinetic symptoms in PD (Albin et al., 1989; DeLong, 1990). The pathway-specific alterations in the striatum following the loss of SN DA neurons induce numerous cellular and synaptic changes in the basal ganglia nuclei and extended brain regions, which further drive the abnormal neural activity throughout the cortico-basal ganglia-thalamocortical network in experimental parkinsonism (Gittis et al., 2011; Kita and Kita, 2011; Fieblinger et al., 2014; Chu et al., 2015, 2017; Mathai et al., 2015; Shen et al., 2015; Parker et al., 2016, 2018; Sharott et al., 2017; McIver et al., 2019; Willard et al., 2019).

The primary motor cortex (M1) plays essential and complex roles in motor control and motor learning and is a key node in the cortico–basal ganglia–thalamocortical network (Shepherd, 2013; Ebbesen and Brecht, 2017; Ebbesen et al., 2018). M1 is a laminar structure and contains a heterogeneous group of neurons that differ in gene expression, morphology, connectivity, and electrophysiological properties (Sheets et al., 2011; Oswald et al., 2013; Shepherd, 2013; Suter et al., 2013; Economo et al., 2018). These neurons can be classified into the intratelencephalic neurons (ITNs) distributed across the layers (L) 2-6 and the pyramidal tract neurons (PTNs) that mainly locate within the L5b (Oswald et al., 2013; Shepherd, 2013). Both PTNs and ITNs project to the striatum and receive basal ganglia feedbacks through the transition of the motor thalamus (Bodor et al., 2008; Kita and Kita, 2011; Kress et al., 2013; Lee et al., 2020).

In physiological state, M1 network dynamics plays an essential role in the execution and coordination of complex movements and the acquisition of motor skills (Guo et al., 2015a; Kawai et al., 2015; Sreenivasan et al., 2016; Barthas and Kwan, 2017; Ebbesen et al., 2017; Wang et al., 2017; Economo et al., 2018; Sauerbrei et al., 2020). In parkinsonian state, M1 exhibits an aberrant oscillation and bursting pattern of activity at both individual neuron and population levels (Goldberg et al., 2002; Mallet et al., 2008; Pasquereau and Turner, 2011; Shimamoto et al., 2013; Hemptinne et al., 2015; Pasquereau et al., 2016), which disrupt its normal function in motor control and motor learning.

The abnormal neuronal activity in the M1 has been hypothesized to a consequence of pathological basal ganglia output (Hosp et al., 2011, 2015; Pasquereau and Turner, 2011; Guo et al., 2015b; Pasquereau et al., 2016), but the cellular adaptations could also play a key role in such abnormal activities. Moreover, compelling evidence suggests that PTNs and ITNs may adapt differently to the degeneration of midbrain DA neurons (Pasquereau and Turner, 2011; Pasquereau et al., 2016).Thus, we hypothesized that loss of SN DA neurons alters the intrinsic properties of M1 pyramidal neurons in a cell-type-specific manner, and that the intrinsic adaptations contribute to the abnormal neuronal activity of M1 in parkinsonian state. We addressed these hypotheses using electrophysiology and retrograde tracing approach in mice with 6-hydroxydopmaine (6-OHDA) lesion, an established model of parkinsonism.

Material and Methods

Animals

Mice were housed up to four animals per cage under a 12-h light/12-h dark cycle with access to food and water ad libitum in accordance with Van Andel Institute IACUC and NIH guidelines for care and use of animals.

Stereotaxic surgery

Mice were deeply anesthetized under 2% isoflurane, were placed in a stereotaxic frame (Kopf), and were supported by a thermostatic heating pad. To induce degeneration of SN DA neurons, 6-OHDA (3-4 mg/mL, 1.0 μL) was injected unilaterally into the medial forebrain bundle (MFB, from bregma (in mm): AP, −0.7, ML, +1.2, DV, −4.7) over 10 min using a 10-μl syringe (Hamilton) and a motorized microinjector (Stoelting, Wood Dale, IL, USA) (Chu et al., 2017). Controls were injected in the same location with vehicle. Desipramine (25 mg/kg) and pargyline (50 mg/kg) were subcutaneously injected 30-40 min prior to 6-OHDA injection, to enhance the sensitivity and toxicity of 6-OHDA (Chu et al., 2015, 2017). To label and identify PTNs and ITNs in the M1, animals also received red or green Retrobeads (0.3 μL, Lumafluor, Inc) injected into the ipsilateral pontine nuclei (from bregma (in mm): AP, −5.0; ML, +0.6; DV, −5.0; 0.3 μl per injection) or the contralateral dorsolateral striatum (from bregma (in mm): AP, +0.4; ML, −2.0; DV, −2.8). The surgical wound was closed using sutures and mice were allowed to recover after surgery in a heated cage with access to food and water at the cage-floor level. Motor cortex slices containing the forelimb regions from 6-OHDA- or vehicle-injected mice were prepared 3-4 weeks after surgery for ex vivo electrophysiology (days post-surgery: control = 27 [22, 28] days, n = 19 mice; 6-OHDA = 24 [22, 27] days, n = 19 mice).

Slice preparation

Mice were deeply anesthetized with intraperitoneal avertin (250 mg/kg) and then were perfused transcardially with ice-cold, sucrose-based, artificial cerebrospinal fluid (aCSF) containing (in mM) 230 sucrose, 26 NaHCO3, 10 glucose, 10 MgSO4, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2. Next, coronal brain slices (250 μm) containing M1 forelimb regions were prepared in the same slicing solution using a vibratome (VT1200S; Leica Microsystems Inc., Buffalo Grove, IL, USA). Brain slices were kept in normal aCSF (in mM: 126 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 1 sodium pyruvate and 0.005 L-glutathione) equilibrated with 95% O2 and 5% CO2 for 30 min at 35 °C and then held at room temperature until electrophysiological recording.

Ex vivo electrophysiology recording

Brain slices were transferred into a recording chamber perfused at a rate of 4 mL/min with synthetic interstitial fluid (in mM: 126 NaCl, 26 NaHCO3, 10 glucose, 3 KCl, 1.6 CaCl2, 1.5 MgSO4, 1.25 NaH2PO4) equilibrated with 95% O2 and 5% CO2 at 35 °C via a feedback-controlled in-line heater (TC-324C, Warner Instruments). DNQX (20 μM), D-APV (50 μM), and SR-95531 (10 μM) were routinely added to block synaptic transmission mediated by ionotropic glutamatergic and GABAergic receptors. Neurons were visualized and recorded under gradient contrast SliceScope 6000 (Scientifica, UK) with infrared illumination using a CCD camera (SciCam Pro, Scientifica, UK) and motorized micromanipulators (Scientifica, UK). Individual neurons labeled with Retrobeads in the M1 L5b were identified using a 60X water immersion objective lens (Olympus, Japan) and targeted for whole-cell patch-clamp recording, using a MultiClamp 700B amplifier and a Digidata 1500B digitizer under the control of pClamp11 (Molecular Devices, San Jose, USA). Data were collected at a sampling rate of 20-50 KHz. Borosilicate glass pipettes for patch clamp recordings (4-7 MV) were pulled using a micropipette puller (P1000, Sutter Instruments). Pipette capacitance was compensated for before the formation of the whole-cell configuration. Series resistance (Rs) was regularly monitored and compensated for with the bridge balance circuit in current clamp mode. Liquid junction potential (about 11 mV) was corrected.

Retrobeads-labeled neurons in the L5b of M1 were recorded in whole-cell current-clamp mode to study their intrinsic properties. In these experiments, glass pipettes were filled with a potassium gluconate–based internal solution of (in mM) 140 K-gluconate, 3.8 NaCl, 1 MgCl2, 10 HEPES, 0.1 Na4-EGTA, 2 ATP-Mg, and 0.1 GTP-Na, pH 7.3, osmolarity 290 mOsm. The resting membrane potential (Vm) was recorded once the whole-cell configuration was obtained. The intrinsic properties and excitability of M1 pyramidal neuron subtypes were studied by injecting a family of current steps ranging from −80 pA to 720 pA in 40-pA increments and with a duration of 1 s. Current injections were from Vm. Input resistance was determined by measuring the steady-state voltage responses to a series of 1-s hyperpolarizing currents.

The rheobase, as an indicator of neuronal excitability, was defined as the current intensity to elicit an action potential (AP) during a 1-s current injection. AP waveforms at the rheobase were systematically analyzed and quantified. Specifically, the threshold of AP was determined as the voltage level at which dV/dt exceeded 20 mV/ms. AP amplitude was defined as the voltage difference between the threshold and the peak voltage. AP half-width was measured as the time difference at 50% of AP amplitude. Fast and medium afterhyperpolarizations (fAHPs and mAHPs) were measured as the negative voltage peaks relative to the threshold within 2-5 ms and 20-50 ms from AP threshold, respectively (Villalobos et al., 2004; Bean, 2007). To quantify spike trains, spike frequency adaptation was measured as the ratio between the last inter-spike-interval and the average of the first two inter-spike-intervals. The gain of the spike-current curve was defined as the slope of the instantaneous spike-current curve.

Immunohistochemistry

Striatal immunoreactivity of tyrosine hydroxylase (TH) was assessed to validate the degeneration of the nigrostriatal DA projections in vehicle- and 6-OHDA-injected mice. Briefly, brain tissues were first fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH7.4, at 4 °C for 12 hours before rinsing in phosphate-buffered saline (PBS, 0.05 M; pH 7.4). Tissues was re-sectioned at 70 μm using a vibratome (Leica VT1000S; Microsystems Inc.). Immunochemical detection of TH was conducted in PBS containing 0.2% Triton X-100 (Fisher Scientific) and 2% normal donkey serum (Sigma-Aldrich, St. Louis, MO). Brain sections were incubated in primary antibody (mouse anti-TH, 1:1000, cat#: MAB318, Sigma-Aldrich, St. Louis, MO, USA) for 48 h at 4 °C or overnight at room temperature (RT), washed in PBS, and then incubated in the secondary antibody (donkey anti-mouse Alexa Fluor 488, cat#: 715-545-150 or donkey anti-mouse Alexa Fluor 594, cat#715-585-150; 1: 500, Jackson ImmunoReseach) for 90 minutes at RT before washing with PBS. Brain sections were mounted on glass slides using VECTASHIELD antifade mounting medium (cat#: H-1000, Vector Laboratories, Burlingame, CA, USA) and were cover-slipped. TH immunoreactivity was imaged using an Olympus BX63F microscope equipped with an Olympus DP80 camera or a confocal laser scanning microscope (A1R; Nikon, Melville, USA). The intensity of TH immunofluorescence was quantified using ImageJ (NIH). All the mice with 6-OHDA injection showed > 80% TH immunoreactivity loss in the ipsilateral dorsal striatum versus contralateral dorsal striatum.

Experimental design and statistical analysis

Nineteen male and 19 female adult (4-5 months old) C57BL/6 mice were used in the study. Data were analyzed in Clampfit (Molecular Devices) and ImageJ (NIH). Statistics were done using Prism8 (GraphPad Software, San Diego, CA, USA). To minimize the assumption of the data normality, we used non-parametric, distribution-independent Mann-Whiney U (MWU) or Wilcoxon signed rank (WSR) tests for paired or non-paired data comparisons, respectively. The Holm-Bonferroni correction was applied for multiple comparisons. All tests were two-tailed, and an α-level of 0.05 was used to determine statistical significance. Results are reported as median and interquartile range. Boxplots illustrate the median (central line), interquartile range (box), and 10-90% range (whiskers) of data. Data are available upon request.

Results

Cell-type-specific decrease in the intrinsic excitability of M1 L5b pyramidal neurons following the loss of midbrain DA neurons

We compared the intrinsic excitability of projection-defined pyramidal neurons in the L5b of M1 from vehicle-injected controls (hereinafter, “controls”) and from mice with 6-OHDA lesion (hereinafter, “6-OHDA mice”). To distinguish M1 pyramidal neurons based on long-range projections, Retrobeads were stereotaxically injected into the ipsilateral pontine nuclei (Fig. 1A) and the contralateral striatum (Fig. 2A) to retrogradely label PTNs (Fig. 1B) and ITNs (Fig. 2B), respectively. The intrinsic electrophysiological properties of M1 PTNs and ITNs were assessed using whole-cell current-clamp recording in the presence of antagonists of ionotropic glutamatergic and GABAergic receptors, including DNQX (20 μM), D-APV (50 μM), and SR95531 (10 μM). All recordings were conducted between 3 and 4 weeks post-surgery, when the level of midbrain DA degeneration and functional adaptations in the brain have reached maximum and been stabilized (Vila et al., 2000; Viaro et al., 2011). The data are reported as median and interquartile range in the text and figures.

Figure 1.
Figure 1. Decreased intrinsic excitability of PTNs in the L5b of M1 following the loss of midbrain DA neurons. A and B)

Representative graphs showing the injection site of red Retrobeads in the pontine nuclei (A) and a retrogradely labeled PTN in L5b of M1 (B). C and D) Representative spike trains of PTNs evoked by somatic current injections from controls and 6-OHDA mice (C), and the frequency-current relationship of the PTNs from controls and 6-OHDA mice (D). E-G) Summarized results showing a reduced gain of spike-current curve (E), a decreased firing frequency in response to maximal current injections (i.e., 720 pA) (F), and an enhanced spike adaptations (G) in PTNs from 6-OHDA mice relative to those from controls. *, p < 0.05, MWU.

Figure 2.
Figure 2. Intact intrinsic excitability of ITNs in the M1 L5b following the loss of midbrain DA neurons. A and B)

Representative graphs showing the injection site of green Retrobeads in the dorsal striatum (A) and retrogradely labeled M1 L5b ITNs (B). C and D) Representative spikes trains evoked by somatic current injections into ITNs from control and 6-OHDA mice (C), and the frequency-current relationship of ITNs from controls and 6-OHDA mice (D). E-G) Boxplots showing an unaltered gain of spike-current curve (E), firing frequency in response to maximal current injections (i.e., 720 pA) (F), and spike adaptation (G) between ITNs from 6-OHDA mice and controls. ns, not significant, MWU.Cell-type-specific changes in the passive membrane properties of M1 L5b pyramidal neurons following the loss of midbrain DA neurons.

The intrinsic excitability of M1 pyramidal neuron subtypes was assessed by somatic injection of a range of depolarizing currents in controls and 6-OHDA mice (Figures 1C, D). We found that the gain of spike-current curve (control = 78.2 [67.4, 91.25] spikes/nA, n = 25 neurons/5 mice; 6-OHDA = 52.0 [44.7, 72.2] spikes/nA, n = 25 neurons/4 mice; p = 0.0015, MWU; Figures 1E) was significantly decreased in M1 PTNs from 6-OHDA mice relative to those from controls. The same reduction was also detected for the spike frequency (e.g. frequency at 720 pA current injection, control = 55 [47, 59] Hz, n = 25 neurons/5 mice; 6-OHDA = 38 [29, 53] Hz, n = 25 neurons/4 mice; p = 0.0016, MWU; Figure 1F). M1 PTNs could sustain persistent firing with minimal spike adaptation during repetitive firing in the physiological state (Figure 1C) (Oswald et al., 2013; Suter et al., 2013). However, following the loss of midbrain DA neurons, PTNs showed significantly enhanced spike adaptation. For instance, the spike adaptation of a 10- to 15-Hz spike train, control = 1.06 [0.97, 1.26], n = 25 neurons/5 mice; 6-OHDA = 1.38 [1.17, 1.60], n = 25 neurons/4 mice; p = 0.0001, MWU (Figures 1C, G). These results suggest a significant reduction in the intrinsic excitability of M1 PTNs following the loss of midbrain DA neurons.

In contrast, M1 ITNs showed comparable spike-current relationships between the controls and 6-OHDA mice. This was reflected by three measures, namely, a) the unaltered gain of spike-current curve (control = 78 [54, 93] spikes/nA, n = 38 neurons/6 mice; 6-OHDA = 77 [67, 88] spikes/nA, n= 35 neurons/6 mice; p = 0.94, MWU; Figure 2D, E); b) the spike frequency at maximal (720 pA) current injection (control = 53 [38, 64] Hz, n = 38 neurons/6 mice; 6-OHDA = 50 [40, 58] Hz, n = 35 neurons/6 mice; p = 0.4, MWU; Figure 2D, F); and c) spike adaptations (control = 1.36 [1.08, 1.67], n= 38 neurons/6 mice; 6-OHDA = 1.20 [1.02, 1.61], n = 35 neurons/6 mice; p = 0.24, MWU; Figure 2C, G). Altogether, our results thus suggest that the intrinsic excitability of PTNs, but not ITNs, was significantly decreased following the loss of midbrain DA neurons, that is, the M1 L5b pyramidal neurons exhibited cell-type-specific adaptations in their intrinsic excitability in the parkinsonian state.

In the absence of significant synaptic inputs, PTNs in 6-OHDA mice and controls had similar resting membrane potentials (Vm, control = −79.3 [−81.6, −77.9] mV, n = 25 neurons/5 mice; 6-OHDA = −79.7 [−81.5, −78.4] mV, n=25 neurons/4 mice; p = 0.85, MWU, Figure 3A). However, PTNs from 6-OHDA mice had an increased input resistance (Rm, control = 69.3 [60.1, 81.5] MV, n = 25 neurons/5 mice; 6-OHDA = 109.6 [73.0, 136.4] MV, n= 25 neurons/4 mice; p = 0.0028, MWU; Figures 3A, B). In contrast, ITNs showed a hyperpolarized Vm in 6-OHDA mice (control = −77.7 [−80.2, −75.0] mV, n=38 neurons/6 mice; 6-OHDA = −79.6 [−82.1, −77.2] mV, n = 35 neurons/6 mice; p = 0.012, MWU; Figure 3D). ITNs in 6-OHDA mice also had an unaltered Rm (control = 89.7 [60.4, 121.3] MV, n = 38 neurons/6 mice; 6-OHDA = 70.9 [54.7, 93] MV, n = 35 neurons/6 mice; p = 0.067, MWU; Figure 3E). Changes in Vm and Rm were associated with a significant increase in rheobase in ITNs (control= 80.8 [51.3, 125] pA, n = 36 neurons/6 mice; 6-OHDA = 125 [86, 191] pA, n = 35 neurons/6 mice; p = 0.002, MWU; Figure 3F). PTNs had unaltered rheobase following the loss of midbrain DA neurons (control = 82 [47, 163] pA, n= 25 neurons/5 mice; 6-OHDA = 79 [56, 120] pA, n = 25 neurons/4 mice; p = 0.645, MWU; Figure 3C). Thus, the loss of midbrain DA neurons induced distinct adaptations in passive membrane properties of PTNs and ITNs in M1.

Figure 3.
Figure 3. Distinct changes in the passive membrane properties of PTNs and ITNs in the M1 L5b following the loss of midbrain DA neurons.

A-C) Boxplots showing changes in the resting membrane potential (A), input resistance (B), and rheobase (C) of PTNs from 6-OHDA mice relative to those from controls. D-F) Boxplots showing changes in in the resting membrane potential (D), input resistance (E), and rheobase (F) of ITNs from 6-OHDA mice relative to those from controls. ns, not significant; *, p < 0.05, MWU.

Cell-type-specific changes in action potential waveforms of M1 L5b pyramidal neurons following the loss of midbrain DA neurons

The shape of the action potential is determined by the interaction of numerous voltage-gated ion channels and has profound effects on firing rate and pattern of neurons (Bean, 2007). We therefore analyzed the morphology of AP waveforms of PTNs (Figure 4A) and ITNs (Figures 4G) from controls and 6-OHDA mice. Following the loss of the midbrain DA neurons, M1 PTNs showed a striking AP broadening (AP half-width, controls = 0.69 [0.62, 0.75] ms, n = 25 neurons/5 mice; 6-OHDA = 0.85 [0.68, 1.23] ms, n = 25 neurons/4 mice; p = 0.005, MWU; Figure 4B). The broadened AP of PTNs was associated with a significantly prolonged AP rise time (controls = 0.22 [0.19, 0.24] ms, n = 25 neurons/5 mice; 6-OHDA = 0.25 [0.22, 0.32] ms, n = 25 neurons/4 mice; p < 0.002, MWU; Figures 4C) and also a prolonged decay time (controls = 0.60 [0.53, 0.68] ms, n = 25 neurons/5 mice, 6-OHDA = 0.80 [0.58, 1.3] ms, 25 neurons/4 mice; p = 0.0035, MWU; Figure 4D). Further, the maximal rates of AP depolarization and repolarization of M1 PTNs also greatly decreased. The maximal AP rise rate in controls = 358 [306, 437] mV/ms, n = 25 neurons/5 mice, while the maximal AP rise rate in 6-OHDA mice = 295 [245, 339] mV/ms, n= 25 neurons/4 mice; p = 0.0008, MWU (Figure 4E). The maximal AP decay rate, controls = −112 [−104, −130] mV/ms, n = 25 neurons/5 mice, and 6-OHDA = −84 [−64, −106] mV/ms, n = 25 neurons/4 mice; p = 0.0001, MWU (Figure 4F). Thus, the decreased excitability of PTNs is largely caused by an abnormal AP broadening, involving impaired AP depolarization and repolarization.

Figure 4.
Figure 4. Distinct changes in AP waveforms of PTNs and ITNs in the M1 L5b following loss of midbrain DA neurons.

A) Top, representative AP waveforms of PTNs from controls and 6-OHDA mice. APs were aligned at threshold and overlaid for comparison. Bottom, the corresponding dV/dt traces plotted against time, showing depolarizing and repolarizing rates of APs. B) Boxplot showing broadened AP width in PTNs from 6-OHDA mice relative to those from controls. C and D), Boxplots showing prolonged AP rise time (C) and decay time (D) in PTNs from 6-OHDA mice relative to those from controls. E and F), Boxplots showing decreased AP rise rate (E) and decay rate (F) in PTNs from 6-OHDA mice relative to those from controls. G) Top, representative AP waveforms of ITNs from controls and 6-OHDA mice. APs were aligned at threshold and overlaid for comparison. Bottom, the corresponding dV/dt traces plotted against time, showing depolarizing and repolarizing rates of APs. H) Boxplot showing similar AP width in ITNs from 6-OHDA mice relative to those from controls. I-J) Boxplots showing a shorter AP rise time (I) but similar decay time (J) in ITNs from 6-OHDA mice relative to those from controls. K-L) Boxplots showing an increased AP rise rate (K) but similar decay rate (L) in ITNs from 6-OHDA mice relative to those from controls. ns, not significant; *, p < 0.05, MWU.

In contrast, ITNs from controls and 6-OHDA mice showed similar AP width (control = 0.73 [0.64, 0.81] ms; n = 38 neurons/6 mice, 6-OHDA = 0.70 [0.61, 0.75] ms, n = 36 neurons/6 mice; p = 0.2, MWU; Figure 4H). The similar AP width of ITNs was associated with an unaltered decay time and decay rate. The decay time for controls = 0.67 [0.58, 0.77] ms, n = 38 neurons/6 mice, while for 6-OHDA = 0.63 [0.57, 0.70] ms, n = 35 neurons/6 mice; p = 0.15, MWU (Figure 4J). The decay rate values were, for controls = −102 [−82, −122] mV/ms, n = 38 neurons/6 mice, and for 6-OHDA = −112 [−100, −122] mV/ms, n = 35 neurons/6 mice; p = 0.1, MWU (Figure 4L). Moreover, ITNs from 6-OHDA mice exhibited a faster AP depolarization relative to those from controls, as reflected by a shorter rise time (controls = 0.24 [0.21, 0.26) ms, n = 38 neurons/6 mice; 6-OHDA = 0.21 [0.19, 0.24] ms, n = 35 neurons/6 mice; p = 0.03, MWU; Figure 4I] and also a faster rise rate (control = 314 [276, 383] mV/ms, n = 38 neurons/6 mice; 6-OHDA = 377 [329, 431] mV/ms, n = 35 neurons/6 mice; p = 0.009, MWU; Figure 4K). Thus, these results suggest that ITNs of 6-OHDA mice had subtler changes in AP waveforms and that efficient AP depolarization is likely a compensatory mechanism for the hyperpolarized Vm and reduced Rm (Figures 3D, E), leading to an unaltered superathreshold excitability (Figures 2C, D).

Progressive alterations in AP waveforms of PTNs during repetitive firing following the loss of SN DA neurons

In the physiological state, the burst pattern of activity of M1 PTNs is closely linked to movement parameters, but this specificity of movement encoding is disrupted in PD (Pasquereau et al., 2016; Economo et al., 2018; Sauerbrei et al., 2020). In addition, PTNs show a decreased intraburst firing rate in MPTP-treated animals (Pasquereau and Turner, 2011). We predicted that the altered intrinsic properties of PTNs contribute to these in vivo observations in the parkinsonian state. Thus, spike trains containing 10-15 spikes were elicited from M1 PTNs in controls and 6-OHDA mice, and the waveform of the first and the last APs within a spike train were analyzed to study progressive changes during repetitive firing.

The literature suggests that M1 PTNs have the capacity for high-frequency AP firing (300-450 Hz) (Suter et al., 2013). We also noted constant AP waveforms during repetitive firing in the control mice (Fig. 5A). Specifically, the PTNs from control mice showed subtle AP broadening during repetitive firing (AP width in controls, the first AP = 0.68 [0.60, 0.72] ms, the last AP = 0.69 [0.60, 0.75] ms, n = 25 neurons/5 mice; p = 0.013, WSR; Figure 5C). They also showed an unaltered decay rate (the first AP = −114 [−110, −131] mV/ms, the last AP = −115 [−105, −132] mV/ms, n = 25 neurons/5 mice; p = 0.57, WSR; Figure 5E) and an unaltered decay time (the first AP = 0.59 [0.53, 0.68] ms, the last AP = 0.57 [0.50, 0.68] ms, n = 25 neurons/5 mice; p = 0.26, WSR; Figure 5G) between the first and the last APs during repetitive firing. In addition, the AP threshold of M1 PTNs from controls was also constant between the first and the last AP (threshold, the first AP = −53.5 [−57.2, −51.4] mV, and the last AP = −52.3 [−54.9, −50.9] mV; n = 25 neurons/5 mice; p = 0.26, WSR; Figure 5I). The constant AP parameters of PTNs during repetitive firings are key to delivering motor commands to subcortical premotor centers with temporal precision during movement initiation and execution.

Figure 5.
Figure 5. Progressive changes in AP waveforms of M1 PTNs during repetitive firing.

A-B) APs from a 10- to 15-Hz spike train were superimposed for PTNs from control (A) or 6-OHDA mice (B). APs were aligned at threshold, and the solid and dashed lines indicated the first and the last APs from the spike train, respectively. All the other APs were shown as transparent lines. C-D) Summarized graphs showing the alterations of AP width between the first and the last APs. Each line in (C) connects the AP width of the first and the last APs from the same spike train. Filled and open circles indicate the median plus interquartile range of the first and the last AP width of PTNs, respectively. (D) Boxplots showing the AP width difference between the first and the last APs was greater in PTNs from 6-OHDA mice relative to those from controls. E-F) Summarized graphs showing alterations of AP decay rate between the first and the last APs (E) and greater changes of AP decay rate in PTNs from 6-OHDA mice relative to those from controls (F). G-H) Summarized graphs showing alterations of AP decay time between the first and the last APs (G), and greater changes of AP decay time in PTNs from 6-OHDA mice relative to those from controls (H). I-J) Summarized graphs showing alterations of AP threshold between the first and the last APs (I), and greater changes of AP threshold in PTNs from 6-OHDA mice relative to those from controls (J). ns, not significant; *, p < 0.05, WSR.

In contrast, PTNs from 6-OHDA mice showed progressive AP broadening during repetitive firing (the first AP width = 0.78 [0.67, 0.97] ms, the last AP = 0.88 [0.70, 1.08] ms, n = 25 neurons/4 mice; p = 0.0001, WSR; Figure 5C). This AP broadening was associated with a significantly decreased AP decay rate (the first AP = −91.6 [−69.3, −114.7] mV/ms, the last AP = −78.7 [−63.2, −104.4] mV/ms, n = 25 neurons/4 mice; p = 0.0001, WSR; Figure 5E) and decay time (the first AP = 0.72 [0.61, 1.0] ms, the last AP = 0.77 [0.61, 1.20] ms, n = 25 neurons/4 mice; p = 0.016, WSR; Figure 5G). Moreover, PTNs in 6-OHDA mice had a progressive increase in AP threshold during repetitive firing (the first AP = −53.7 [−59.1, −50.8] mV, the last AP = −51.1 [−56.6, −44.8] mV, n = 25 neurons/4 mice; p = 0.0001, WSR; Figure 5I).

When the difference between the first and last APs was analyzed, we found greater differences in the above parameters in PTNs from 6-OHDA mice than those from controls. The AP width(last-first) for controls was 0.006 (−0.004, 0.04) ms, n = 25 neurons/5 mice, while for 6-OHDA mice was 0.05 (0.01, 0.10) ms, n = 25 neurons/4 mice; p = 0.016, MWU (Figure 5D). The AP decay rate(last-first) for the controls was 2.44 [− 3.66, 5.56] mV/ms, n = 25 neurons/5 mice, while the value for 6-OHDA mice was 7.94 [4.58, 11.90] mV/ms, n = 25 neurons/4 mice; p = 0.0004, MWU (Figure 5F). The AP decay time(last-first) for controls was −0.015 (−0.045, 0.016) ms, n = 25 neurons/5 mice, while the value for 6-OHDA mice was 0.059 (−0.03, 0.12) ms, n = 25 neurons/4 mice; p = 0.018, MWU (Figure 5H). Finally, the AP threshold(last-first) value for controls was 0.40 [−1.65, 2.78] mV, n = 25 neurons/5 mice, while the value for 6-OHDA mice was 2.96 [1.05, 4.07] mV, n = 25 neurons/4 mice; p < 0.006, MWU (Figure 5J). These results suggest that following the loss of midbrain DA neurons the altered intrinsic properties of M1 PTNs impair their capability to sustain constant AP waveforms during repetitive firing, which likely compromises their capacity of fast firing and the temporal precision of motor commands for motor control (Suter et al., 2013; Sauerbrei et al., 2020).

Impaired activity of BK channels underlies the decreased intrinsic excitability of M1 PTNs following the loss of midbrain DA neurons

AP width is determined by the time and velocity of depolarization and repolarization (Bean, 2007). Our results above indicated that broadened APs of PTNs following the loss of DA neurons are largely attributed to prolonged AP repolarization. Furthermore, following the loss of midbrain DA neurons, the amplitude of the fast afterhyperpolarization (fAHP) in PTNs diminished (controls = 12.6 [9.8, 14.5] mV, n = 25 neurons/5 mice; 6-OHDA = 9.1 [6.0, 12.3] mV, n = 25 neurons/5 mice; p = 0.003, MWU; Figures 6A, B), but the amplitude of median afterhyperpolarization (mAHP) was similar to that of controls (controls = 21.7 [16.6, 24.4] mV, n = 25 neurons/5 mice; 6-OHDA = 23.0 [20.1, 26.4] mV, n = 25 neurons/5 mice; p = 0.63, MWU; Figure 6C). BK currents contribute to fAHPs, AP repolarization and/or AP broadening during repetitive neuronal firing in multiple brain regions (Faber and Sah, 2003; Bean, 2007; Contet et al., 2016). The reduced fAHP amplitude in PTNs from 6-OHDA mice indicates that altered BK channel activity probably underlies broadened AP of PTNs in 6-OHDA mice.

Figure 6.
Figure 6. Decreased BK channel activity underlies AP broadening and impaired intrinsic excitability of PTNs following loss of midbrain DA neurons.

A) Representative AP waveforms showing diminished fAHPs (arrow) in PTNs from 6-OHDA mice, relative to those from controls. B-C) Boxplot showing decreased amplitude of fAHPs (B) and unaltered mAHPs (C) in PTNs from 6-OHDA mice relative to those from controls. D) Representative APs waveforms of PTNs from controls and 6-OHDA mice in the presence of BAPTA. APs were aligned at the threshold and overlaid for comparison. E) Boxplot showing reversal of broadened AP of PTNs in 6-OHDA mice relative to controls after intracellular BAPTA dialysis. F) Summarized graph showing unaltered excitability of PTNs from 6-OHDA mice relative to those from controls in the presence of BAPTA. The number of APs elicited by 400-, 600-, and 800-pA somatic current injections were compared. G and H) Representative APs waveforms before and after paxillin application in PTNs from controls (G) and 6-OHDA mice (H). APs were aligned at the threshold and overlaid for comparison. I) Summarized graph showing paxillin broadened APs of PTNs from both controls and 6-OHDA mice. J) Summarized graph showing that paxillin significantly decreased the number of spikes (evoked by injecting 720-pA current) of PTNs from controls but did not affect those from 6-OHDA mice. Application of paxillin occluded the decreased number of spikes of PTNs from 6-OHDA mice relative to those from controls. Filled and open symbols in (I and J) indicate the values of before and after paxillin application, respectively. ns, not significant; *, p < 0.05, WSR or MWU.

BK channel activation requires both membrane depolarization and Ca2+ influx during APs. Therefore, we decreased the activation of BK channels by intracellularly dialyzing a high-affinity Ca2+ chelator, BAPTA (10 mM), and determined its effect on the AP width of PTNs from both controls and 6-OHDA mice. In the presence of BAPTA, the AP of PTNs from 6-OHDA mice was slightly and statistically significant narrower (half-width: controls = 0.99 [0.92, 1.17] ms, n = 40 neurons/3 mice; 6-OHDA = 0.95 [0.83, 1.03] ms, n = 39 neurons/3 mice; p = 0.029, MWU; Figures 6D, E). In addition, the PTNs from both groups also showed similar intrinsic excitability in the presence of BAPTA (Figure 6F). For example, the number of spikes in response to maximal current injection at 800 pA for controls = 42 [28, 55] spikes, n = 40 neurons/3 mice, while the value for 6-OHDA = 40 [31, 49] spikes, n = 39 neurons/3 mice; p = 0.84, MWU (Figure 6F).

Furthermore, we blocked BK channels activity by a selective BK channel blocker, paxillin (10 μM), through bath application and we found that it significantly prolonged the APs of PTNs in both groups (Figure 6G, H). The half-width of controls before and after paxillin were 0.77 [0.64, 0.81] ms, and 0.84 [0.8, 0.96] ms, respectively (n = 15 neurons/3 mice; p = 0.0001, WSR; Figures 6I). For PTNs from 6-OHDA mice, the half-width before and after paxillin were 0.82 [0.75, 0.87] ms and 0.89 [0.78, 0.98] ms, respectively (n= 14 neurons/3 mice; p = 0.0009, WSR; Figures 6I). With the blockade of BK channels by paxillin, the AP width was not statistically different between PTNs from controls and 6-OHDA mice (MWU, p = 0.62; Figure 6G, I). In addition, paxillin significantly decreased the intrinsic excitability of PTNs from controls (the number of spikes in response to 480-pA current injection, baseline = 36 [32, 48] Hz, paxillin = 31 [17, 43] Hz; n = 15 neurons/ 3 mice; p = 0.0002, WSR; Figure 6J). In contrast, paxillin did not affect the number of spikes in PTNs of 6-OHDA mice (spike frequency at 480-pA current injection, baseline = 32 [27, 39] Hz, paxillin = 29 [28, 39] Hz; n = 14 neurons/ 3 mice; p = 0.71, WSR; Figure 6J). In the presence of paxillin, there is no difference in the intrinsic excitability of PTNs between 6-OHDA mice and controls (MWU, p = 0.7; Figure 6J). Together, these results suggested that impaired BK channel function underlies the broadened APs and decreased intrinsic excitability of PTNs following the loss of midbrain DA neurons.

Intrinsic adaptations of M1 pyramidal neurons were not due to the decreased DA neuromodulation in M1

M1 receives dopaminergic inputs from the ventral tegmental area and these neurons are partially degenerate in 6-OHDA mice (Hosp et al., 2011, 2015; Hosp and Luft, 2013). Thus, we predicted that loss of direct mesocortical DA neuromodulation contributes to the intrinsic adaptations of PTNs and ITNs in M1. To test this prediction, we activated DA receptors on PTNs and ITNs through bath perfusion of DA (10 μM) and determined its impact on the intrinsic properties of M1 PTNs and ITNs from both controls and 6-OHDA mice. The activation of DA receptors by DA produced negligible effects to the intrinsic excitability of M1 PTNs in controls (e.g., the firing frequency at a 360-pA current injection, baseline = 37 [30, 47] Hz; DA = 34 [20, 39] Hz; n = 10 neurons/3 mice; p = 0.25, WSR; Figures. 7A, B). In addition, the application of DA (10 μM) did not produce significant effects to the intrinsic excitability of M1 PTNs in 6-OHDA mice either (e.g., the firing frequency at a 360-pA current injection, baseline = 26 [20, 33] Hz, DA = 27 [19, 36] Hz, n = 14 neurons/3 mice; p = 0.72, WSR; Figures. 7C, D). Furthermore, DA (10 μM) did not alter the AP width of PTNs in both group either (controls: baseline = 0.72 [0.56, 0.8] ms, DA = 0.71 [0.55, 0.84] ms, n = 10 neurons/3 mice, p = 0.32, WSR; 6-OHDA: baseline = 0.74 [0.62, 0.85] ms, 6-OHDA DA = 0.74 [0.57, 0.88] ms, n = 14 neurons/3 mice, p = 0.24, WSR). These data suggested that restoration of local DA neuromodulation cannot rescue the impaired intrinsic excitability and AP broadening of M1 PTNs following the loss of midbrain DA neurons.

Figure 7.
Figure 7.

DA application could not rescue intrinsic adaptations in PTNs and ITNs of M1 following loss of midbrain DA neurons. A-B) Representative AP traces showing the effect of DA application (10 μM) on the number of APs of PTNs from controls (A) and the summarized results (B). C-D) Representative AP traces showing the effect of DA application on the number of APs of PTNs from 6-OHDA mice (C) and the summarized results (D). EG) Summarized graphs showing the impact of DA (10 μM) on intrinsic properties of ITNs from controls and 6-OHDA mice, including the Vm (E), Rm (F), rheobase (G) of ITNs from controls and 6-OHDA mice. Filled and open circles in (E-G) indicate the values of before and after DA application, respectively Each line connected the rheobase before (filled circle) and after (open circle) DA application. *, p < 0.05, WSR.

Next, we tested whether the altered passive membrane properties of M1 ITNs of 6-OHDA mice (Figure 3) are caused by the loss of local DA neuromodulation. In control mice, the activation of DA receptors by DA (10 μM) did not produce a significant effect on the Vm (baseline = −78 [−81, −74] mV, DA = −78 [−80, −75] mV, n = 10 neurons/2 mice; p > 0.99, WSR; Figure 7E) or on the Rm of ITNs (baseline = 88 [51, 109] MV, DA = 86 [50, 108] MV, n = 10 neurons/2 mice; p = 0.16, WSR; Figure 7F). However, the rheobase of ITNs was significantly increased (baseline = 66 [43, 112] pA, DA = 116 [77, 212] pA, n = 10 neurons/ 2 mice; p = 0.027, WSR; Figure 7G). On the other hand, in ITNs of 6-OHDA mice, the application of DA (10 μM) did not alter the Vm (baseline = −79 [−82, −76] mV, DA = −77 [−80, −75] mV, n = 14 neurons/3 mice; p = 0.22, WSR; Figure 7E); the Rm (baseline = 65 [53, 88] MV, DA = 64 [53, 75] MV, n = 14 neurons/3 mice; p = 0.06, WSR; Figure 7F); or the rheobase (baseline = 95 [80, 145] pA, DA = 90 [34, 161] pA, n = 14 neurons/3 mice; p = 0.33, WSR; Figure 7G). Thus, the loss of DA neuromodulation did not play a major role in the cell-type-specific intrinsic adaptations of M1 pyramidal neurons in the parkinsonian state.

Discussion

We systematically studied adaptative changes in the intrinsic properties of M1 pyramidal neurons in experimental parkinsonism. We found cell-type-specific decrease of the intrinsic excitability PTNs and adaptive changes in the passive membrane properties of ITNs in M1. We also found that prolonged AP repolarization led to AP broadening and resulted in the decreased intrinsic excitability of M1 PTNs. Further, we revealed that impaired BK channel function was the main ionic mechanism underlying the altered AP waveforms and intrinsic excitability of M1 PTNs. Last, we showed that the intrinsic adaptations in M1 PTNs and ITNs were not caused by a loss of mesocortical dopaminergic neuromodulation. Our findings strongly suggest that the loss of midbrain DA neurons not only alters basal ganglia activity, but also triggers intrinsic adaptations in M1 circuitry, which contribute to the reduced motor cortical output and motor deficits in the parkinsonian state.

Compelling evidence supports an abnormal rate and pattern of activity in M1 in parkinsonian state, which could be caused by loss of direct mesocortical DA projection, the pathological basal ganglia output associated with nigrostriatal degeneration, or both. (Goldberg et al., 2002; Pasquereau and Turner, 2011; Pasquereau et al., 2016). To distinguish between these possibilities, we employed ex vivo electrophysiology to study the intrinsic properties of projection-defined M1 pyramidal neurons. Our findings that PTNs, but not the ITNs, in the L5b of M1 had decreased intrinsic excitability in 6-OHDA mice are consistent with the earlier findings in MPTP-treated primates, showing a cell-type-specific reduction of the firing frequency of M1 pyramidal neurons (Pasquereau and Turner, 2011). Given the innervation of subcortical motor centers by M1 PTNs, these results suggested that intrinsic adaptations of M1 pyramidal neurons contribute to an insufficient motor cortical outputs following the loss of midbrain DA neurons, in addition to an elevated basal ganglia inhibition as proposed by the classical model (Albin et al., 1989; DeLong, 1990; Galvan and Wichmann, 2008). We postulate that these adaptations exacerbate the hypokinetic parkinsonian motor deficits.

Distinct changes in the intrinsic excitability of M1 pyramidal neurons cannot be explained by alterations in their passive membrane properties. For example, we found an increased Rm of PTNs in 6-OHDA mice, which might be a homeostatic compensation for, rather than a cause of, their decreased intrinsic excitability. It is possible that the increased Rm makes M1 PTNs more sensitive to and more easily to be entrained by synaptic inputs in the parkinsonian state, including the abnormal basal ganglia signals through the thalamocortical projections. The increased sensitivity of PTNs to synaptic inputs may also disrupts their selectivity in encoding movements. In contrast, ITNs were hyperpolarized and showed a trend of decreased input resistance following the loss of midbrain DA neurons, but these changes were compensated for by other adaptions (e.g., an increased Nav availability for AP generation), so they did not significantly alter the superathreshold excitability. Together, our data suggested that, intrinsic adaptations play an important role in the cell-type-specific alterations of the activity of M1 pyramidal neurons following the loss of midbrain DA neurons. The molecular mechanisms underlying cell-type specific adaptations in M1 in parkinsonian state should be determined in future studies.

APs of PTNs were significantly broadened following the loss of DA neurons and it was largely due to a prolonged repolarization (Figures 4A-E). AP width has significant impact on Ca2+ influx during APs, which further controls the quantity and kinetics of neurotransmitter release and synaptic plasticity at axon terminal (Bean, 2007). Thus, we suggest that AP broadening is a compensatory mechanism for the decreased intrinsic excitability of M1 PTNs in parkinsonian state in order to maintain stable motor cortical outputs, though this possibility should be further studied. We further showed that the AP broadening in PTNs from 6-OHDA mice was occluded in the presence of BAPTA and paxillin, indicating a key role of BK channels in the intrinsic adaptations of M1 PTNs in parkinsonian state (Shao et al., 1999; Faber and Sah, 2003; Bean, 2007). Indeed, it has been proposed that impaired function or inactivation of BK channels that underlies AP repolarization and/or fAHPs could slow the de-inactivation of Nav channels during AP and reduce Nav availability for the subsequent AP generation, leading to a decreased excitability (Bean, 2007; Jaffe et al., 2011). A progressive and accumulative Nav inactivation is consisted with our observations that PTNs in 6-OHDA mice showed stronger spike adaptation during repetitive firing relative to those from controls (Fleidervish et al., 1996), though the contribution of the other mechanisms such as the M-current can’t be discounted (Buchta et al., 2017; Parrilla-Carrero et al., 2018). It is intriguing that in the presence of BAPTA PTNs from 6-OHDA mice exhibited narrower APs than those from controls (Figures 6D, E). It indicates the involvement of other ionic mechanisms in controlling of AP repolarizationof PTNs, e.g., Kv conductance (e.g., Kv3 and Kv4) (Rudy and McBain, 2001; Pathak et al., 2016; Soares et al., 2017).

An acute application of DA did not rescue altered intrinsic excitability of PTNs and had minimal effects to intrinsic properties of ITNs in 6-OHDA mice. This indicated that loss of dopaminergic neuromodulation does not play a major role in the intrinsic adaptations of M1 pyramidal neurons in parkinsonian state, which is consistent with recent findings that dopaminergic modulation of pyramidal neurons is mainly mediated indirectly by parvalbumin-expressing interneurons in M1 (Vitrac et al., 2014; Cousineau et al., 2020). Thus, we propose that circuitry mechanisms underlie cellular adaptions in M1 in parkinsonian state. For example, recent work highlighted the critical role indirect pathway SPN hyperactivity in driving cellular and synaptic adaptations in basal ganglia nuclei, such as the subthalamic nucleus (Chu et al., 2017; Sharott et al., 2017; Ryan et al., 2018; McIver et al., 2019). Thus, it is likely that the pathological basal ganglia outputs following loss of DA that drive adaptive changes in the intrinsic properties of M1 pyramidal neurons, which are mediated by thalamocortical projections.

The exaggerated rhythmic activity and aberrant oscillations in the basal ganglia circuitry are closely related to the expression of motor symptoms in PD (Bevan et al., 2002; Little and Brown, 2014; Hemptinne et al., 2015). Indeed, the motor cortex exhibits abnormal neural activity in parkinsonian state at both the single-cell and population levels (Goldberg et al., 2002; Pasquereau and Turner, 2011; Hemptinne et al., 2015; Pasquereau et al., 2016). Moreover, both experimental and computation studies have indicated that cerebral cortical inputs play a crucial role in orchestrating pathological activity in basal ganglia nuclei (e.g., the external global pallidus and the subthalamic nucleus) (Bevan et al., 2002; Magill et al., 2004; Kita and Kita, 2011; Corbit et al., 2016; Chu et al., 2017; Sharott et al., 2017). Recent studies further showed that electrical or optogenetic stimulation of the motor cortical pyramidal neurons and/or their projections produced therapeutic effects in parkinsonian animals (Gradinaru et al., 2009; Li et al., 2012; Hemptinne et al., 2015; Sanders and Jaeger, 2016; Magno et al., 2019). Thus, an in-depth understanding of motor cortical adaptations in PD at the cellular and circuitry levels will help to design better therapeutic approaches with fewer off-target effects.

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This work was supported by fund from Van Andel Institute (H.Y.C). The authors thank Mr. David Nadziejka for technical editing.

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Posted October 21, 2020.
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Cell-Type-Specific Decrease of the Intrinsic Excitability of Motor Cortical Pyramidal Neurons in Parkinsonian Mice
Liqiang Chen, Yerim Kim, Hong-Yuan Chu
bioRxiv 2020.10.20.347732; doi: https://doi.org/10.1101/2020.10.20.347732
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