Fig 1.
Validation of Chat-Cre, D1-Cre, and D2-Cre mouse lines.
(A) Experimental design for axonal tracing. AAV-DIO-EmGFP virus was injected into the dorsal striatum of ChAT-Cre, D1-Cre, or D2-Cre mice. Histology was performed two weeks later. (B) Following the injection of AAV-DIO-EmGFP virus into the dorsal striatum of a D1-Cre mouse, we observed strong terminal fluorescent signals in the GPi/EP and the SNr. For clarity, EmGFP signals are shown in white. (C) AAV-DIO-EmGFP virus injection into the dorsal striatum of a D2-Cre mouse produced EmGFP+ terminal signals mainly in the GPe. (D) Infusion of AAV-DIO-EmGFP viral vectors in the dorsal striatum of a ChAT-Cre mouse resulted in EmGFP expression (green) exclusively in ChAT-immunopositive (red) neurons. Abbreviations: GPe, external globus pallidus; GPi/EP, internal globus pallidus/entopeduncular nucleus; SNr, substantia nigra, reticular part.
Fig 2.
The method of rabies-based retrograde tracing.
(A) AAV virus and rabies virus for transsynaptic retrograde tracing. Double-floxed EGFP-TVA and rabies glycoprotein (RG) under control of CAG were respectively packaged into AAV viral vectors. The rabies virus without glycoprotein (SADΔG) was equipped with the mCherry fluorescent protein. (B) Experimental design. On Day 1, AAV-DIO-EGFP-TVA and AAV-DIO-RG viral vectors were injected into the dorsal striatum of ChAT-Cre, D1-Cre, or D2-Cre mice. Two weeks later, SADΔG-mCherry was injected into the same site of the dorsal striatum. Histology was performed after one more week. The starter cells are labeled both in green and red, whereas the input neurons are labeled only in red. (C) Confirmation of effective transsynaptic labeling using rabies virus. Sequential injections of AAV-DIO-EGFP-TVA, AAV-DIO-RG and SADΔG-mCherry viruses into the dorsal striatum of a D1-Cre mouse labeled starter cells (green) and input neurons (red; left panel). Same virus injections did not label any neurons in a wild-type C57BL/6J mouse (right panel). (D) Zoom-in view of starter cells and transsynaptically labeled cells in the dorsal striatum of the D1-Cre mouse shown in (C). (E) Transsynaptic retrograde labeling neurons in the cingulate cortex (cg).
Fig 3.
Monosynaptic inputs onto D1, D2, and ChAT neurons in the dorsal striatum.
(A) Coronal representation of retrograde monosynaptic labeling of input neurons onto D1 MSNs, D2 MSNs, and cholinergic interneurons. (B) Sagittal brain sections of an infected ChAT-Cre mouse, showing innervations to cholinergic neurons in the dorsal striatum. Abbreviations: Au, auditory cortex; BL, Basolateral amygdaloid nucleus; Cg, cingulate cortex; CL, centrolateral thalamic nucleus; CPu, caudate putamen; DRN, dorsal raphe nucleus; GPe, external globus pallidus; LO, lateral orbital cortex; LPMR, lateral posterior thalamic nucleus, mediorostral part; M2, secondary motor cortex; PC, paracentral thalamic nucleus; PF, parafascicular thalamic nucleus; PtA, parietal association cortex; RS, retrosplenial cortex; S1, primary somatosensory cortex; STh, subthalamic nucleus; VO, ventral orbital cortex; V1, primary visual cortex; V2, secondary visual cortex; VL, ventrolateral thalamic nucleus.
Fig 4.
Quantitative analysis of whole-brain monosynaptic inputs to the dorsal striatum D1, D2, and ChAT neurons.
(A) We clustered input area into the dorsal striatum, cortex, thalamus, and other nuclei. Proportions of input neurons from the dorsal striatum and the cortex are significantly different between D1/D2 projection neurons and cholinergic interneurons. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two tailed t-test; n = 4 mice. (B) Proportion of specific extrastriatal areas for each striatal cell type.
Fig 5.
Strong retrograde labeling in representative brain areas following rabies virus infection of striatal D1 MSNs, D2 MSNs, and cholinergic interneurons.
(A) The cingulate cortex and secondary motor cortex (Cg/M2). (B) The secondary visual cortex (V2). (C) The centrolateral/paracentral thalamic nucleus (CL/PL). (D) The external globus pallidus (GPe). (E) The substantia nigra compacta (SNc). (F) The dorsal raphe nucleus (DRN).
Fig 6.
The majority of the intrastriatal input neurons targeting the ChAT neurons are non-cholinergic.
Following the injection of rabies virus and its helper virus, retrograde labeled neurons (red) and cholinergic neurons (green) did not overlap. Additionally, the intrastriatal input neurons and ChAT neurons exhibited distinct morphology and soma sizes.
Fig 7.
Summary of the inputs to the dorsal striatum cholinergic interneurons and D1/D2 projection neurons.
Blue, green, and red lines indicate monosynaptic inputs to striatal ChAT, D1, and D2 neurons, respectively. Thickness of the lines represents proportional input strength (cell numbers) from given brain areas. Abbreviations: CPu, caudate putamen; GPe, external globus pallidus; GPi/EP, internal globus pallidus/entopeduncular nucleus; SNc, substantia nigra, compact part; SNr, substantia nigra, reticular part; STN, subthalamic nucleus;.