Journal: bioRxiv

Article Title: Projections between the globus pallidus externa and cortex span motor and non-motor regions

doi: 10.1101/2024.11.08.622712

Figure Lengend Snippet: a, left, Schematic of experimental approach: CTb was injected into one of four cortical regions – motor cortex (n=3 mice), sensory cortex (n=3 mice), insular cortex (n=3 mice), and frontal cortex (n=4 mice) – in wild-type mice to retrogradely label pallidocortical neurons in GPe for in vitro whole-cell recordings. right, Example image of a CTb-positive (green) and biocytin-positive (magenta) cell. Biocytin was introduced into the cell during the whole-cell recording for post-hoc identification. b, Example current clamp recordings from pallidocortical neurons projecting to each of the four cortical regions. A hyperpolarizing and a supra-threshold depolarizing response is shown for each cell. c, Quantification of passive properties of pallidocortical neurons according to cortical projection target: resting membrane potential (resting membrane V), sag potential (sag V), membrane resistance (Rm) and current-voltage relationship. Total number of recorded cells for each region: motor cortex: 17, sensory cortex: 22, insular cortex: 24, frontal cortex: 20. Mean and SEM are shown. There was a significant difference in sag potential between groups for sag potential (Kruskal-Wallis (K-W)=9.102, p=0.028, significant for motor cortex vs insular cortex p=0.0180 after multiple comparison correction). No significant differences between groups for other passive properties were identified. d, Quantification of active properties of pallidocortical neurons according to cortical projection target: current step required to generate a first action potential (1st AP current step), mean interspike interval (ISI), mean action potential rate of voltage change (mean max dVdT), mean adaptation ratio (first AP interval/last AP interval). Mean and SEM are shown. There was a significant difference between groups for mean ISI (Kruskal-Wallis (K-W)=16.65, p=0.0008, p=0.0213, significant for motor cortex vs insular cortex (p=0.0107) and sensory cortex vs insular cortex (p=0.0014), after multiple comparison corrections). No significant differences between groups after multiple comparison correction for other active properties. e , Principal component analysis (PCA) of electrophysiological properties (n=13 mice, 83 cells). left, Scree plot results. Percent variance in the data explained by the first four principal components. middle, K-means clustering for k=2, inset: Elbow plot of Within-Cluster Sum of Squares (WCSS) for different values of k. The maximum of the ratio of the second derivative/first derivative was used to identify the optimal value for k (the inflection point of the curve). Data points are color-coded by cluster assignment. The plot illustrates the distribution of data across clusters. right , Biplot of the first two principal components (PC1 and PC2) showing variable coefficients and scores. Points represent the scores for each cell on PC1 and PC2 and are colored according to cortical projection target. Red lines indicate the direction and magnitude of variable coefficients, the line length is proportional to the contribution of each variable to the principal components.

Article Snippet: For whole cell voltage clamp recording, patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3, Warner Instruments) were filled with internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1).

Techniques: Injection, In Vitro, Membrane, Comparison

Journal: bioRxiv

Article Title: Projections between the globus pallidus externa and cortex span motor and non-motor regions

doi: 10.1101/2024.11.08.622712

Figure Lengend Snippet: a, Anatomic tracing of cortical inputs to GPe. left, Schematic of experimental approach: non-pseudotyped G-deleted RVs encoding either GFP or tdTomato was injected into GPe (CVS-N2c-dG-GFP) and striatum (CVS-N2c-dG-tdTomato) of the right hemisphere of wild type mice (n=3). right, Example images of cortex ipsilateral and contralateral to the injection site. inset , zoom of cortical region. b, Proportion of all cells innervating GPe and striatum ipsilateral to the injection site (n=2 mice, mean (+/- SEM) total cells for GPe=72400 (13563), mean (+/- SEM) total cells for striatum=39834 (1754). c, Proportion of ispsilateral cells innervating GPe vs striatum, stratified by broad brain regions (n=2 mice). There was a significant difference between GPe and striatum for cortical input (p<0.0001) and striatal input (p<0.0001) after multiple comparison correction (two-way ANOVA with Šídák’s multiple comparison test). Key, STR: striatum, CX: cortex, TH: thalamus, MB: midbrain, HY: hypothalamus, STN: subthalamic nucleus, GPe: globus pallidus externa, GPi: globus pallidus interna, HPF: hippocampal formation, COA: cortical amygdala area, P: pons. d, Proportion of all ipsilateral cortical cells innervating GPe vs striatum, stratified by cortical layer (n=2 mice). There was a significant difference between GPe and striatum for layers 2-3 (p=0.0001), and layer 5 (p=0.0014) after multiple comparison correction (two-way ANOVA with Šídák’s multiple comparison test). e, Schematic for experimental electrophysiological verification of cortical innervation to GPe in wild type mice: CTb was injected into cortex and SNr to retrogradely label GPe neurons innervating cortex or SNr respectively. ChR2 was injected into cortex at the same site as CTb. Whole-cell voltage clamp recordings were obtained from CTb labeled or unlabeled GPe neurons and used to record synaptic currents evoked by stimulation of cortical axons with a single 2 ms blue light pulses. f , Example histological image of an acute brain slice used for electrophysiological analyses showing a biocytin and CTb-labeled cortex-projecting neuron analyzed by whole-cell recording. g , Synaptic currents evoked by ChR2 stimulation of cortical axons recording in three different GPe neuron types: unlabeled (n=51 cells, 14 mice), cortex-projecting (pallidocortical, n=42 cells, 15 mice), and SNr-projecting (pallidonigral, n=15 cells, 5 mice). The proportions of responding vs non-responding cells are shown below the traces. h, Summary of EPSC amplitude and onset latency for all responding neurons. Mean and SEM are shown. There was no significant difference between neuron types for EPSC amplitude (Kruskal-Wallis test K-W=3.914, p=0.1413) or latency (K-W=0.6003, p=0.7407). i, Example histology of ChR2 and CTb injections in insular cortex and motor cortex are shown. Synaptic currents evoked by ChR2 stimulation of axons from insular cortex (n=56 cells, 20 mice) and motor cortex (n=52 cells, 14 mice) to all GPe neuron types. j , Summary of EPSC amplitude and onset latency for all responding neurons. Mean and SEM are shown. EPSCs evoked by insular cortex stimulation to GPe neurons were significantly larger those evoked by motor cortex inputs (two-tailed Mann-Whitney U test, U=312, p=0.0139). There was no significant difference between onset latency (two-tailed Mann-Whitney U test, U=390, p=0.1670).

Article Snippet: For whole cell voltage clamp recording, patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3, Warner Instruments) were filled with internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1).

Techniques: Injection, Comparison, Labeling, Slice Preparation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Projections between the globus pallidus externa and cortex span motor and non-motor regions

doi: 10.1101/2024.11.08.622712

Figure Lengend Snippet: a, Schematic of experimental approach: ChR2 was injected into motor or insular cortex of ChAT-i-Cre x Ai14 ( tdTom reporter ) , Pvalb-i-Cre x Ai14 (tdTom reporter ) , or FoxP2-i-Cre mice. An AAV carrying a Cre -dependent fluorophore was injected into GPe of FoxP2-i-Cre mice. tdTom-labeled (ChAT+ or Pvalb+) or fluorophore-labeled (FoxP2+) neurons were voltage-clamped in GPe. 2ms pulses of 470 nm light were delivered to stimulate cortical inputs to GPe. Example histological images are shown, biocytin indicates a recorded neuron. b, Synaptic currents evoked by ChR2 stimulation of cortical inputs to ChAT+, Pvalb+, and FoxP2+ neurons are shown. Proportion of responding vs non-responding cells are shown below traces. Number of mice/cells: ChAT n=2 mice, 18 cells; PV n=2 mice, 16 cells; FoxP2 n=2 mice, 3 cells. c, Synaptic potentials evoked by ChR2 stimulation of cortical inputs to ChAT+, Pvalb+, and FoxP2+ neurons. Number of mice/cells: ChAT n=2 mice, 6 cells, PV n=2 mice, 5 cells, FoxP2 n=2 mice, 2 cells. d, Summary of EPSC amplitude and peak depolarization evoked by ChR2 stimulation for all neurons for which both voltage- and current-clamp recordings were obtained. Number of mice/cells: ChAT n=2 mice, 6 cells, PV n=2 mice, 5 cells, FoxP2 n=2 mice, 2 cells. Mean and SEM are shown. No significant differences were found between groups. e, Proportion of neurons that fired an action potential in response to ChR2 stimulation of cortical input for each cell-type. f, Mean pre-light and post-light interspike interval (ISI) was measured in 4 ChAT + neurons and 5 Pvalb + neurons that were spontaneously spiking (no FoxP2+ neurons were spontaneously spiking). Example traces are shown for each cell type (timing of 470 nm 2ms light pulse is indicated by the dashed vertical line). There was no significant difference in interspike interval after light for ChAT+ neurons (Two-tailed paired t-test: t=1.303, df=3, p=0.2835). There was a significant difference in interspike interval after light for Pvalb+ neurons (Two-tailed paired t-test: t=2.910, df=4, p=0.0437).

Article Snippet: For whole cell voltage clamp recording, patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3, Warner Instruments) were filled with internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1).

Techniques: Injection, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Projections between the globus pallidus externa and cortex span motor and non-motor regions

doi: 10.1101/2024.11.08.622712

Figure Lengend Snippet: a, mean leak current for all neurons shown in traces in , mean and SEM are shown. ChAT+ n=2 mice, 18 cells. Pvalb+ n=2 mice, 16 cells, FoxP2+ n=2 mice, 2 cells. There was a significant difference in leak current between cell types (one-way ANOVA: F (2, 33)=9.527, p=0.0005, multiple comparison correction: ChAT+ vs FoxP2+ p=0.0004; Pvalb+ vs FoxP2 + p=0.0068. b, Leak current and resting membrane potential for neurons used for quantifying paired voltage clamp and current clamp responses to light stimulation in , mean and SEM are shown. ChAT+ n=2 mice, 6 cells. Pvalb+ n=2 mice, 5 cells, FoxP2+ n=2 mice, 2 cells. There was a significant difference in leak current between cell types (Kruskal-Wallis test: K-W=10.15, p=0.0002, multiple comparison correction: ChAT+ vs FoxP2+ p=0.0139). There was a significant difference in resting membrane potential between cell types (K-W=9.738, p=0.0003, multiple comparison correction: ChAT+ vs Pvalb+: p=0.0059).

Article Snippet: For whole cell voltage clamp recording, patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3, Warner Instruments) were filled with internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1).

Techniques: Comparison, Membrane

Journal: bioRxiv

Article Title: Projections between the globus pallidus externa and cortex span motor and non-motor regions

doi: 10.1101/2024.11.08.622712

Figure Lengend Snippet: a, left, Schematic of experimental approach: Cre- dependent ChR2 was injected into the cortex of Rbp4-Cre mice. Neurons in the SNr were patched during stimulation of ChR2 with 2 ms pulses of 470 nm light. right, Example histological images of the cortical injection site and patched neurons in the SNr are shown. Anti-tyrosine hydroxylase (TH) stain is used to indicate the location of dopamine neurons in the substantia nigra pars compacta (SNc) which is dorsal to the SNr. b, Voltage clamp responses of SNr neurons to stimulation of cortical axons in the SNr in the absence of any pharmacologic agents, and in the presence of TTX+4AP (to isolate monosynaptic inputs) and glutamatergic receptor blockers (NBQX and CPP) to block glutamatergic input. Proportion of neurons responding to cortical input is shown below the traces. c, Quantification of EPSC amplitude and EPSC latency in all responding cells. Mean and SEM are shown, n=18 neurons, 5 mice. d, EPSC amplitude in the absence and presence of glutamatergic blockers, n=5 neuron pairs from 4 mice (Wilcoxin matched-pairs signed rank test: W=15.00, p=0.0625). e, ChR2 was injected either into motor cortex or insular cortex to compare inputs to SNr from two different cortical sites. f, Voltage clamp responses to blue light stimulation of motor cortex and insular cortex inputs. Proportion of responding neurons are shown below the traces. g , Quantification of EPSC amplitude and latency. Mean and SEM are shown. Motor cortex n=15 cells, 2 mice, insular cortex n=18 cells, 3 mice. There was a significant difference between motor cortex and insular cortex EPSC amplitude (two-tailed unpaired t-test: t=2.195, df=16, p=0.0433), but not EPSC latency (Mann Whitney Test: U=30, p=0.4063). h, Summary of intrinsic properties of all SNr neurons.

Article Snippet: For whole cell voltage clamp recording, patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3, Warner Instruments) were filled with internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1).

Techniques: Injection, Staining, Blocking Assay, Two Tailed Test, MANN-WHITNEY