Table 1 Experimental design and animal groups

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 Glu^TNC-CGRP^VN 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 AAV_retro-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