Striatal Direct Pathway Targets Npas1+ Pallidal Neurons

Abstract

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (^DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (^DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1⁺ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1⁺ projection was dramatically strengthened. As ^DLSdSPN-Npas1⁺ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states.SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease.

Keywords: 6-OHDA; GABAergic inhibition; arkypallidal neurons; body kinematics; hypokinesia; movement dynamics.

Figure 1.

Optogenetic stimulation of SPN subtypes induces unique changes in locomotor speed. a, Left, A schematic diagram showing injection in the DMS and DLS. Cre-inducible (CreOn) ChR2-eYFP and eYFP AAV injections were targeted to the DMS (in mm as follows: 0.9 rostral, 1.4 lateral, 3.4 and 3.0 ventral from bregma) or the DLS (in mm as follows: 0.7 rostral, 2.3 lateral, 3.4 and 3.0 ventral from bregma) of Adora2a^Cre, Drd1a^Cre, or Tac1^Cre mice. Right, Coronal brain sections showing the viral spread in the dStr. Green represents ChR2-eYFP. Blue represents DAPI. Tracks produced by the fiber cannulae are marked by arrowheads. Scale bar applies to both images. b, An open-field arena (28 cm × 28 cm) was used for examining the locomotor activity of test subjects (see Materials and Methods). c–g, Changes in speeds are shown with light delivery (blue horizontal lines) in the DMS (orange) and DLS (purple) of Adora2a^Cre, Drd1a^Cre, and Tac1^Cre mice that expressed ChR2 or eYFP. h, Changes in speeds are shown with light delivery (blue horizontal lines) in the medial (left) and lateral (right) SNr of Drd1a^Cre mice that expressed ChR2. CreOn-ChR2-eYFP AAV injections were targeted to the DMS and the DLS, respectively. Solid trace is the population mean calculated from all mice; shading indicates the SEM.

Figure 2.

Optogenetic stimulation of SPN subtypes produces unique changes in movement metrics. a, Left, A heatmap summarizing motor responses of mice to optogenetic stimulation of ^DLSdSPNs (red), ^DMSdSPNs (blue), ^DLSiSPNs (green), and ^DMSiSPNs (purple). Twenty-five movement metrics were measured to fully capture the behavioral structures. Each of the 25 rows represents the fold change of movement metrics. Warm colors (red) represent positive changes. Cool colors (blue) represent negative changes. Rows and columns were sorted using hierarchical clustering. Dendrograms are divided into two main arms; metrics on the upper arm are positively correlated with “total frequency,” whereas metrics on the lower arm are negatively correlated with “total frequency.” Each column is a mouse; 48 mice were used in this and all subsequent analyses in b–e (^DLSdSPNs = 13 mice, ^DMSdSPNs = 9 mice, ^DLSiSPNs = 15 mice, ^DMSiSPNs = 11 mice). Right, A heatmap summarizing motor responses of mice to optogenetic stimulation of genetically defined neurons in the GPe. The neurons of interest are Foxp2⁺ (pink), Kcng4⁺ (purple), Npas1⁺ (green), and PV⁺ (orange). The plot was reproduced from Cui et al. (2021). b, Mean changes in the event frequency of motionless, fine movement, rearing, and locomotion on optogenetic stimulation (blue horizontal lines) of ^DMSdSPNs, ^DLSdSPNs, ^DMSiSPNs, and ^DLSiSPNs. Scale bar applies to all traces. c, Slope graphs showing the fraction of time spent for motionless, fine movement, and locomotion in mice and the effect with optogenetic stimulation of selective neuron types. Each connected line indicates a mouse. d, A correlation matrix constructed using data from Adora2a^Cre and Drd1a^Cre mice transduced with CreOn-ChR2-eYFP AAV. Eighteen parameters were included in this matrix. Blue represents positive correlations. Brown represents negative correlations. Inset, Principal component analysis plots showing the distribution of ^DMSdSPNs (blue), ^DLSdSPNs (red), ^DMSiSPNs (purple), and ^DLSiSPNs (green). Fold changes of 23 movement metrics with optogenetic stimulation were used in this analysis. e, A correlation matrix constructed from fold changes in movement metrics following optogenetic stimulation of ^DMSdSPNs, ^DLSdSPNs, ^DMSiSPNs, and ^DLSiSPNs. Blue represents positive correlations. Brown represents negative correlations.

Figure 3.

Extended optogenetic interrogation confirms distinct behavior roles of ^DMSdSPNs and ^DLSdSPNs. a, Mean changes in the event frequency of motionless, fine movement, rearing, and locomotion on optogenetic stimulation (blue horizontal lines) of ^DMSdSPNs and ^DLSdSPNs terminals in the SNr. Scale bar applies to all traces. b, Changes in speeds are shown with light delivery (blue horizontal lines) in the DMS and DLS of Drd1a^Cre mice (top), DLS of Adora2a^Cre mice (bottom right) with GtACR2 expression. No changes in speeds were observed in the absence of opsin expression (bottom left). Solid trace is the population mean calculated from all mice; shading indicates the SEM.

Figure 4.

dSPNs send terminating axons to the GPe. a, Top left, A confocal micrograph showing retrogradely labeled SPNs in the dStr from a Cre-reporter (R26^LSL-tdTomato) mouse. A Cre-expressing lentivirus and CTb 488 were injected into the GPe. tdTomato⁺ and CTb 488⁺ neurons (white circles) were visible. Bottom left, Unbiased quantification of GPe-projecting neurons across the entire brain. Each marker represents a mouse (n = 8 mice). Arrow points to the data (red) from the dStr. Right, Representative two-photon images from coronal sections show that GPe-projecting neurons were found in the dStr, central amygdala (CeA), parafascicular nucleus (PF), and subthalamic nucleus (STN). Inset in the first image indicates the location of the injection site. Scale bar in the third image applies to the bottom three images. b, Left, High-magnification images show that terminals (eGFP⁺, white) from iSPNs or dSPNs were abundant in the GPe. CreOn-mRuby2-T2A-Synaptophysin-eGFP AAV was injected into the dStr of Adora2a^Cre (top), Drd1a^Cre (middle), and Tac1^Cre (bottom) mice to visualize SPN terminals. Maximal projections from 10 optical sections are shown. Inset, mRuby2⁺ dSPNs in the dStr. Top and middle right, Immunohistological analyses showing that eGFP⁺ boutons (green) were in proximity with VGAT and gephyrin (magenta), as shown in white in three orthogonal planes. Insets, Magnified views of areas within the dotted square outlines. Bottom right, Quantification of eGFP⁺ bouton density in the GPe from Adora2a^Cre (n = 12 ROIs), Drd1a^Cre (n = 10 ROIs), and Tac1^Cre (n = 12 ROIs) mice. c, Left, A low-magnification micrograph from a sagittal brain section demonstrating the target of retrograde tracers CTb 488 and CTb 647 into the GPe and SNr, respectively. The Drd1a^tdTomato allele was used to decipher the identity of SPNs. Right, Representative high-magnification images show that CTb 488 and CTb 647 were detected in the same tdTom⁺ neurons in the dStr. White circles represent colocalization. d, A low-magnification micrograph from a sagittal brain section shows the expression of eYFP in the dStr as well as its projection targets, including the GPe, GPi, and SNr following injections of CreOn-Flp CAV into the SNr in combination with CreOn-FlpOn-ChR2-eYFP AAV into the dStr of a Drd1a^Cre mouse. CTb 647 was coinjected with CreOn-Flp CAV to visualize the injection site. Inset, The eYFP⁺ axons in the GPe. BLA, Basolateral amygdalar nucleus; BSTa, bed nuclei of the stria terminalis, anterior division; cc, corpus callosum; CeA, central amygdala; CLA, claustrum; cpd, cerebral peduncle; Ctx, cortex; DR, dorsal raphe nucleus; fr, fasciculus retroflexus; GPi, internal globus pallidus; GU, gustatory areas; MOp, primary motor area; MOs, secondary motor area; MRN, midbrain reticular nucleus; opt, optic tract; PAG, periaqueductal gray; PF, parafascicular nucleus; PO, posterior complex of the thalamus; PPN, pedunculopontine nucleus; PRNc, pontine reticular nucleus, caudal part; PRNr, pontine reticular nucleus, rostral part; SCm, superior colliculus, motor-related; SI, substantia innominata; SNc, substantia nigra pars compacta; SSp-bfd, primary somatosensory area, barrel field; SSp-ll, primary somatosensory area, lower limb; SSp-m, primary somatosensory area, mouth; SSp-n, primary somatosensory area, nose; SSp-tr, primary somatosensory area, trunk; SSp-ul, primary somatosensory area, upper limb; SSs, supplemental somatosensory area; STN, subthalamic nucleus; VAL, ventral anterior-lateral complex of the thalamus; VISC, visceral area; VM, ventromedial thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; Th, thalamus; ZI, zona incerta.

Figure 5.

dSPN and iSPN inputs to GPe have unique properties. a, Left, Confocal micrographs of two neighboring brain sections showing the iSPN projection to the GPe. The dStr of an Adora2a^Cre mouse was transduced with a CreOn-ChR2-eYFP AAV. The association of enkephalin (top) and substance P (bottom) with eYFP-labeled axonal fibers in the GPe was assessed with immunofluorescence labeling. Right, High-magnification images showing the spatial relationship between enkephalin (magenta, top) and substance P (magenta, bottom) with iSPN axons (green) in the GPe. Rectangular images represent orthogonal projections. Crosshairs indicate the projected planes. Insets, Magnified views of areas within the dotted square outlines. b, Left, Confocal micrographs of two neighboring brain sections showing the dSPN projection to the GPe. The dStr of a Drd1a^Cre mouse was transduced with a CreOn-ChR2-eYFP AAV. The association of enkephalin (top) and substance P (bottom) with eYFP-labeled axonal fibers in the GPe was assessed with immunofluorescence labeling. Right, High-magnification images represent the spatial relationship between enkephalin (magenta, top) or substance P (magenta, bottom) and dSPN axons (green). Insets, Magnified views of areas within the dotted square outlines. c, Top, Two representative voltage-clamp recordings showing striatopallidal (dStr-GPe) IPSCs in control (gray) condition and in the presence of quinpirole (10 μm, black). IPSCs were evoked with optogenetics; light was delivered in the dStr. Drd1a^Cre and Adora2a^Cre mice were used to examine the properties of dSPN and iSPN inputs, respectively. dSPN-GPe IPSCs (top) and iSPN-GPe IPSCs (bottom) are shown. The recordings were obtained from an Npas1⁺ and a PV⁺ neuron, respectively. Bottom, The relative iSPN-GPe IPSC amplitude (mean ± SEM) was plotted versus time (n = 12 neurons). Horizontal black line indicates the timing of quinpirole application. d, Top, Box plots summarize the effect of quinpirole on dStr-GPe IPSC amplitude. Left, All recorded neurons for dSPN or iSPN input (dSPN input = 6 neurons, iSPN input = 16 neurons). Right, iSPN input broken down by neuron types (PV⁺ = 4 neurons, Npas1^– = 7 neurons, Npas1⁺ = 5 neurons). Bottom, Quantification of eGFP⁺ bouton density in the GPe shown in e (DMS: Adora2a^Cre = 6 ROIs, Drd1a^Cre = 4 ROIs, Tac1^Cre = 6 ROIs, DLS: Adora2a^Cre = 6 ROIs, Drd1a^Cre = 6 ROIs, Tac1^Cre = 6 ROIs). e, Representative high-magnification images showing SPN terminals (eGFP⁺, white) in the GPe. CreOn-mRuby2-T2A-Synaptophysin-eGFP AAV was injected into the dStr subregions of Adora2a^Cre, Drd1a^Cre, and Tac1^Cre mice. Terminals from iSPNs (Adora2a^Cre) or dSPNs (Drd1a^Cre and Tac1^Cre) from the DMS (left) or DLS (right) were abundant in the GPe. Images from medial and intermediate levels of the GPe are shown for terminals from the DMS and DLS, respectively. Maximal intensity from 10 optical sections is shown for each example.

Figure 6.

Topographical organization of dStr-GPe-dStr projections. a, Representative epifluorescence images showing eGFP signals from CreOn-mRuby2-T2A-Synaptophysin-eGFP AAV injections into the DMS or DLS of Adora2a^Cre (left) and Drd1a^Cre (right) mice. b, Graphical representation of terminals from iSPNs and dSPNs across five different sagittal planes. AAV was injected into Adora2a^Cre (left), Drd1a^Cre (middle), and Tac1^Cre (right) mice. Low-magnification epifluorescence images (see a) of eGFP-labeled terminals were vectorized. Orange line segments indicate terminals from the DMS. Purple line segments indicate terminals from the DLS. Scale bar applies to all panels. ac, Anterior commissure; ec, external capsule, GPi, internal globus pallidus; ic, internal capsule. c, Top left, Low-magnification image showing fluorescence signals following a striatal CTb 647 injection. Top right, Normalized counts (mean ± SEM) for CTb-labeled (CTb⁺) Npas1⁺ neurons in the GPe are shown. Neurons were retrogradely labeled from DMS (orange, n = 148 neurons, 12 sections) and DLS (purple, n = 241 neurons, 12 sections) injections. Arrowheads on the horizontal axis indicate injection locations. Bottom, CTb⁺ (magenta) and Npas1⁺ (green) neurons were seen in the GPe. White circles represent colabeling. Inset, Representative example showing the colabeling of CTb 647 and Npas1 in a GPe neuron.

Figure 7.

dSPNs strongly innervate Npas1⁺ neurons. a, Top, Low-magnification images showing GPe starter cells (left, white) and striatal input cells (right, green) in coronal brain sections from Npas1^Cre (left) or Pvalb^Cre (right) mice injected with hSyn-DIO-mRuby2-TVA-RVG AAV and RbV-expressing eGFP sequentially into the GPe. Bottom, Representative high-magnification images showing that striatal input cells (eGFP⁺, cyan) from Pvalb^Cre (left) or Npas1^Cre (right) mice were positive for Drd1a (yellow) or Drd2 (magenta) mRNA. Blue represents DAPI. b, Left, Representative traces showing IPSCs recorded in PV⁺ or Npas1⁺ neurons with optogenetic stimulation of dSPNs (top) or iSPNs (bottom) in the DLS. Traces from five stimulus intensities (2.4–56.7 mW/mm²) are shown. Right, Input-output relationship for corresponding inputs. Mean and SEM are displayed. A full list of median values, sample sizes, and statistical comparisons at different stimulus intensities for discrete inputs is shown in Table 3. Response rate for each input is shown in Table 5. c, Summary of anatomical (see a; Pvalb^Cre = 5 mice, Npas1^Cre = 5 mice) and functional (see b; dSPN-PV⁺ = 8 neurons, dSPN-Npas1⁺ = 28 neurons, iSPN-PV⁺ = 12 neurons, iSPN-Npas1⁺ = 20 neurons) connectivity for discrete dStr-GPe inputs. The IPSC amplitudes at maximal stimulus intensity are plotted. d, Top, Representative raster plots showing that stimulation (10 Hz for 2 s) of iSPNs or dSPNs in the DLS strongly suppressed firing of PV⁺ or Npas1⁺ neurons, respectively. Blue bars represent the period of blue light stimulation. Bottom, Summary of changes in baseline activity with stimulation of discrete dStr-GPe inputs (dSPN-PV⁺ = 9 neurons, dSPN-Npas1⁺ = 11 neurons, iSPN-PV⁺ = 9 neurons, iSPN-Npas1⁺ = 11 neurons).

Figure 8.

dSPN inputs are selectively strengthened following chronic 6-OHDA lesion. a, Connection strengths from dSPNs (left) and iSPNs (right) to PV⁺ neurons and Npas1⁺ neurons across two DLS (top) and DMS domains (bottom) were assessed with both somatic (i.e., intrastriatal, dStr) and terminal (i.e., intrapallidal, GPe) stimulation. Data from naïve (black) and 6-OHDA-lesioned (red) mice are shown. Results are summarized as box plots. For a full listing of IPSC amplitudes and sample sizes, see Tables 3 and 4. b, Representative voltage-clamp recordings show that IPSCs arose from dSPNs in PV⁺ neurons (top) and Npas1⁺ neurons (bottom). A range of stimulus intensities (2.4–56.7 mW/mm²) were tested. Note the increase in IPSC amplitude in 6-OHDA mice (red, right) compared with that from naïve (black, left). c, GABA_A receptor dependency of the IPSCs was tested with a GABA_A receptor antagonist, SR95531 (10 μm). Each marker represents a cell (dSPN-Npas1⁺ = 4 neurons, iSPN-PV⁺ = 5 neurons). d, The spatial specificity of the optogenetic stimulation was assessed with the applications of TTX (1 μm, light green) and its coapplication with 4-aminopyridine (4-AP 100 μm, dark green). Each marker represents a cell (dSPN-Npas1⁺: dStr stim = 5 neurons, GPe stim = 5 neurons, iSPN-PV⁺: dStr stim = 7 neurons, GPe stim = 3 neurons). e, Confocal micrographs showing the innervation of dSPN axons in the GPe from naïve (left) and 6-OHDA-lesioned (right) mice. To visualize dSPN axons, Drd1a^Cre mice were transduced with CreOn-ChR2-eYFP AAV. Intermediate (top) and medial (bottom) levels are shown. f, Representative high-magnification images show dSPN bouton density in the GPe from naïve (left) and 6-OHDA-lesioned (right) mice. dSPN boutons were represented by VGAT⁺ (magenta) and ChR2-eYFP⁺ (green) puncta. Breakout panels represent orthogonal xz projection (bottom) and yz projection (right). Crosshairs indicate the pixel of interest. White represents the colocalization of the signals. Insets, Magnified views of areas within the dotted square outlines. g, Quantification of the data shown in e and f: axonal density (naïve = 4 mice, 6-OHDA = 6 mice), eYFP⁺-VGAT⁺ puncta density (naïve = 6 sections, 6-OHDA = 10 sections), and eYFP⁺-gephyrin⁺ puncta density (naïve = 6 sections, 6-OHDA = 8 sections).

Figure 9.

Schematic summary of proposed mechanisms for the observed motor responses. Npas1⁺ neurons and PV⁺ neurons in the GPe are the principal recipients of dSPN input and iSPN input, respectively. Npas1⁺ neurons in turn project to the dStr and the SNc (Abecassis et al., 2020; Cui et al., 2021; Evans et al., 2020). PV⁺ neurons influence the output of SNr neurons via their direct projection and indirect projection through the subthalamic nucleus. Selective activation of dSPNs (e.g., with optogenetic stimulation) leads to either motor promotion or suppression, depending on the balance of these two striatopallidal subcircuits. The coordination between the two subcircuits can be fine-tuned by local collaterals at the dStr, GPe, and SNr levels. Lateral inhibition between dSPNs and iSPNs is asymmetrical; iSPNs unidirectionally inhibit dSPNs (Taverna et al., 2008). PV⁺ neurons emit numerous local collaterals, thus providing additional crosstalk between the two striatopallidal subcircuits Cui et al. (2021). Local inhibitory circuits in the SNr have also been described; in particular, they can play important roles in regulating information transfer across the mediolateral axis (Brown et al., 2014). The same circuit motifs are conserved across the mediolateral extent of the basal ganglia. The diagrams serve as a working model that explains the behavioral effects observed in this study: DMS, naïve (a), DLS, naïve (b), and DLS, parkinsonian state (c). Stimulation of ^DMSdSPNs has a strong motor-promotion effect as inhibition of Npas1⁺ neuron activity promotes the release of dopamine as a result of increased SNc neuron firing. In addition, as there are fewer PV⁺ neurons in the medial GPe (Kita, 1994; Hontanilla et al., 1998; Mastro et al., 2014; Hernandez et al., 2015; Abecassis et al., 2020), there is relatively weak engagement of the iSPN-PV⁺-SNr pathway. Furthermore, it likely strongly engages the canonical direct-pathway (from dSPNs to SNr) (not shown). Stimulation of ^DLSdSPNs has a strong motor-suppression effect as there are more PV⁺ neurons in the lateral GPe. This favors engagement of the iSPN-PV⁺-SNr pathway and hence a net motor suppression. Following the loss of dopamine neurons, output from both dSPNs and Npas1⁺ neurons is strengthened (Glajch et al., 2016). Despite the lack of changes in the connection strength of the iSPN-PV⁺ pathway, the strong disinhibition of iSPNs by Npas1⁺ neurons is expected to lead to strong disinhibition of the iSPN-PV⁺-SNr pathway, thus contributing to the hypokinetic symptoms of PD.