1. Volume 22, Issue 9, Pages 2370-2382 (February 2018)
Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons 
Shigeki Kato, Ryoji Fukabori, Kayo Nishizawa, Kana Okada, Nozomu Yoshioka, Masateru Sugawara, Yuko Maejima, Kenju Shimomura, Masahiro Okamoto, Satoshi Eifuku, Kazuto Kobayashi 
Cell Reports 
Volume 22, Issue 9, Pages (February 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 2370-2382DOI: (10.1016/j.celrep.2018.02.016)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Expression of IL-2Rα-GFP Transgene in Neurons Innervating the DS
(A) Coordinates of the HiRet vector injection. The anteroposterior coordinates (mm) from bregma are shown.
(B) GFP immunohistochemistry showing IL-2Rα-GFP expression in the DS around the injection sites.
(C) Cresyl violet staining and NeuN immunostaining of striatal sections.
(D) GFP immunohistochemistry demonstrating transgene expression through retrograde transport into the brain regions. Right: magnified views of the rectangles in the left photos.
(E) Double-fluorescence histochemistry with Alexa Fluor 555-conjugated CTb and immunostained GFP. Confocal microscopic images of CL sections are shown. CTb-positive signals, GFP-positive signals, and merged images are indicated in red, green, and yellow, respectively.
fr, fasciculus retroflexus; SNr, substantia nigra pars reticulata. Scale bars: 1 mm (A and B), 200 μm (C and D), and 50 μm (E).
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4. Figure 2
Selective Elimination of CL Neurons Innervating the DS by IT Treatment
(A) Coordinates for IT injection. The anteroposterior coordinate (mm) from bregma is shown. Scale bar: 1 mm.
(B) Impact of IT treatment on cells transduced in a retrograde manner by the HiRet vector. Mice (n = 4 for each group) received a bilateral injection of the HiRet-IL-2Rα-GFP or HiRet-GFP vector into the DS, and then they were treated with IT or PBS by injection into the unilateral CL. GFP immunohistochemistry of CL sections was performed, and representative images of the staining are shown.
(C) Cell counts of GFP-immunopositive cells per section. Data are presented as mean ± SEM. ∗p < 0.001 compared with each of the HiRet-GFP/PBS, HiRet-GFP/IT, and HiRet-IL-2Rα-GFP/PBS groups (Bonferroni’s test).
(D) Cresyl violet staining of sections prepared from the vector-injected mice.
(E) GFP immunohistochemistry of sections prepared from the injected mice.
(F) Anterograde tracing of axon terminals arising from the CL. The vector-injected animals were treated unilaterally with IT or PBS into the CL and used for BDA injection into the CL. Striatal sections were prepared and stained for BDA. Lateral images are magnified views of the rectangles in the medial images, and camera lucida drawings of labeled axonal terminals are shown beneath the magnified views. LV, lateral ventricle.
Scale bars: 1 mm (A) and 200 μm (B and D–F).
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5. Figure 3
Attenuation of Striatal LFP Evoked by Optical Stimulation in Mice Lacking CL Thalamostriatal Neurons
(A) Diagram of the recording of optically evoked LFP. Optical stimulation was delivered through an optical fiber stuck in the CL. LFP and spiking activity were recorded through a stainless-steel electrode in the DS and CL.
(B) Histological sections showing the locations of the recorded electrode (left) and optical fiber (right). The locations of the electrode tips are indicated by arrowheads. Scale bar: 1 mm.
(C and D) Recordings of neuronal activities evoked by optical stimulation. Mice (n = 5 or 6 for each group) received bilateral injections of the HiRet-Cre vector in the DS and of the AAV-DIO-IL-2Rα/ChR2/YFP vector in the CL. Then, they were injected with IT solution or PBS in the CL.
(C) Peristimulus time histograms of the CL and DS neurons. The waveforms in the insets are the spike shapes.
(D) LFP in the DS evoked by optical CL stimulation. The waveform of each condition was calculated by averaging the evoked potentials to 200 stimulation trains. Grand averaged waveforms of the IT-treated and PBS-treated animals are shown, with negative voltages upward. Bold and sharp lines indicate the mean values and SEMs, respectively. The amplitude of the peak was significantly reduced in the IT-treated group (arrow).
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6. Figure 4
Acquisition and Performance of Visual Discrimination in Mice Lacking CL Thalamostriatal Neurons
(A) Strategy for visual stimulus-dependent two-choice reaction time task.
(B–D) Acquisition of visual discrimination. Mice (n = 7 for each group) were bilaterally injected with the HiRet-IL-2Rα-GFP vector into their DS, bilaterally injected with IT or PBS into their CL, and then tested for acquisition in the task. The correct response ratio (B), correct response time (C), and omission ratio (D) in each session block comprising three consecutive sessions are plotted.
(E–G) Performance of visual discrimination. Mice (n = 10 or 12 for each group) were injected with the vector into the DS and trained to perform the task. After achievement of the 70% criterion, the mice were injected with IT or PBS into their CL and then tested for the task performance. The correct response ratio (E), correct response time (F), and omission ratio (G) in each session are plotted. The values during the pretreatment phase (Pre) are the averages of 3 days before the IT treatment.
Data are presented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 compared with the HiRet-IL-2Rα-GFP/PBS group (Bonferroni’s test).
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7. Figure 5
Behavioral Flexibility in Animals with Deleted Thalamostriatal Neurons
(A) Reversal learning. Mice (n = 11 or 12 for each group) were treated serially with the HiRet-IL-2Rα-GFP vector in the DS and with IT or PBS in the CL and then tested for the reversal task on the basis of place discrimination.
(B) Attentional set-shifting. Mice (n = 10 or 11 for each group) were treated serially with the vector in their DS and with IT or PBS in their CL and then were tested for the set-shifting task.
Data are presented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 compared with the HiRet-IL-2Rα-GFP/PBS group (Bonferroni’s test). Insets show session number to reach the criteria. ∗p < 0.05 and ∗∗p < 0.01 compared with the HiRet-IL-2Rα-GFP/PBS group (Student’s t test).
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8. Figure 6
Spatial Working Memory Performance in Mice Lacking CL Thalamostriatal Neurons
(A) Strategy for delayed matching-to-position task.
(B) Performance of delayed matching-to-position task. Mice (n = 10 or 12 for each group) were injected with the HiRet-IL-2Rα-GFP vector into the DS, and then they were injected with IT or PBS into their CL and tested for the task. Data are presented as mean ± SEM.
(C) Strategy for delayed alternation task with a T maze.
(D) Performance of delayed alternation task. Mice (n = 7 for each group) were treated as mentioned and used for the task.
Data are presented as mean ± SEM.
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9. Figure 7
Impaired Behavioral Flexibility Caused by Chemogenetic Suppression of the CL Thalamostriatal Activity
(A) Strategy for pathway-specific chemogenetic manipulation. Mice received an injection of the HiRet-Cre vector in the DS and of the AAV-DIO-hM4Di/GFP vector in the CL. Then, they were systemically administered CNO, resulting in the silencing of selective neural pathways.
(B) GFP immunohistochemistry showing transgene expression in the CL. Light image indicates a magnified view of the rectangle in the left image. Scale bar: 200 μm.
(C) Slice electrophysiology showing CNO responsiveness. Representative trace for spontaneous and evoked action potentials in CL neurons is indicated. Dotted line indicates 0 mV level. Histograms represent membrane potentials and firing frequency of neurons expressing hM4Di before (Pre) and after (Post) the application of CNO (10 μM) (n = 5).
(D) Performance of visual discrimination. Mice (n = 6 for each group) that received vector injections were trained to perform the task. After achievement of the 70% criterion, the mice were administered i.p. CNO (5 mg/kg) or PBS 30 min before the behavioral test and then performed the task. The correct response ratio, correct response time, and omission ratio for each session are plotted. The values during the pretreatment phase (Pre) are the averages of 3 days before drug treatment.
(E) Reversal learning task. The injected mice (n = 8 for each group) were administered i.p. CNO (5 mg/kg) or PBS 30 min before the behavioral test and then performed the reversal learning task.
(F) Attentional set-shifting. Mice (n = 6 for each group) were treated as mentioned and tested regarding performance of the attentional set-shifting task.
Data are presented as mean ± SEM. ∗p < 0.05 compared with the control group (Bonferroni’s test). Insets show the number of sessions required to reach the criteria. ∗p < 0.05 and ∗∗p < compared with the control group (Student’s t test).
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