The Tail of the Mouse Striatum Contains a Novel Large Type of GABAergic Neuron Incorporated in a Unique Disinhibitory Pathway That Relays Auditory Signals to Subcortical Nuclei

Morphological features of SMI-32-positive large neurons in the TS. A, Double-immunofluorescence image consists of SMI-32 (green) and PV (blue) immunoreactivities. SMI-32-immunoreactive filamentous structures are clearly seen inside both soma and dendrites. Scale bar, 20 µm. B, C, Comparison of type 1 (B, arrow) and type 2 (C, arrowheads) neurons. The latter are much smaller in soma size and weaker in staining intensity. Image in C was taken from the rostral striatum where numerous bundles of fibers passing through are also labeled. A similar staining pattern in fiber bundles is also visible in the dorsal half of the striatum in Figure 1A. Scale bar, 100 µm. D, Reconstruction of three cells contained in Figure 1G. Most of the dendrites are directed to the intermediate (I) division of the trilaminar part. Multiple contours in the intermediate division are tracings of outlines showing areas of low TH signal, acquired from serial 40-µm-thick sections. Scale bar, 200 µm. E, Distribution of the two types of neurons along the rostrocaudal axis. Type 1 neurons are located only in caudal locations, whereas type 2 neurons are found at both rostral and caudal levels. F, Quantitative analysis of the cell body perimeter. Type 1 neurons (n = 32 cells) were significantly larger than type 2 neurons (n = 246 cells; ***p < 0.001, Welch's t test). G, Quantitative analysis of immunoreactivity in somata. Gray levels of type 1 neurons (n = 32 cells) were significantly higher than those of type 2 neurons (n = 246 cells; ***p < 0.001, Welch's t test). Error bars show SDs.

Quantification of the length of dendritic segments of TSL neurons located in each division of the TS. Data are expressed as the proportion of dendritic length in each division to the total dendritic length of each cell. Medially oriented dendrites entering the GP are also included in this quantification.

Colocalization of other neurochemicals. A–C, Comparison between TSL neurons and ChAT-positive neurons. Dual-immunofluorescence image in A consists of ChAT (magenta) and SMI-32 (green) immunoreactivities. There is no overlap between SMI-32-positive neurons (arrows) and ChAT-positive neurons. Another distinctive feature is that SMI-32 neurons are larger than ChAT-positive neurons, as shown in B. Scale bars: A, 50 µm; B, 20 µm. C, Quantitative analysis of the soma size of the two populations. The mean size of SMI-32 neurons (n = 42) was significantly larger than that of ChAT-positive neurons (n = 147 cells: ***p < 0.001, Welch's t test). D, E, PV immunoreactivity in TSL neurons. Dual-immunofluorescence image in D consists of SMI-32 (green) and PV (magenta) immunoreactivities; the former is shown separately in E as a monochrome image. SMI-32-positive TSL neurons (arrows) show immunoreactivity for PV, but not all PV neurons are immunoreactive for SMI-32, as indicated by arrowheads. Scale bar, 50 µm. F–I, Dual-immunofluorescence images in F and H consist of SMI-32 (green) and GAD (magenta) immunoreactivities, with the latter shown in G and I as monochrome images. GAD immunoreactivity inside the soma indicates that this cell is a GABAergic neuron. Note the dense accumulation of GAD-immunoreactive boutons on surfaces of both soma and dendrites at proximal (F, G) and distal (H, I) sites. Scale bars, 10 µm.

Glutamatergic input structures of TSL neurons. A, Triple-immunofluorescence images (left column) consist of PV (green), VGluT1 (blue), and VGluT2 (red) immunoreactivities, which are shown in the middle and right columns with a different color set using green for PV and magenta for VGluT1 and VGLuT2 to see the mode of contact more clearly. VGluT1-positive boutons rarely contact TSL neurons, whereas VGluT2-positive boutons make contact with both soma and dendrites of a TSL neuron. Arrows indicate PV-positive boutons abutting on TSL neurons. Scale bars: left column, 10 µm; middle and right columns, 5 µm. B–D, Comparisons of the numerical density of bouton contacts on soma (B), proximal dendrite (C), and distal dendrite (D) between VGluT1-positive and VGluT2-positive boutons. ***p < 0.0001, t test.

Anterograde tracer labeling to determine the source of VGluT2-positive boutons on TSL neurons. A, Injection of phal (red) is into the medial division of the MG, which is located just medial to the dorsal (MGD) and ventral (MGV) divisions of the MG. IP, interpeduncular nucleus; PAG, periaqueductal gray; RN, red nucleus; SC, superior colliculus; SN, substantia nigra. Scale bar, 1 mm. B1, SMI-32 immunoreactivity in the trilaminar part of the TS. The arrow indicates a TSL neuron located in the medial (M) division, which extends dendrites to the intermediate (I) division. The frame area is superimposed on the phal image in B2 and enlarged in C. Scale bar, 100 µm. B2, Axons and axon terminals originating from the MGM are distributed broadly in the GP as well as in the trilaminar part of the TS. C, High-power triple fluorescence image consisting of SMI-32 (green), Phal (red), and VGluT2 (blue) immunoreactivities. Two large boutons make contact with a dendrite of a TSL neuron. Scale bar, 10 µm. Insets, Enlargement of the circled area. The Phal-positive bouton makes direct contact with the dendrite and shows VGluT2 immunoreactivity. Scale bar, 1 µm.

SP- and Enk-immunoreactive boutons on TSL neurons. A, Triple-immunofluorescence image for PV (green), SP (blue), and Enk (red), showing bouton contacts on soma and dendrite. Scale bar, 10 µm. B, C, A different color set consisting of magenta (SP and Enk) and green (PV) is used. Note the sparse contacts made by SP-positive boutons (B), which are contrasted with dense contacts by Enk-positive boutons (C). D–G, Framed area is further examined in a correlative CLSM-EM quantitative analysis. D, Enk-positive boutons (magenta) indicated by arrows (1–7) make contact with a PV-positive dendrite (green). Two boutons (arrowheads, a, b) are detached from the dendrite and are excluded from the counting. Crossed arrow in D indicates PV-positive axonal bouton on the dendrite. Asterisks in D and E indicate the same dendrite. Scale bar, 5 µm. E, An EM image of the same structure that is shown in D. Enk and PV immunoreactivities are shown using DAB-nickel and DAB, respectively. Because of the omission of detergent from the incubating medium, DAB signals in EM sections cut at the deeper part of the specimen are generally weakened, and thus PV immunoreactivity in the dendrite can be seen only at the bottom left part (PV+). All seven axon terminals (arrows 1–7) corresponding to boutons in D form symmetrical synapses with the TSL dendrite (inset), whereas two boutons (arrowheads, a, b) do not make contact with the dendrite. Scale bar: D, 2 µm; (in D) D inset, 0.2 µm. F, G, Quantitative analyses of bouton (D) and synaptic (E) contacts indicate that the densities of contacts by Enk-positive elements are significantly higher than those by SP-positive elements in both the light (F) and electron (G) microscopic analyses.

Encasement of TSL neurons by Enk-positive boutons. A, Low-power image of Enk immunoreactivity. The GP is a target region of Enk-positive indirect pathway neurons; thus, it shows intense immunoreactivity for Enk. Three divisions of the trilaminar part [medial (M), intermediate (I), lateral (L)] are characterized by differential immunoreactivity for Enk with graded intensity from the medial to lateral. Thus, the M division generally shows very low immunoreactivity for Enk, but intense labeling along string-like structures goes through the medial division toward the intermediate division, as seen in a framed area. Scale bar, 100 µm. B, Double-immunofluorescence image of the framed area in A, consisting of SMI-32 (green) and Enk (red) immunoreactivities. Numerous Enk-positive boutons accumulate around TSL neurons. Note that these boutons not only densely cover SMI-32-positive somata and dendrites, but also are distributed in the surrounding area. Scale bar, 20 µm. C–E, Double-immunofluorescence image in the intermediate division of the trilaminar part. Again, numerous Enk-positive boutons not only make direct contact with the distal segments of dendrites but also distribute diffusely in surrounding tissue close to the dendrites. Scale bar, 20 µm. F–I, Accumulation of Enk-positive boutons is very selective, avoiding a SMI-32-negative PV neuron (arrowhead) located close to TSL neurons (arrows). Scale bar, 20 µm.

Electron micrographs of a dendrite of a TSL neuron and surrounding structures double immunolabeled for PV and Enk, the immunoreactivities of which are visualized using DAB and DAB-nickel. A, A PV-positive dendrite receives many symmetrical synaptic contacts from both Enk-positive (asterisks) and Enk-negative (open circles) axon terminals. It also receives asymmetrical synapses (squares). Framed areas (b–d and f) are enlarged in B–D and F. B–D, Enk-positive axon terminals (asterisks) form symmetrical synapses (arrowheads) with the dendrite of the TSL neuron shown in A. Immunonegative axon terminals (open circles) also form symmetrical synapses (arrowheads). In D, a rare occasion of a spine extending from the dendrite of the TSL neuron is shown by the arrow. E1, Enlargement of the framed area in D. The spine (sp 1) receives a symmetrical synapse (arrowhead) from an axon terminal on the right side (open circle). The left-sided terminal (square) forms asymmetrical synapses (white arrowheads) with the dendritic shaft of the TSL neuron and another spine of unidentified origin (sp 2). E2, Enlargement of E1. Double arrows indicate adherens junction. F, The TSL dendrite receives asymmetrical synapses (white arrowheads). G1–G11, Serial ultrathin sections of the framed area (g) in A. Enk-positive elements (asterisks) contain numerous synaptic vesicles but do not form synapses with surrounding structures including a neighboring spine (sp), which is targeted by Enk-positive (dot) and Enk-negative (open circle) axon terminals forming symmetrical synapses (arrowheads).

Immunoelectron micrographs of a soma of a TSL neuron and surrounding structures double immunolabeled for PV and Enk in the medial division of the TS. A, A large soma is lightly stained for PV using DAB as a chromogen, whereas Enk immunoreactivity is detected in axon terminals surrounding the soma, using DAB-nickel as the second chromogen showing denser signals. The perimeter of the soma is 115 μm, indicating that this cell is a TSL neuron (Figure 4C). The large perikaryon is filled with organelles such as numerous mitochondria (m) and rough endoplasmic reticulum (rER). Axon terminals surrounding the soma include both Enk-positive (asterisks) and negative ones, part of which in framed areas are enlarged in B and C. Arrows indicate Enk-positive elements around the soma with some distance away from it. B, Enk-positive axon terminals make direct contacts with the soma, forming symmetrical synapses (arrowheads). C, A PV-positive large axon terminal (white dot) forms a symmetrical synapse (arrowhead) with the soma of TSL neuron that is also labeled for PV.

Visualization of an MSN located in the intermediate division of the trilaminar part of the TS using GAD67-CrePR knock-in mouse. A, Low-magnification image of the trilaminar part, labeled for tdTaomato (red), Enk (green), and PV (blue). A cell showing intense immunoreactivity for tdTomato is located in the intermediate division (i). G, Globus pallidus; m, medial division; l, lateral division. Scale bar, 200 µm. B, Enlargement of the tdTomato-positive cell. Note the presence of massive spines along dendrites, indicating that this cell is an MSN. An axon (ax) emitting from the cell body bifurcates many times and arborizes around the cell to form local axon collaterals. Arrow indicates a point where the axon was cut at the section surface. The corresponding position in a neighboring section is shown by arrow in C1. Scale bar, 50 µm. C1–C3, Continuation of the axonal arborization originating from the cell in B. C1, C2, The axon forms many large boutons (C1) that surround and make contact with a dendrite of a TSL neuron (C2). C3, In triple-immunofluorescence image, numerous Enk-positive elements accumulate around the dendrite, a characteristic profile of TSL dendrite. Scale bar, 10 µm. D, E, Enlargement of the boutons indicated by arrows in C2, viewed from three different angles. The images clearly indicate that Enk-positive elements are contained inside the tdTomato-positive boutons. Scale bar, 1 µm. F, A reconstruction of the tdTomato-labeled MSN from serial sections (soma and dendrites, green; axon, dark blue). Dendrites of four TSL neurons (TSL 1–4) are also reconstructed, and spheres of different colors show the sites of contacts between the MSN axon and individual dendrites. The dendrite shown in C belongs to TSL1, with which the axon makes as many as 15 contacts (red spheres). Scale bar, 100 µm.

A reconstruction of a tdTomato-labeled MSN that is located in the intermediate division and is different from the cell demonstrated in Figure 11. Soma and dendrites are shown in orange, and axon is shown in dark blue. Axon makes as many as 17 contacts (green spheres) with the dendrite of TSL2, which is the same dendrite shown in Figure 11F. Note that an axon of this MSN also targets the proximal dendrite of TSL 5 in the medial division of the trilaminar part, where the axon makes 13 contacts with the dendrite. Scale bar, 100 µm.

Anterograde tracer study applied to GAD67-CrePR knock-in mouse. A–H, Triple-immunofluorescence images consist of tdTomato (red), phal (green), and PV (blue) immunoreactivities (A, E), which are shown separately in B and F, C and G, and D and H, respectively. A–D, Insets, Injection site centered into the A1. m, Medial division; i, intermediate division; l, lateral division. Scale bars: (in A) A–D, 200 µm; (in A, inset) A–D, Insets, 400 µm. E–H, Enlargement of the framed area in A. Axons labeled for phal make direct contacts (arrowheads) with a dendrite of an MSN that has many spines. Scale bar, 10 µm.

Retrograde tracer studies. A, D, E, F, After the injection of FG into the rostral part of the ZI (D), sections were double immunostained for SMI-32 (green) and FG (magenta; A), which are separately shown in E and F. Retrograde labeling was observed in a soma of TSL neuron (arrow) as well as several neurons in the GP and surrounding area (arrowheads). B, C, Labeling in the TSL neuron is enlarged and shown separately for SMI-32 (B) and FG (C). G, J, K, L, After the injection of FG into the VM of the thalamus (VM; J), sections were double immunostained for SMI-32 (green) and Fluoro-Gold (magenta; G), which are separately shown in K and L. Retrograde labeling was observed in a soma of TSL neuron (arrow). H, I, Labeling in TSL neuron is enlarged and shown separately for SMI-32 (H) and FG (I). Scale bars: A, E–G, K, L, 100 µm; C, I, 20 µm; in D, J, 500 µm.