Materials_NN2 and_CJC Methods_NN2 
Animals_NN2 
Male_AJ0 Sprague_NP0 Dawley_NP0 rats_NN2 (_PUL initial_AJ0 weight_NN1 250_CRD –_PUN 300_CRD g_NN0 ;_PUN Charles_NP0 River_NP0 ,_PUN Raleigh_NP0 ,_PUN NC_NP0 ,_PUN USA_NP0 )_PUR were_VBD single-housed_AJ0 under_PRP a_AT0 12-h_NN1 reverse_NN1 light_NN1 /_PUN dark_AJ0 cycle_NN1 (_PUL ZT0_NP0 =_NP0 19_CRD :_PUN 00_CRD )_PUR and_CJC had_VHD access_NN1 to_PRP food_NN1 and_CJC water_NN1 ad_NN1 libitum_NN1 ._SENT Animals_NN2 were_VBD housed_VVN in_PRP a_AT0 temperature-_AJ0 and_CJC humidity-controlled_AJ0 animal_NN1 facility_NN1 with_PRP AAALAC_AJ0 accreditation_NN1 ._SENT All_DT0 experiments_NN2 were_VBD approved_VVN by_PRP the_AT0 Institutional_AJ0 Animal_NN1 Care_NN1 and_CJC Use_VVB Committee_NN1 at_PRP Texas_NP0 A_ZZ0 &_NP0 M_ZZ0 University_NN1 and_CJC conducted_VVD according_VVG to_PRP specifications_NN2 of_PRF the_AT0 National_AJ0 Institutes_NN2 of_PRF Health_NN1 as_CJS outlined_VVN in_PRP the_AT0 Guide_NN1 for_PRP the_AT0 Care_NN1 and_CJC Use_NN1 of_PRF Laboratory_NN1 Animals_NN2 ._SENT 
Viruses_NN2 
The_AT0 transduction_NN1 and_CJC monosynaptic_AJ0 spread_NN1 of_PRF glycoprotein_NN1 (_PUL G_ZZ0 )_PUR -deleted_AJ0 rabies_NN1 virus_NN1 (_PUL RV_NP0 )_PUR with_PRP EnvA_NP0 pseudotyping_NN1 (_PUL EnvA-RV-EGFP_NP0 )_PUR is_VBZ limited_VVN to_PRP neurons_NN2 expressing_VVG TVA_NN1 receptor_NN1 and_CJC rabies_NN1 glycoprotein_NN1 (_PUL RG_NP0 )_PUR in_PRP a_AT0 Cre-dependent_AJ0 manner_NN1 (_PUL Wickersham_NP0 et_NP0 al._NP0 ,_PUN 2007a_CRD ,_PUN b_ZZ0 ;_PUN Watabe-Uchida_NP0 et_NP0 al._NP0 ,_PUN 2012_CRD )_PUR ._SENT We_PNP targeted_VVD specific_AJ0 PL_NP0 neurons_NN2 by_PRP injecting_VVG either_DT0 NAc_AJ0 core_NN1 or_CJC RMTg_NN1 with_PRP retrograde_NN1 Cre-expressing_NN1 virus_NN1 (_PUL CAV2-Cre_NP0 )_PUR and_CJC injecting_VVG PL_NP0 with_PRP Cre-dependent_NP0 helper_NN1 AAV_NP0 viruses_NN2 (_PUL TVA_NP0 and_CJC RG_NP0 )_PUR and_CJC EnvA-RV-EGFP_NN1 ._SENT 
EnvA-RV-EGFP_NN1 (_PUL titer_NN1 of_PRF 4.3_CRD ×_PRP 10e8_CRD )_PUR was_VBD provided_VVN by_PRP Ed_NP0 Callaway_NP0 via_PRP the_AT0 Gene_NP0 Transfer_NN1 ,_PUN Targeting_VVG ,_PUN and_CJC Therapeutics_NN1 Facility_NN1 at_PRP the_AT0 Salk_NP0 Institute_NN1 (_PUL Wickersham_NP0 et_NP0 al._NP0 ,_PUN 2007a_CRD )_PUR ._SENT AAV-TVA_NP0 (_PUL AAV1-EF1a-FLEX-TVAmCherry_NP0 ,_PUN a_AT0 titer_NN1 of_PRF 4_CRD ×_PRP 10e12_CRD )_PUR and_CJC AAV-RG_NP0 (_PUL AAV1-CA-FLEX-RG_NP0 ,_PUN a_AT0 titer_NN1 of_PRF 4_CRD ×_PRP 10e12_CRD )_PUR were_VBD obtained_VVN through_PRP the_AT0 viral_AJ0 vector_NN1 core_NN1 at_PRP the_AT0 University_NN1 of_PRF North_NP0 Carolina_NP0 (_PUL developed_VVN by_PRP Watabe-Uchida_NP0 et_NP0 al._NP0 ,_PUN 2012_CRD )_PUR ._SENT CAV2-Cre_NP0 (_PUL CAV2-CMV-Cre_NP0 recombinase_NN1 )_PUR was_VBD provided_VVN by_PRP Eric_NP0 J_NP0 ._SENT Kremer_NP0 at_PRP the_AT0 Institut_NP0 de_NP0 Genetique_NP0 Moleculaire_NP0 de_NP0 Montpellier_NP0 (_PUL Kremer_NP0 et_NP0 al._NP0 ,_PUN 2000_CRD )_PUR ._SENT We_PNP diluted_VVD CAV2-Cre_NP0 1_CRD :_PUN 10_CRD in_PRP 10_CRD %_NN0 glycerol_NN1 in_PRP PBS_NP0 for_PRP a_AT0 final_AJ0 titer_NN1 of_PRF 10.3_CRD ×_PRP 10e11_CRD ppml_NN0 ,_PUN based_VVN on_PRP preliminary_AJ0 experiments_NN2 indicating_VVG cell_NN1 toxicity_NN1 at_PRP higher_AJC titers_NN2 ._SENT All_DT0 viral_AJ0 procedures_NN2 were_VBD approved_VVN by_PRP the_AT0 Institutional_AJ0 Biosafety_NN1 Committee_NN1 at_PRP Texas_NP0 A_ZZ0 &_NP0 M_ZZ0 University_NN1 and_CJC conducted_VVD according_VVG to_PRP specifications_NN2 of_PRF the_AT0 National_AJ0 Institutes_NN2 of_PRF Health_NN1 ._SENT 
Stereotaxic_AJ0 Surgery_NN1 
Rats_NN2 were_VBD anesthetized_AJ0 with_PRP vaporized_AJ0 isoflurane_NN1 (_PUL induced_VVN at_PRP 5_CRD %_NN0 ,_PUN maintained_VVN at_PRP 1_CRD –_PUN 2_CRD %_NN0 )_PUR ,_PUN given_VVN the_AT0 analgesic_AJ0 ketoprofen_NN1 (_PUL 2_CRD mg_NN0 /_PUN kg_NN0 ,_PUN s.c._NN0 )_PUR ,_PUN and_CJC placed_VVN in_PRP a_AT0 stereotaxic_AJ0 frame_NN1 (_PUL Kopf_NP0 ,_PUN Tujunga_NP0 ,_PUN CA_NP0 ,_PUN USA_NP0 )_PUR ._SENT A_AT0 skin_NN1 incision_NN1 was_VBD made_VVN over_PRP the_AT0 skull_NN1 and_CJC holes_NN2 were_VBD drilled_VVN in_PRP the_AT0 skull_NN1 over_PRP the_AT0 target_NN1 sites_NN2 ._SENT Intracranial_AJ0 injections_NN2 were_VBD made_VVN using_VVG a_AT0 pulled_AJ0 glass_NN1 micropipette_NN1 and_CJC Nanoject_NN1 2010_CRD injector_NN1 (_PUL World_NN1 Precision_NN1 Instruments_NN2 ,_PUN Sarasota_NP0 ,_PUN FL_AJ0 ,_PUN USA_NP0 )_PUR ._SENT After_PRP the_AT0 injection_NN1 ,_PUN the_AT0 pipette_NN1 was_VBD slowly_AV0 raised_VVN from_PRP the_AT0 brain_NN1 and_CJC the_AT0 skin_NN1 was_VBD stapled_VVN closed_AJ0 ._SENT 
For_PRP conventional_AJ0 retrograde_NN1 tracing_NN1 ,_PUN rats_NN2 were_VBD given_VVN unilateral_AJ0 microinjections_NN2 of_PRF 100_CRD nl_NN0 of_PRF 2_CRD %_NN0 Fluoro-Gold_NN1 (_PUL FG_NP0 ,_PUN Fluorochrome_AJ0 ,_PUN Denver_NP0 ,_PUN CO_NN1 ,_PUN USA_NP0 )_PUR into_PRP unilateral_AJ0 NAc_NP0 core_NN1 (_PUL AP_NP0 +1.8_CRD ,_PUN ML_NN0 +2.6_CRD ,_PUN DV_CRD –_PUN 7.3_CRD from_PRP bregma_NN1 ,_PUN 6_CRD °_NN0 )_PUR and_CJC 100_CRD nl_NN0 of_PRF 0.2_CRD %_NN0 cholera_NN1 toxin_NN1 subunit_NN1 b_ZZ0 (_PUL CTB_NP0 ,_PUN List_VVB Biological_AJ0 Laboratories_NN2 ,_PUN Campbell_NP0 ,_PUN CA_NP0 ,_PUN USA_NP0 )_PUR into_PRP RMTg_NP0 (_PUL AP_NP0 –_PUN 7.2_CRD ,_PUN ML_NN0 +1.9_CRD ,_PUN DV_CRD –_PUN 7.6_CRD from_PRP dura_NN1 ,_PUN 10_CRD °_NN0 )_PUR ._SENT FG_NP0 was_VBD used_VVN only_AV0 in_PRP NAc_NP0 because_CJS it_PNP tended_VVD to_TO0 cause_VVI toxicity_NN1 in_PRP RMTg_NP0 ,_PUN evident_AJ0 by_PRP circling_VVG of_PRF animals_NN2 after_PRP unilateral_AJ0 injection_NN1 ._SENT Rats_NN2 were_VBD sacrificed_VVN at_PRP least_AV0 1_CRD week_NN1 after_PRP injections_NN2 ._SENT 
For_PRP monosynaptic_AJ0 retrograde_NN1 tracing_NN1 ,_PUN rats_NN2 were_VBD given_VVN two_CRD surgeries_NN2 ._SENT During_PRP the_AT0 first_ORD stereotaxic_AJ0 surgery_NN1 ,_PUN CAV2-Cre_NP0 (_PUL 1_CRD ,_PUN 000_CRD nl_NN0 )_PUR plus_PRP biotin_NN1 dextran_NN1 (_PUL 10_CRD K_ZZ0 MW_NN0 ,_PUN 0.2_CRD %_NN0 final_AJ0 volume_NN1 ,_PUN for_PRP visualization_NN1 of_PRF injection_NN1 site_NN1 )_PUR was_VBD injected_VVN unilaterally_AV0 into_PRP either_DT0 NAc_NP0 core_NN1 (_PUL AP_NP0 +1.8_CRD ,_PUN ML_NN0 +2.6_CRD ,_PUN DV_CRD –_PUN 7.3_CRD from_PRP bregma_NN1 ,_PUN 6_CRD °_NN0 )_PUR or_CJC RMTg_NP0 (_PUL AP_NP0 –_PUN 7.2_CRD ,_PUN ML_NN0 +1.8_CRD ,_PUN DV_CRD –_PUN 7.6_CRD from_PRP dura_NN1 ,_PUN 10_CRD °_NN0 )_PUR ,_PUN and_CJC a_AT0 mixture_NN1 of_PRF AAV-RG_NP0 and_CJC AAV-TVA_NP0 (_PUL 1_CRD ,_PUN 000_CRD nl_NN0 of_PRF a_AT0 1_CRD :_PUN 1_CRD mix_NN1 )_PUR was_VBD injected_VVN into_PRP ipsilateral_AJ0 PL_NP0 (_PUL AP_NP0 +3.1_CRD ,_PUN ML_NN0 +1.2_CRD ,_PUN DV_CRD –_PUN 3.8_CRD from_PRP skull_NN1 ,_PUN 12_CRD °_NN0 )_PUR ._SENT Three_CRD weeks_NN2 later_AV0 during_PRP a_AT0 second_ORD stereotaxic_AJ0 surgery_NN1 ,_PUN EnvA-RV-EGFP_NP0 (_PUL 1_CRD ,_PUN 000_CRD nl_NN0 )_PUR was_VBD injected_VVN into_PRP the_AT0 same_DT0 location_NN1 in_PRP ipsilateral_AJ0 PL._NN1 Rats_NN2 were_VBD sacrificed_VVN 7_CRD days_NN2 after_PRP rabies_NN1 injections_NN2 ._SENT 
Tissue_NN1 Processing_NN1 
Rats_NN2 were_VBD deeply_AV0 anesthetized_AJ0 with_PRP isoflurane_NN1 and_CJC then_AV0 transcardially_AV0 perfused_VVN with_PRP 0.9_CRD %_NN0 NaCl_NN1 followed_VVN by_PRP 10_CRD %_NN0 neutral-buffered_AJ0 formalin_NN1 via_PRP a_AT0 peristaltic_AJ0 pump_NN1 (_PUL Cole_NP0 Parmer_NP0 ,_PUN Vernon_NP0 Hills_NP0 ,_PUN IL_NP0 ,_PUN USA_NP0 )_PUR ._SENT Brains_NN2 were_VBD collected_VVN ,_PUN post-fixed_AJ0 in_PRP 10_CRD %_NN0 formalin_NN1 overnight_AV0 at_PRP 4_CRD °_NN0 C_ZZ0 ,_PUN placed_VVN in_PRP 20_CRD %_NN0 sucrose_NN1 in_PRP phosphate-buffered_AJ0 saline_NN1 (_PUL PBS_NP0 )_PUR with_PRP 0.02_CRD %_NN0 sodium-azide_NN1 at_PRP 4_CRD °_NN0 C_ZZ0 for_PRP ≥2_CRD days_NN2 ,_PUN frozen_VVN in_PRP dry_AJ0 ice_NN1 ,_PUN cut_VVN into_PRP 40-μm_AJ0 thick_AJ0 coronal_AJ0 sections_NN2 on_PRP a_AT0 cryostat_NN1 ,_PUN and_CJC collected_VVN into_PRP PBS-azide_NN1 for_PRP storage_NN1 before_PRP immunohistochemistry_NN1 (_PUL IHC_NP0 )_PUR ._SENT For_PRP IHC_NN1 ,_PUN all_DT0 incubations_NN2 and_CJC rinses_NN2 took_VVD place_NN1 at_PRP room_NN1 temperature_NN1 on_PRP a_AT0 shaker_NN1 ._SENT Sections_NN2 were_VBD rinsed_VVN in_PRP PBS_NP0 three_CRD times_NN2 between_PRP steps_NN2 ._SENT 
For_PRP FG_NP0 and_CJC CTB_NP0 tracing_NN1 ,_PUN free-floating_AJ0 sections_NN2 were_VBD incubated_VVN overnight_AV0 in_PRP goat_NN1 anti-CTB_NN1 (_PUL 1_CRD :_PUN 50_CRD K_ZZ0 ,_PUN List_VVB Biological_AJ0 Labs_NN2 Cat#_NP0 703_CRD ,_PUN RRID_NP0 :_PUN AB_10013220_CRD )_PUR in_PRP PBS_NP0 with_PRP 0.25_CRD %_NN0 Triton_NP0 X-100_CRD (_PUL PBST_NP0 ,_PUN Sigma_NN1 –_PUN Aldrich_NP0 )_PUR ,_PUN and_CJC then_AV0 incubated_VVN for_PRP 1_CRD h_ZZ0 in_PRP Alexa_NP0 Fluor_NP0 594-conjugated_AJ0 donkey_NN1 anti-goat_NN1 (_PUL 1_CRD :_PUN 500_CRD in_PRP PBST_NP0 ,_PUN Jackson_NP0 Immunoresearch_NP0 Labs_NN2 cat#_NN1 705-585-147_CRD ,_PUN RRID_NP0 :_PUN AB_2340433_CRD )_PUR ._SENT Sections_NN2 were_VBD mounted_VVN onto_PRP glass_NN1 slides_NN2 ,_PUN coverslipped_AJ0 with_PRP ProLong_VVB Diamond_NP0 Antifade_NP0 (_PUL Thermo_NP0 Fisher_NP0 Scientific_AJ0 ,_PUN Fair_AJ0 Lawn_NN1 ,_PUN NJ_NP0 ,_PUN USA_NP0 )_PUR ,_PUN sealed_VVN with_PRP clear_AJ0 nail_NN1 polish_NN1 ,_PUN and_CJC stored_VVN at_PRP 4_CRD °_NN0 C_ZZ0 ._SENT 
For_PRP RV_NP0 tracing_NN1 ,_PUN one_PNI well_AV0 (_PUL every_AT0 12th_ORD section_NN1 )_PUR was_VBD used_VVN for_PRP mCherry_NN1 /_PUN EGFP_NN1 fluorescence_NN1 (_PUL for_PRP counting_VVG starter_NN1 cells_NN2 )_PUR ,_PUN and_CJC one_PNI well_AV0 (_PUL every_AT0 12th_ORD section_NN1 )_PUR was_VBD used_VVN for_PRP IHC_NN1 to_TO0 visualize_VVI mCherry_NN1 /_PUN EGFP_NN1 with_PRP DAB_NP0 (_PUL for_PRP counting_VVG inputs_NN2 to_PRP PL_NP0 )_PUR ._SENT For_PRP fluorescence_NN1 ,_PUN sections_NN2 were_VBD mounted_VVN onto_PRP glass_NN1 slides_NN2 ,_PUN coverslipped_AJ0 with_PRP ProLong_VVB Diamond_NN1 Antifade_NN1 ,_PUN sealed_VVN with_PRP clear_AJ0 nail_NN1 polish_NN1 ,_PUN and_CJC stored_VVN at_PRP 4_CRD °_NN0 C_ZZ0 ._SENT For_PRP DAB_NP0 ,_PUN free-floating_AJ0 sections_NN2 were_VBD incubated_VVN for_PRP 15_CRD min_NN0 in_PRP 0.3_CRD %_NN0 H2O2_CRD in_PRP PBS_NP0 ,_PUN overnight_AV0 in_PRP mouse_NN1 anti-DsRed_AJ0 (_PUL 1_CRD :_PUN 2_CRD K_ZZ0 in_PRP PBST_NP0 ,_PUN Takara_NP0 Bio_NP0 Cat#_NP0 632392_CRD ,_PUN RRID_NP0 :_PUN AB_2801258_CRD )_PUR ,_PUN 1_CRD h_ZZ0 in_PRP biotinylated_AJ0 donkey_NN1 anti-mouse_NN1 (_PUL 1_CRD :_PUN 500_CRD in_PRP PBST_NP0 ,_PUN Jackson_NP0 Immunoresearch_NP0 Labs_NN2 Cat#_NP0 715_CRD –_PUN 065_CRD –_PUN 151_CRD ,_PUN RRID_NP0 :_PUN AB_2340785_CRD )_PUR ,_PUN and_CJC 45_CRD min_NN0 in_PRP ABC_NP0 (_PUL Vector_NN1 Elite_NN1 Kit_NN1 ,_PUN 1_CRD :_PUN 500_CRD in_PRP PBST_NP0 ,_PUN Vector_NN1 )_PUR ._SENT The_AT0 reaction_NN1 was_VBD visualized_VVN via_PRP incubation_NN1 for_PRP 10_CRD min_NN0 in_PRP 0.025_CRD %_NN0 3_CRD ,_PUN 3′-diaminobenzidine_NN1 (_PUL DAB_NP0 )_PUR ,_PUN 0.05_CRD %_NN0 nickel_NN1 ammonium_NN1 sulfate_NN1 ,_PUN and_CJC 0.015_CRD %_NN0 H2O2_CRD (_PUL to_TO0 yield_VVI black_AJ0 DAB_NP0 color_NN1 )_PUR ._SENT Sections_NN2 were_VBD then_AV0 incubated_VVN overnight_AV0 in_PRP rabbit_NN1 anti-GFP_NN1 (_PUL 1_CRD :_PUN 50_CRD K_ZZ0 in_PRP PBST_NP0 ,_PUN Abcam_NP0 Cat#_NP0 ab290_CRD ,_PUN RRID_NP0 :_PUN AB_303395_CRD )_PUR ,_PUN 1_CRD h_ZZ0 in_PRP biotinylated_AJ0 donkey_NN1 anti-rabbit_NN1 (_PUL 1_CRD :_PUN 500_CRD in_PRP PBST_NP0 ,_PUN Jackson_NP0 Immunoresearch_NP0 Labs_NN2 Cat#_NP0 711-065-152_CRD ,_PUN RRID_NP0 :_PUN AB_2340593_CRD )_PUR ,_PUN 45_CRD min_NN0 in_PRP ABC_NP0 (_PUL Vector_NN1 Elite_NN1 Kit_NN1 ,_PUN 1_CRD :_PUN 500_CRD in_PRP PBST_NP0 ,_PUN Vector_NN1 )_PUR ,_PUN and_CJC visualized_VVD via_PRP the_AT0 same_DT0 DAB_NP0 reaction_NN1 but_CJC without_PRP nickel_NN1 (_PUL to_TO0 yield_VVI brown_AJ0 DAB_NP0 color_NN1 )_PUR ._SENT Sections_NN2 were_VBD mounted_VVN onto_PRP Superfrost_AJ0 Plus_NN1 slides_NN2 ,_PUN dried_AJ0 ,_PUN counterstained_VVN with_PRP Methyl_NP0 Green_NP0 (_PUL 0.5_CRD %_NN0 in_PRP sodium_NN1 acetate_NN1 buffer_NN1 ,_PUN Sigma_NN1 –_PUN Aldrich_NP0 )_PUR ,_PUN and_CJC coverslipped_VVD with_PRP Permount_NP0 (_PUL Thermo_NP0 Fisher_NP0 Scientific_AJ0 ,_PUN Fair_AJ0 Lawn_NN1 ,_PUN NJ_NP0 ,_PUN USA_NP0 )_PUR ._SENT 
Data_NN0 Analysis_NN1 
Fluorescent_AJ0 and_CJC brightfield_NN1 images_NN2 were_VBD acquired_VVN using_VVG an_AT0 Olympus_NP0 BX51_NN1 microscope_NN1 ._SENT For_PRP CTB_NP0 and_CJC FG_NP0 tracing_NN1 ,_PUN neurons_NN2 were_VBD counted_VVN in_PRP PL_NP0 on_PRP two_CRD sections_NN2 per_PRP rat_NN1 ._SENT Four_CRD rats_NN2 were_VBD excluded_VVN from_PRP CTB_NP0 /_PUN FG_NP0 analysis_NN1 due_AJ0 to_PRP small_AJ0 /_PUN missing_AJ0 RMTg_NN1 injections_NN2 ._SENT One_CRD rat_NN1 was_VBD removed_VVN from_PRP CTB_NP0 /_PUN FG_NP0 analysis_NN1 after_PRP being_VBG identified_VVN as_PRP a_AT0 significant_AJ0 outlier_AJC (_PUL Grubb_NP0 's_POS method_NN1 )_PUR due_AJ0 to_PRP a_AT0 higher_AJC percentage_NN1 of_PRF double_AJ0 labeling_NN1 ._SENT The_AT0 remaining_AJ0 data_NN0 were_VBD analyzed_VVN with_PRP a_AT0 paired_AJ0 t-test_NN1 ._SENT For_PRP rabies_NN1 tracing_NN1 ,_PUN all_DT0 EGFP-labeled_AJ0 neurons_NN2 were_VBD counted_VVN on_PRP all_DT0 sections_NN2 stained_VVN with_PRP DAB_NP0 (_PUL every_AT0 12th_ORD section_NN1 )_PUR and_CJC categorized_VVN into_PRP brain_NN1 regions_NN2 according_VVG to_PRP boundaries_NN2 identified_VVN by_PRP Paxinos_NP0 and_CJC Watson_NP0 (_PUL 2007_CRD )_PUR ._SENT RMTg_NN1 boundaries_NN2 were_VBD defined_VVN (_PUL Figures_NN2 1B_CRD ,_PUN 2C_NN1 )_PUR according_VVG to_PRP Smith_NP0 et_NP0 al._NP0 (_PUL 2019_CRD )_PUR ._SENT A_AT0 neuron_NN1 was_VBD only_AV0 counted_VVN if_CJS a_AT0 soma_NN1 was_VBD clearly_AV0 visible_AJ0 ._SENT Fluorescent_AJ0 sections_NN2 were_VBD used_VVN to_TO0 count_VVI all_DT0 double-labeled_AJ0 neurons_NN2 (_PUL EGFP_NP0 +_NP0 mCherry_NP0 )_PUR on_PRP every_AT0 12th_ORD section_NN1 ._SENT Expression_NN1 of_PRF EGFP_NN1 alone_AV0 indicated_VVN input_NN1 cells_NN2 ,_PUN and_CJC expression_NN1 of_PRF both_DT0 mCherry_NN1 and_CJC EGFP_NN1 indicated_VVD starter_NN1 cells_NN2 ._SENT Inputs_NN2 were_VBD normalized_VVN within_PRP each_DT0 animal_NN1 and_CJC calculated_VVN as_PRP a_AT0 percentage_NN1 of_PRF total_AJ0 inputs_NN2 per_PRP rat_NN1 ._SENT Brain_NN1 regions_NN2 were_VBD only_AV0 included_VVN in_PRP analysis_NN1 and_CJC figures_NN2 if_CJS normalized_AJ0 inputs_NN2 were_VBD ≥0.2_CRD %_NN0 in_PRP ≥2_CRD rats_NN2 ._SENT Data_NN0 were_VBD analyzed_VVN using_VVG two-way_AJ0 ANOVAs_NN2 (_PUL with_PRP repeated_AJ0 measures_NN2 when_CJS appropriate_AJ0 )_PUR and_CJC Sidak_NP0 post_NN1 hoc_NN1 analyses_NN2 ._SENT 
Results_NN2 
Conventional_AJ0 Retrograde_NN1 Tracing_VVG in_PRP PL_NP0 Targets_NN2 
Sparse_AJ0 double_AJ0 labeling_NN1 was_VBD seen_VVN in_PRP PL_NP0 (_PUL Figure_NN1 1A_CRD )_PUR when_CJS conventional_AJ0 retrograde_NN1 tracers_NN2 (_PUL FG_NP0 and_CJC CTB_NP0 )_PUR were_VBD injected_VVN into_PRP ipsilateral_AJ0 NAc_NP0 core_NN1 and_CJC RMTg_NP0 (_PUL Figure_NN1 1B_CRD ;_PUN n_ZZ0 =_NN1 11_CRD )_PUR ._SENT We_PNP observed_VVD NAc-projecting_NP0 PL_NP0 neurons_NN2 in_PRP layers_NN2 II_CRD /_PUN III_CRD and_CJC upper_AJ0 layer_NN1 V_ZZ0 ,_PUN whereas_CJS RMTg-projecting_AJ0 neurons_NN2 were_VBD located_VVN in_PRP deeper_AJC layer_NN1 V_ZZ0 (_PUL Figure_NN1 1A_CRD )_PUR ._SENT Double-labeled_AJ0 neurons_NN2 (_PUL projecting_VVG to_PRP both_DT0 NAc_AJ0 core_NN1 and_CJC RMTg_NP0 )_PUR represented_VVD only_AV0 0.5_CRD %_NN0 ±_NN0 0.1_CRD of_PRF PL_NP0 neurons_NN2 projecting_VVG to_PRP NAc_NP0 core_NN1 and_CJC 1.1_CRD %_NN0 ±_NN0 0.3_CRD of_PRF PL_NP0 neurons_NN2 projecting_VVG to_PRP RMTg_NP0 (_PUL Figure_NN1 1C_CRD ;_PUN t_ZZ0 (_PUL 10_CRD )_PUR =_NP0 3.004_CRD ,_PUN p_ZZ0 =_NN1 0.013_CRD )_PUR ._SENT These_DT0 data_NN0 indicate_VVB that_CJT PL_NP0 neurons_NN2 projecting_VVG to_PRP NAc_NP0 core_NN1 vs._PRP RMTg_NP0 are_VBB predominantly_AV0 separate_AJ0 subpopulations_NN2 ._SENT 
Monosynaptic_AJ0 Retrograde_NN1 Tracing_VVG in_PRP PL_NP0 Subpopulations_NN2 
To_TO0 determine_VVI whether_CJS these_DT0 PL_NP0 subpopulations_NN2 differ_VVB in_PRP terms_NN2 of_PRF input_NN1 specificity_NN1 ,_PUN we_PNP conducted_VVD monosynaptic_AJ0 retrograde_NN1 tracing_VVG in_PRP each_DT0 projection_NN1 subpopulation_NN1 via_PRP retrograde_NN1 Cre_NN1 delivery_NN1 (_PUL Figure_NN1 2A_NN1 )_PUR ._SENT CAV2-Cre_NP0 was_VBD injected_VVN into_PRP either_DT0 NAc_NP0 core_NN1 (_PUL n_ZZ0 =_NN1 4_CRD )_PUR or_CJC RMTg_NP0 (_PUL n_ZZ0 =_NP0 4_CRD ;_PUN Figures_NN2 2B_AJ0 ,_PUN C_ZZ0 )_PUR ,_PUN and_CJC Cre-dependent_AJ0 helper_NN1 AAVs_NN2 driving_VVG expression_NN1 of_PRF TVA-mCherry_NP0 and_CJC RG_NP0 were_VBD injected_VVN into_PRP PL._NN1 Three_CRD weeks_NN2 later_AV0 ,_PUN EnvA-RV-EGFP_NP0 was_VBD injected_VVN into_PRP PL._NN1 Rabies_NN1 only_AV0 infects_VVZ and_CJC monosynaptically_AV0 spreads_VVZ from_PRP neurons_NN2 expressing_VVG both_AV0 TVA_NP0 and_CJC RG_NP0 ._SENT 
We_PNP mapped_VVD and_CJC counted_VVD the_AT0 number_NN1 of_PRF starter_NN1 cells_NN2 (_PUL mCherry_NP0 +_NP0 EGFP_NP0 )_PUR using_VVG fluorescent_AJ0 labeling_NN1 (_PUL representative_AJ0 images_NN2 in_PRP Figures_NN2 2D_NN0 –_PUN G_ZZ0 )_PUR ._SENT We_PNP counted_VVD the_AT0 number_NN1 of_PRF input_NN1 cells_NN2 (_PUL EGFP_NP0 )_PUR across_PRP different_AJ0 brain_NN1 regions_NN2 using_VVG immunohistochemistry_NN1 with_PRP DAB_NP0 labeling_NN1 (_PUL representative_AJ0 images_NN2 in_PRP Figure_NN1 4_CRD )_PUR ._SENT Starter_NN1 cells_NN2 projecting_VVG to_PRP either_DT0 NAc_AJ0 core_NN1 or_CJC RMTg_NP0 were_VBD primarily_AV0 in_PRP PL_NP0 ,_PUN but_CJC also_AV0 in_PRP neighboring_AJ0 infralimbic_AJ0 cortex_NN1 (_PUL IL_NP0 ;_PUN Figure_NN1 3A_CRD )_PUR ._SENT Although_CJS the_AT0 average_AJ0 number_NN1 of_PRF starter_NN1 cells_NN2 differed_VVN for_PRP NAc_NP0 vs._PRP RMTg_NP0 (_PUL t_ZZ0 (_PUL 6_CRD )_PUR =_NP0 5.50_CRD ,_PUN p_ZZ0 =_NN1 0.0015_CRD )_PUR ,_PUN the_AT0 number_NN1 of_PRF transsynaptically_AV0 labeled_AJ0 cells_NN2 was_VBD correlated_VVN with_PRP the_AT0 number_NN1 of_PRF starter_NN1 cells_NN2 (_PUL Figure_NN1 3B_CRD ;_PUN r_ZZ0 =_NP0 0.91_CRD ,_PUN p_ZZ0 =_NN1 0.0019_CRD )_PUR ._SENT 
Inputs_NN2 were_VBD normalized_VVN within_PRP each_DT0 animal_NN1 and_CJC calculated_VVN as_PRP a_AT0 percentage_NN1 of_PRF total_AJ0 inputs_NN2 per_PRP rat_NN1 (_PUL Figure_NN1 3C_CRD )_PUR ._SENT The_AT0 total_AJ0 ipsilateral_AJ0 inputs_NN2 were_VBD significantly_AV0 greater_AJC than_CJS total_AJ0 contralateral_AJ0 inputs_NN2 (_PUL main_AJ0 effect_NN1 for_PRP contra_NN1 /_PUN ipsi_NN1 :_PUN F_ZZ0 (_PUL 1_CRD ,_PUN 6_CRD )_PUR =_NP0 37_CRD ,_PUN 354_CRD ,_PUN p_ZZ0 <_NN1 0.0001_CRD )_PUR ,_PUN and_CJC the_AT0 two_CRD PL_NN1 subpopulations_NN2 differed_VVN in_PRP terms_NN2 of_PRF ipsilateral_NN1 vs._PRP contralateral_AJ0 inputs_NN2 (_PUL main_AJ0 effect_NN1 for_PRP PL_NP0 subpopulation_NN1 :_PUN F_ZZ0 (_PUL 1_CRD ,_PUN 6_CRD )_PUR =_NP0 14.24_CRD ,_PUN p_ZZ0 =_NP0 0.009_CRD ;_PUN interaction_NN1 :_PUN F_ZZ0 (_PUL 1_CRD ,_PUN 6_CRD )_PUR =_NP0 83.43_CRD ,_PUN p_ZZ0 <_NN1 0.0001_CRD )_PUR ._SENT Therefore_AV0 ,_PUN we_PNP separately_AV0 analyzed_VVD contralateral_AJ0 and_CJC ipsilateral_AJ0 inputs_NN2 when_CJS comparing_VVG afferents_NN2 for_PRP the_AT0 two_CRD PL_NN1 subpopulations_NN2 ._SENT PL_NP0 neurons_NN2 projecting_VVG to_PRP NAc_NP0 core_NN1 vs._PRP RMTg_NP0 were_VBD significantly_AV0 different_AJ0 in_PRP terms_NN2 of_PRF ipsilateral_AJ0 input_NN1 (_PUL two-way_AJ0 ANOVA_NN1 ,_PUN overall_AJ0 effect_NN1 of_PRF PL_NP0 subpopulation_NN1 :_PUN F_ZZ0 (_PUL 1_CRD ,_PUN 6_CRD )_PUR =_NP0 53.44_CRD ,_PUN p_ZZ0 =_NP0 0.0003_CRD ;_PUN overall_AJ0 effect_NN1 of_PRF afferents_NN2 :_PUN F_ZZ0 (_PUL 29_CRD ,_PUN 174_CRD )_PUR =_NP0 86.11_CRD ,_PUN p_ZZ0 <_NP0 0.0001_CRD ;_PUN interaction_NN1 :_PUN F_ZZ0 (_PUL 29_CRD ,_PUN 174_CRD )_PUR =_NP0 5.05_CRD ;_PUN p_ZZ0 <_NN1 0.0001_CRD )_PUR ._SENT We_PNP found_VVD significant_AJ0 differences_NN2 for_PRP ipsilateral_AJ0 PL_NP0 and_CJC IL_NP0 (_PUL p_ZZ0 's_VBZ <_NN1 0.0001_CRD )_PUR ,_PUN and_CJC mediodorsal_AJ0 thalamic_AJ0 nucleus_NN1 (_PUL MD_NP0 ;_PUN p_ZZ0 =_NN1 0.036_CRD )_PUR ._SENT PL_NP0 neurons_NN2 projecting_VVG to_PRP NAc_NP0 core_NN1 vs._PRP RMTg_NP0 were_VBD also_AV0 significantly_AV0 different_AJ0 in_PRP terms_NN2 of_PRF contralateral_AJ0 input_NN1 (_PUL overall_AJ0 effect_NN1 of_PRF PL_NP0 subpopulation_NN1 :_PUN F_ZZ0 (_PUL 1_CRD ,_PUN 6_CRD )_PUR =_NP0 119.9_CRD ,_PUN p_ZZ0 <_NP0 0.0001_CRD ;_PUN overall_AJ0 effect_NN1 of_PRF afferents_NN2 :_PUN F_ZZ0 (_PUL 6_CRD ,_PUN 36_CRD )_PUR =_NP0 60.46_CRD ,_PUN p_ZZ0 <_NP0 0.0001_CRD ;_PUN interaction_NN1 :_PUN F_ZZ0 (_PUL 6_CRD ,_PUN 36_CRD )_PUR =_NP0 34.06_CRD ;_PUN p_ZZ0 <_NN1 0.0001_CRD )_PUR ,_PUN with_PRP significant_AJ0 differences_NN2 for_PRP contralateral_AJ0 PL_NN1 (_PUL p_ZZ0 <_NN1 0.0001_CRD )_PUR ._SENT 
For_PRP both_DT0 populations_NN2 ,_PUN the_AT0 primary_AJ0 source_NN1 of_PRF afferent_AJ0 information_NN1 was_VBD neighboring_AJ0 prefrontal_AJ0 neurons_NN2 in_PRP PL_NP0 and_CJC IL_NP0 (_PUL Figures_NN2 4A_CRD ,_PUN B_ZZ0 )_PUR ._SENT However_AV0 ,_PUN RMTg-projecting_NP0 PL_NP0 neurons_NN2 received_VVD a_AT0 significantly_AV0 higher_AJC percentage_NN1 of_PRF inputs_NN2 from_PRP local_AJ0 ipsilateral_AJ0 PL_NN1 and_CJC IL_NN1 neurons_NN2 (_PUL Figure_NN1 3C_CRD ;_PUN PL_NP0 :_PUN 39.4_CRD %_NN0 ±_NN0 0.2_CRD and_CJC IL_NP0 :_PUN 20.1_CRD %_NN0 ±_NN0 3.8_CRD )_PUR ,_PUN as_CJS compared_VVN to_PRP NAc-projecting_NP0 PL_NP0 neurons_NN2 (_PUL PL_NP0 :_PUN 26.9_CRD %_NN0 ±_NN0 4.0_CRD and_CJC IL_NP0 :_PUN 11.8_CRD %_NN0 ±_NN0 1.3_CRD )_PUR ._SENT We_PNP also_AV0 observed_VVD that_CJT RMTg-projecting_NP0 PL_NP0 neurons_NN2 received_VVD a_AT0 higher_AJC percentage_NN1 of_PRF input_NN1 from_PRP the_AT0 medial_AJ0 orbitofrontal_AJ0 cortex_NN1 (_PUL OFC_NP0 )_PUR ,_PUN while_CJS NAc-projecting_NP0 PL_NP0 neurons_NN2 received_VVD a_AT0 higher_AJC percentage_NN1 of_PRF input_NN1 from_PRP the_AT0 dorsal_AJ0 tenia_NN1 tecta_NN1 ,_PUN lateral_AJ0 OFC_NN1 ,_PUN and_CJC agranular_AJ0 insular_AJ0 cortex_NN1 ._SENT The_AT0 percentage_NN1 of_PRF inputs_NN2 was_VBD similar_AJ0 for_PRP NAc-projecting_NP0 and_CJC RMTg-projecting_NP0 PL_NP0 neurons_NN2 for_PRP secondary_AJ0 motor_NN1 cortex_NN1 ,_PUN anterior_AJ0 olfactory_AJ0 nucleus_NN1 ,_PUN ventral_AJ0 OFC_NN1 ,_PUN dorsal_AJ0 peduncular_AJ0 cortex_NN1 ,_PUN cingulate_NN1 cortex_NN1 areas_NN2 1_CRD and_CJC 2_CRD ,_PUN entorhinal_AJ0 /_PUN ectorhinal_AJ0 /_PUN perirhinal_AJ0 cortex_NN1 ,_PUN and_CJC piriform_AJ0 cortex_NN1 ._SENT Interestingly_AV0 ,_PUN NAc-projecting_NP0 PL_NP0 neurons_NN2 received_VVD a_AT0 significantly_AV0 greater_AJC percentage_NN1 of_PRF contralateral_AJ0 cortical_AJ0 input_NN1 from_PRP PL_NP0 (_PUL 2.92_CRD %_NN0 ±_NN0 0.30_CRD )_PUR as_CJS compared_VVN to_PRP RMTg-projecting_AJ0 PL_NP0 neurons_NN2 (_PUL 0.43_CRD %_NN0 ±_NN0 0.15_CRD )_PUR ._SENT NAc-projecting_NP0 PL_NP0 neurons_NN2 also_AV0 received_VVD a_AT0 greater_AJC percentage_NN1 of_PRF contralateral_AJ0 inputs_NN2 from_PRP IL_NN1 ,_PUN secondary_AJ0 motor_NN1 cortex_NN1 ,_PUN all_DT0 areas_NN2 of_PRF OFC_NN1 (_PUL medial_AJ0 ,_PUN ventral_AJ0 ,_PUN and_CJC lateral_AJ0 )_PUR ,_PUN and_CJC cingulate_NN1 cortex_NN1 area_NN1 1._CRD 
Both_DT0 PL_NP0 subpopulations_NN2 received_VVD prominent_AJ0 afferent_AJ0 information_NN1 from_PRP the_AT0 thalamus_NN1 (_PUL Figures_NN2 4C_CRD ,_PUN D_ZZ0 )_PUR ,_PUN including_PRP subregions_NN2 within_PRP the_AT0 midline_NN1 thalamic_AJ0 nuclei_NN2 (_PUL anterior_AJ0 /_PUN posterior_AJ0 parts_NN2 of_PRF paraventricular_AJ0 ,_PUN paratenial_AJ0 ,_PUN rhomboid_AJ0 ,_PUN reuniens_NN2 ,_PUN ventral_AJ0 reuniens_NN2 )_PUR ,_PUN anterior_AJ0 thalamic_AJ0 nuclei_NN2 (_PUL anteromedial_AJ0 ,_PUN ventrolateral_AJ0 /_PUN dorsomedial_AJ0 parts_NN2 of_PRF anteroventral_AJ0 ,_PUN interanteromedial_AJ0 )_PUR ,_PUN reticular_AJ0 thalamic_AJ0 nucleus_NN1 ,_PUN MD_NN1 (_PUL mediodorsal_AJ0 ,_PUN lateral_AJ0 /_PUN central_AJ0 /_PUN medial_AJ0 parts_NN2 of_PRF mediodorsal_NN1 ,_PUN intermediodorsal_NN1 )_PUR ,_PUN lateral_AJ0 thalamic_AJ0 nuclei_NN2 (_PUL dorsomedial_AJ0 part_NN1 of_PRF lateroventral_AJ0 ,_PUN mediorostral_AJ0 part_NN1 of_PRF lateral_AJ0 posterior_NN1 )_PUR ,_PUN intralaminar_AJ0 thalamic_AJ0 nuclei_NN2 (_PUL paracentral_AJ0 ,_PUN central_AJ0 medial_AJ0 ,_PUN centrolateral_AJ0 ,_PUN and_CJC parafascicular_NN1 )_PUR ,_PUN and_CJC ventral_AJ0 thalamic_AJ0 nuclei_NN2 (_PUL ventral_AJ0 anterior_AJ0 ,_PUN ventromedial_AJ0 ,_PUN ventral_AJ0 posteromedial_NN1 ,_PUN ventrolateral_NN1 ;_PUN Figure_NN1 3C_CRD )_PUR ._SENT NAc-projecting_NP0 PL_NP0 neurons_NN2 received_VVD a_AT0 higher_AJC percentage_NN1 of_PRF inputs_NN2 from_PRP all_DT0 thalamic_AJ0 regions_NN2 ,_PUN as_CJS compared_VVN to_PRP RMTg-projecting_AJ0 PL_NP0 neurons_NN2 ,_PUN with_PRP a_AT0 significant_AJ0 difference_NN1 observed_VVN for_PRP MD_NP0 (_PUL 6.6_CRD %_NN0 ±_NN0 0.7_CRD vs._PRP 2.1_CRD %_NN0 ±_NN0 0.6_CRD )_PUR ._SENT 
