Objective:To anatomically and phenotypically characterize the insular cortex(IC)-nucleus tractus soli-tarii(NTS)neural pathway.Methods:Adult male Sprague-Dawley rats were divided into three experimental cohorts for neural circuit tracing.Anterograde labeling was achieved by injecting anterograde self-complementary adeno-associated viruses(scAAVs)into the IC.Retrograde tracing involved NTS injections of either retrograde scAAVs or FluoroGold(FG),combined with immunofluorescence histochemical staining to identify IC-originating projection neurons.For postsynaptic neurochemical phenotype characterization,IC was injected with AAV2/1-CaMKII-Cre,while a mixture of AAV2/9-Syn-DIO-mCherry and AAV2/9-VGAT1-EGFP was injected into the NTS.The rats were allowed to survive for one week following scAAVs or FG injection or four weeks after recombinase-dependent systems injection.Then the rats were sacrificed,and serial brain sections were prepared for immunofluorescence histochemical staining(brain section containing FG)and subsequent fluorescence/confocal microscopic analysis.Results:(1)Anterograde viral tracing re-vealed dense axonal terminals from the IC projecting to the medial subnucleus of the NTS,while retrograde tracing re-vealed that IC neurons projecting to the NTS were predominantly localized within the dysgranular layer;(2)IC-NTS projection neurons were exclusive glutamatergic(100%,n=3);(3)NTS neurons receiving IC inputs were mainly lo-calized in the medial subnucleus,and were predominantly GABAergic(79.8±3.2%,n=3).Conclusion:The pres-ent results indicate that a descending pathway from excitatory neurons of the IC terminates onto inhibitory neurons of the NTS,which might represent a potential neuromodulatory target for visceral pain disorders.

Objective:To anatomically and phenotypically characterize the insular cortex(IC)-nucleus tractus soli-tarii(NTS)neural pathway.Methods:Adult male Sprague-Dawley rats were divided into three experimental cohorts for neural circuit tracing.Anterograde labeling was achieved by injecting anterograde self-complementary adeno-associated viruses(scAAVs)into the IC.Retrograde tracing involved NTS injections of either retrograde scAAVs or FluoroGold(FG),combined with immunofluorescence histochemical staining to identify IC-originating projection neurons.For postsynaptic neurochemical phenotype characterization,IC was injected with AAV2/1-CaMKII-Cre,while a mixture of AAV2/9-Syn-DIO-mCherry and AAV2/9-VGAT1-EGFP was injected into the NTS.The rats were allowed to survive for one week following scAAVs or FG injection or four weeks after recombinase-dependent systems injection.Then the rats were sacrificed,and serial brain sections were prepared for immunofluorescence histochemical staining(brain section containing FG)and subsequent fluorescence/confocal microscopic analysis.Results:(1)Anterograde viral tracing re-vealed dense axonal terminals from the IC projecting to the medial subnucleus of the NTS,while retrograde tracing re-vealed that IC neurons projecting to the NTS were predominantly localized within the dysgranular layer;(2)IC-NTS projection neurons were exclusive glutamatergic(100%,n=3);(3)NTS neurons receiving IC inputs were mainly lo-calized in the medial subnucleus,and were predominantly GABAergic(79.8±3.2%,n=3).Conclusion:The pres-ent results indicate that a descending pathway from excitatory neurons of the IC terminates onto inhibitory neurons of the NTS,which might represent a potential neuromodulatory target for visceral pain disorders.