| Experiment 1 | Experiment 2 | Experiment 3 |
---|---|---|---|
Aims | To characterize the cell organization of trigeminal nucleus caudalis (TNC) -vestibular nucleus (VN) pathway | To determine the role of glutamatergic trigeminal neurons in migraine-related vestibular dysfunction | To determine whether the GluTNC-CGRPVN pathway are involved in migraine-related vestibular dysfunction |
Subjects | Wild-type rats | chronic-nitroglycerin (NTG) rats | chronic-NTG rats |
Interventions | 1. Retrograde tracer Fluoro-Gold (FG) was injected in the VN area; Anterograde tracer virus H129-G4 was injected into the TNC area | 1. AAV-Ca2 + /calmodulin-dependent protein kinase IIα (CaMKIIα, an enzyme in glutamatergic neurons)-hM4D-mCherry virus was injected in the TNC area; 2. Intraperitoneal injection of CNO or saline | 1. AAV-EF1α-DIO-hM4D-mCherry virus was injected in the TNC area, while AAVretro-Syn-Cre was simultaneously injected in the VN area; 2. Intraperitoneal injection of CNO or saline |
Groups | 1. FG group (n = 5); 2. H129-G4 group (n = 5) | 1. Saline group (n = 6); 2. Chronic-NTG group (n = 6); 3. Chronic-NTG + CNO group (n = 6); 4. Chronic-NTG + AAV-CaMKIIα-hM4D-mCherry + saline (n = 6); 5. Chronic-NTG + AAV-CaMKIIα-hM4D-mCherry + CNO group (n = 6) | 1. Saline group (n = 6); 2. Chronic-NTG group (n = 6); 3. Chronic-NTG + CNO group (n = 6); 4. Chronic-NTG + AAV-EF1α-DIO-hM4D-mCherry + saline (n = 6); 5. Chronic-NTG + AAV-EF1α-DIO-hM4D-mCherry + CNO group (n = 6) |
Observations | 1. TNC area: colocalization of FG + and glutamate/ GABA + neurons; 2. VN area: colocalization of H129-G4 + and calcitonin gene-related peptide (CGRP) CGRP + neurons | 1. Behavior studies; 2. Colocalization of glutamate and AAV-CaMKIIα-hM4D-mCherry in the TNC; 3. C-fos + neurons in VN area; 4. Detection of miniature excitatory postsynaptic currents (mEPSCs) of TNC-projecting VN neurons | 1. Behavior studies |