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Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and <t>AAV1-EF1a-DIO-EYFP</t> were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.
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Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and <t>AAV1-EF1a-DIO-EYFP</t> were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.
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Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and <t>AAV1-EF1a-DIO-EYFP</t> were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.
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Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and AAV1-EF1a-DIO-EYFP were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.

Journal: Cell reports

Article Title: Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

doi: 10.1016/j.celrep.2023.112291

Figure Lengend Snippet: Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and AAV1-EF1a-DIO-EYFP were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-c-Fos (9F6) Cell Signaling Technology Cat# 2250; RRID: AB_2247211 Rabbit polyclonal anti-PACAP-38 Peninsula Laboratories Cat# T-4473; RRID: AB_519166 Sheep polyclonal anti-FoxP2 R&D Systems Cat# AF5647; RRID: AB_2107133 Mouse monoclonal anti-CGRP Abcam Cat# ab81887; RRID: AB_1658411 Alexa Fluor 568-conjugated donkey anti-mouse Invitrogen Cat# A10037; RRID: AB_2534013 Alexa Fluor 594-conjugated donkey anti-sheep Abcam Cat# ab150180; RRID: AB_2716768 Alexa Fluor 568-conjugated goat anti-rabbit Invitrogen Cat# A11011; RRID: AB_143157 Alexa Fluor 647-conjugated donkey anti-rabbit Invitrogen Cat# A31573; RRID: AB_2536183 Bacterial and virus strains AAV-EF1ɑ-DIO-EYFP Addgene Cat# 27056-AAV1; RRID: Addgene_27056 AAV-EF1ɑ-DIO-eNpHR3.0-EYFP Addgene Cat# 26966-AAV1; RRID: Addgene_26966 AAV-EF1ɑ-double floxedhChR2(H134R)-EYFPWPRE-HGHpA Addgene Cat# 20298-AAV1; RRID: Addgene_20298 AAVrg-EF1ɑ-mCherry-IRES-Cre Addgene Cat# 55632-AAVrg; RRID: Addgene_55632 AAV-hSyn-double floxedhChR2(H134R)-EYFP Addgene Cat# 26973-AAV1; RRID: Addgene_26973 AAV-hSyn-eNpHR3.0-EYFP Addgene Cat# 26972-AAV5; RRID: Addgene_26972 AAV-CAG-FLEX-GCaMP6s-WPRE-SV40 Addgene Cat# 100842-AAV1; RRID: Addgene_100842 AAVrg-hSyn-EGFP Addgene Cat# 50465-AAVrg; RRID: Addgene_50465 AAV-hSyn-EGFP Addgene Cat# 50465-AAV5; RRID: Addgene_50465 pENN.AAV-hSyn-Cre-WPRE-hGH Addgene Cat# 105553-AAV1; RRID: Addgene_105553 AAV-hSyn-FLEx-mGFP-2ASynaptophysin-mRuby Addgene Cat# 71760-AAV1; RRID: Addgene_71760 rAAV8-CAG-mCherry-IRES-WGA-Cre UNC vector Core Cat# AV5901FG Chemicals, peptides, and recombinant proteins Normal Donkey Serum Sigma Cat# D9663 Triton X-100 Sigma Cat# X100 DAPI mounting solution Vectorlab Cat# H-1200 Retrobead (red) LumaFluor N/A (Continued on next page) 14 Cell Reports 42, 112291, April 25, 2023

Techniques: Injection, Expressing, Retrograde Tracing, Labeling, Patch Clamp, Two Tailed Test