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, 30 (4), 1178-1194.e3

The Functional Organization of Cortical and Thalamic Inputs Onto Five Types of Striatal Neurons Is Determined by Source and Target Cell Identities

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The Functional Organization of Cortical and Thalamic Inputs Onto Five Types of Striatal Neurons Is Determined by Source and Target Cell Identities

Yvonne Johansson et al. Cell Rep.

Abstract

To understand striatal function, it is essential to know the functional organization of the numerous inputs targeting the diverse population of striatal neurons. Using optogenetics, we activated terminals from ipsi- or contralateral primary somatosensory cortex (S1) or primary motor cortex (M1), or thalamus while obtaining simultaneous whole-cell recordings from pairs or triplets of striatal medium spiny neurons (MSNs) and adjacent interneurons. Ipsilateral corticostriatal projections provided stronger excitation to fast-spiking interneurons (FSIs) than to MSNs and only sparse and weak excitation to low threshold-spiking interneurons (LTSIs) and cholinergic interneurons (ChINs). Projections from contralateral M1 evoked the strongest responses in LTSIs but none in ChINs, whereas thalamus provided the strongest excitation to ChINs but none to LTSIs. In addition, inputs varied in their glutamate receptor composition and their short-term plasticity. Our data revealed a highly selective organization of excitatory striatal afferents, which is determined by both pre- and postsynaptic neuronal identity.

Keywords: IT tract/PT tract; NMDA to AMPA ratio; cholingergic interneuron; corticostriatal pathway; fast-spiking interneuron; low-threshold spiking interneuron; medium spiny neuron; multineuron patch-clamp; striatum; thalamostriatal pathway.

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

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Figure 1
Figure 1
S1 Input Preferentially Excites Striatal MSNs and FSIs (A) Schematic of virus injection in S1 and the recording site in dorsolateral striatum. (B) Confocal image of the injection site in a D2-tdTomato mouse. Red, D2-MSNs; green, virally transduced cells. Scale bar: 1 mm. (C) High magnification of cortex in (B) showing a neurobiotin-filled pyramidal cell at the injection site in S1. Scale bar: 100 μm. (D) High magnification of the striatum in (B) showing representative expression of ChR2-YFP in S1 axon terminals and D2-MSNs in striatum. Scale bar: 100 μm. (E) Left: schematic of the control experiment with virus injection and recording in S1. Center: whole-cell recordings of the pyramidal cell (PYR) shown in (C) and its response to step current injections. Right: light response in the presence of synaptic blockers. Scale bars: 20 mV, 200 pA, 200 ms. (F) Schematic of simultaneous whole-cell recordings of three striatal neurons in a parasagittal slice within the area of S1 axon terminals (green). (G) Triplet whole-cell recordings in striatum. Differential interference contrast (DIC, top), epifluorescent image of YFP-expressing S1 axon terminals (center), and overlay (bottom) of a parasagittal slice with recording pipettes. Scale bar: 500 μm. (H) Schematic of recordings: tdTomato-positive and tdTomato-negative neurons were recorded simultaneously, while S1 fibers were stimulated through the objective. (I) DIC and fluorescent images of simultaneous patch-clamp recordings from two tdTomato-negative cells (putative D1-MSNs) and one tdTomato-positive D2-MSN. Scale bar: 10 μm. (J) Characteristic responses of different striatal neuron types to increasing step current injections. Scale bars: 20 mV, 200 pA, 200 ms. (K) Relative strength of EPSPs in striatal neurons evoked by stimulation of S1 afferents in the presence of gabazine. Left to right: representative traces of triplet recordings. Simultaneously recorded EPSPs are overlaid. D1-MSNs and D2-MSNs are identified in transgenic mouse lines, while the dopamine receptor subtype of "MSNs" is unknown. Scale bars: 2 mV, 20 ms. (L) Summary graph of EPSP amplitudes obtained from pairs and triplets of striatal neurons in response to S1 stimulation. Solid lines indicate paired recordings in which both neurons responded, dashed lines indicate pairs in which only one neuron responded. The proportions of responding cells are shown in pie charts (inserts). Center values represent mean ± SEM (n = 17 D1-MSN-D2-MSN pairs, N = 8; n = 20 FSI-MSN pairs, N = 14; n = 16 LTSI-MSN pairs, N = 12; n = 14 ChIN-MSN pairs, N = 8; two-tailed paired t test/Wilcoxon signed-rank test). See also Figures S1 and S2 and Table S1.
Figure 2
Figure 2
Target Cells and Synaptic Strengths of Ipsi- and Contralateral M1 Inputs Differ (A) Schematic of virus injections in M1 labeling pyramidal cells projecting to both ipsi- and contralateral striatum. (B) Confocal image of the injection site in a D2-tdTomato mouse. Red, D2-MSNs; green, virally transduced cells. Scale bar: 1 mm. (C) Confocal image of YFP-expressing axon terminals (green) innervating ipsi- and contralateral striatum in a D2-tdTomato mouse. Scale bar: 1 mm. (D) High magnification of (C) showing M1 axon terminals projecting to contralateral striatum (IT tract) and two recorded neurons filled with neurobiotin. Scale bar: 250 μm. (E) High magnification of (D) showing two MSNs that responded to contralateral M1 input. Scale bar: 50 μm. (F) Top: schematic of unilateral virus injections in M1 and striatal multi-neuron recordings ipsilateral to the injection site. Bottom: relative strength of EPSPs in striatal neurons evoked by stimulation of M1I afferents in the presence of gabazine. Left to right: representative traces of triplet recordings. Simultaneously recorded EPSPs are overlaid. Scale bars: 2 mV, 20 ms. (G) Same as in (F) for recordings obtained contralateral to the injection in M1. (H) Summary graph of EPSP amplitudes obtained from pairs and triplets of striatal neurons in response to M1I stimulation. Solid lines indicate paired recordings in which both neurons responded; dashed lines indicate pairs in which only one of the two neurons responded. The proportions of responding cells are shown in pie charts (inserts). Center values represent mean ± SEM (n = 16 D1-MSN-D2-MSN pairs, N = 5; n = 15 FSI-MSN pairs, N = 4; n = 18 LTSI-MSN pairs, N = 8; n = 35 ChIN-MSN pairs, N = 14; two-tailed t test). (I) Same as in (H) for recordings obtained contralateral to the injection in M1 (n = 10 D1-MSN-D2-MSN pairs, N = 3; n = 12 FSI-MSN pairs, N = 5; n = 20 LTSI-MSN pairs, N = 9; n = 19 ChIN-MSN pairs, N = 7; two-tailed paired t test/Wilcoxon signed-rank test). See also Figures S1 and S2 and Table S1.
Figure 3
Figure 3
Thalamic Input Excites All Striatal Cell Types Except of LTSIs (A) Schematic of virus injection in PF and its projections innervating striatum. (B) PF projection neurons express ChR2. Top: response of a neuron in PF to step current injections. Bottom: light responses in the presence of synaptic blockers. Scale bars: 20 mV, 200 pA, 200 ms. (C) Confocal image of the injection site in a D2-tdTomato mouse. Red, D2-MSNs; green, virally transduced cells. Scale bar: 1 mm. (D) Confocal image of YFP-expressing axon terminals (green) innervating ipsilateral striatum. Scale bar: 1 mm. (E) Relative strength of EPSPs recorded in striatal neurons during stimulation of PF afferents in the presence of gabazine. Left to right: representative traces of triplet recordings. Simultaneously recorded EPSPs are overlaid. Scale bars: 2 mV, 20 ms. (F) Summary graph of EPSP amplitudes obtained from pairs and triplets of striatal neurons in response to M1I stimulation. Solid lines indicate paired recordings in which both neurons responded, dashed lines indicate pairs in which only one of the two neurons responded. The proportions of responding cells are shown in pie charts (inserts). Center values represent mean ± SEM (n = 23 D1-MSN-D2-MSN pairs, N = 5; n = 19 FSI-MSN pairs, N = 9; n = 21 LTSI-MSN pairs, N = 7; n = 20 ChIN-MSN pairs, N = 8; two-tailed paired t test/Wilcoxon signed-rank test). See also Figures S1 and S2 and Table S1.
Figure 4
Figure 4
Synaptic Strength and Innervation of Four Inputs to Five Striatal Cell Types (A) Comparison of the relative synaptic strength and innervation probability provided by each input to different striatal cell types. Top: schematic of the four inputs, including their injection sites in S1, M1, and PF and their corresponding recording sites in striatum. Center: relative strength of responses in D2-MSNs compared with D1-MSNs and FSIs, LTSIs, and ChINs compared with MSNs. Each circle represents the ratio of the EPSP amplitudes of two simultaneously recorded responding neurons. Center values represent mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test). The proportions of responding cells are shown in pie charts (inserts). Bottom: schematic illustrating which cell types are most robustly excited by each input. (B) Comparison of the relative synaptic strength and innervation probability of different inputs for each striatal cell type. Top: relative strength as shown in (A) rearranged to compare how different inputs excite and innervate each cell type. The statistics reflect how strong responding neurons are excited by each input (one-way ANOVA with Tukey’s multiple comparison test). The proportions of responding cells are shown in pie charts (inserts). Bottom: schematic summarizing which input is most robustly exciting each cell type.
Figure 5
Figure 5
Short-Term Plasticity Varies in an Input- and Cell Type-Specific Manner (A) Representative whole-cell recording of EPSPs in an LTSI evoked by optogenetic stimulation (20 Hz) of striatal input in the presence of gabazine. Scale bars: 2 mV, 100 ms. (B) High magnification of (A): EPSPs were fitted with a double-exponential function (red), and their amplitudes were extracted after subtracting the decay of preceding EPSPs. Scale bars: 2 mV, 20 ms. (C) Extracted EPSP amplitudes were normalized to the first pulse, and the paired-pulse ratio (PPR) and the steady-state ratio (SSR) were extracted. (D) Quantification of responses of paired D1- and D2-MSNs (left, n = 28 pairs, N = 13) and interneuron-MSN pairs (right, n = 16 FSI-MSN pairs, N = 12; n = 7 LTSI-MSN pairs, N = 4) to 20 Hz stimulation of S1 input. MSNs recorded in parallel with interneurons are not shown. Example traces are shown in inserts for all responding cell types (top right, scale bars: 2 mV, 100 ms). Data are presented as mean ± SEM (†p < 0.05, °p < 0.01, p < 0.001; one-way ANOVA for repeated measures corrected with Dunnett’s multiple comparison test). (E) Comparison of PPR and SSR of data presented in (D) across cell types. Circles represent individual cells; center values represent mean ± SEM (p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA corrected with Tukey’s multiple comparison test). D1-MSNs were recorded with D2-MSNs, and all interneurons were recorded with MSNs (#p < 0.05, ##p < 0.01, ###p < 0.001; two-tailed paired t test/Wilcoxon signed-rank test). (F) Same as in (D) for stimulation of M1I input (left: n = 16 D1-MSN-D2-MSN pairs, N = 4; right: n = 13 FSI-MSN pairs, N = 3; n = 17 LTSI-MSN pairs, N = 7; n = 13 ChIN-MSN pairs, N = 8). (G) Same as in (E) for stimulation of M1I input. (H) Same as in (D) for stimulation of M1C input (left: n = 9 D1-MSN-D2-MSN pairs, N = 3; right: n = 11 FSI-MSN pairs, N = 5; n = 12 LTSI-MSN pairs, N = 8) (I) Same as in (E) for stimulation of M1C input. (J) Same as in (D) for stimulation of PF input (left: n = 21 D1-MSN-D2-MSN pairs, N = 5; right: n = 17 FSI-MSN pairs, N = 8; n = 18 ChIN-MSN pairs, N = 8) (K) Same as in (E) for stimulation of PF input. See also Figure S5 and Table S1.
Figure 6
Figure 6
Cell Type-Specific Expression of Glutamatergic Receptors (A) Extraction of the NMDA to AMPA ratio with cesium-based intracellular solution in the presence of gabazine: average response (dark gray) and individual trials (pale gray) in a D2-MSN showing the measurement of the AMPA current (IAMPA) and the NMDA current (INMDA). Scale bars: 200 pA, 50 ms. (B) Representative average AMPA and NMDA currents obtained in striatal neurons evoked by activating S1 (top), M1I (center), and PF (bottom) inputs. Scale bars: 200 pA, 50 ms. The fractions of ChINs that responded, failed to respond, or expressed silent synapses are shown in pie charts (right). (C) Summary graph of the NMDA to AMPA ratio of responding neurons (n = 10–25 D1- and D2-MSNs per input, N = 2–6; n = 8–16 FSIs per input, N = 3–8; n = 4–14 ChINs per input, N = 2–6; one-way ANOVA with Tukey’s multiple comparison test for S1, M1I M1C, and PF inputs). (D) Scatterplot of absolute AMPA and NMDA currents of responding neurons. Responses to different inputs are pooled for each cell type. (E) I-V curves of AMPA-mediated currents in MSNs and FSIs in response to activation of M1I inputs (n = 11 MSNs, n = 9 FSIs, N = 6; two-tailed unpaired t test). Recordings were obtained with a spermine-based intracellular solution in the presence of gabazine and D-APV. Insets show representative AMPA currents from MSNs and FSIs normalized to the response at −70 mV. Scale bar: 10 ms. (F) Same as in (E) for activation of PF inputs (n = 7 MSNs, n = 6 FSIs, N = 5; two-tailed t test). See also Figure S6 and Table S1.
Figure 7
Figure 7
Opposing Effects of Thalamic and M1I Input on Activity of ChINs and LTSIs (A) Representative traces of a simultaneous recording from an MSN (whole-cell mode, top) and an adjacent ChIN (cell-attached mode, below) during 20 Hz stimulation of M1I inputs in a ChAT-tdTomato mouse. Scale bars: 2 mV, 200 ms. A raster plot and a histogram of the spikes recorded in the ChIN are shown at the bottom. Recordings were done in modified artificial cerebrospinal fluid (ACSF) containing 5 mM potassium to increase spontaneous activity. (B) Same as in (A) for stimulation of PF inputs. Scale bars: 0.5 mV, 200 ms. (C) Average population response following M1I stimulation. The histogram shows the spiking of individual ChINs in gray and the average response in purple (20 ms bins). The insert shows spiking before (t1), during (t2), and after (t3) stimulation and during the recovery pulse (t4). Gray circles represent individual ChINs: colored circles represent the mean ± SEM. All ChINs were recorded in parallel with one or two responding neurons (n = 8 ChINs; n = 11 pairs, N = 5 ChAT-tdTomato mice; repeated-measures one-way ANOVA with Tukey’s multiple comparison test). (D) Same as in (C) for PF-evoked changes in ChIN firing (n = 6 ChINs; n = 8 pairs, N = 2 ChAT-tdTomato mice). (E) Same as in (A) for a simultaneous recording of an MSN, together with an LTSI in response to optogenetic stimulation of M1I. Scale bars: 1 mV, 200 ms. Recordings were done in modified ACSF containing 5 mM potassium and gabazine. (F) Same as in (E) for stimulation of PF inputs. Scale bars: 1 mV, 200 ms. (G) Average population response as in (C) for changes in LTSI firing following M1I stimulation. All LTSIs were recorded in parallel with one or two responding neurons (n = 5 LTSIs; n = 9 pairs, N = 2 SOM-tdTomato mice). (H) Same as in (G) for PF-evoked changes in LTSI firing (n = 7 LTSIs; n = 9 pairs, N = 3 SOM-tdTomato mice). See also Figure S7 and Table S1.

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