Fig. 4: Monosynaptic structural and functional connection between LS and PVT neurons with TMN histaminergic neurons.
From: A whole-brain male mouse atlas of long-range inputs to histaminergic neurons
a Experimental scheme for labeling monosynaptic downstream neurons in TMN from long-range projection of LS GABAergic neurons. rAAV-VGAT1-CRE-EGFP-WPRE-hGH polyA mixed with rAAV-CAG-DIO-mWGA-mCherry was injected into the bilateral LS of HDC; AI47 mouse brain. b Representative images of mWGA-mCherry (monosynaptic input from LS) labeled neurons co-located with GFP (n = 3). c Experimental scheme for labeling monosynaptic downstream neurons in TMN from long-range projection of PVT glutamatergic neurons. rAAV-VGLUT2-CRE-WPRE-hGH polyA mixed with rAAV-CAG-DIO-mWGA-mCherry was injected into the PVT of HDC; AI47 mouse brain. d Representative images of mWGA-mCherry (monosynaptic input from PVT) labeled neurons co-located with GFP (n = 3). e Experimental scheme for viruses injection and electrophysiological test of histaminergic neurons. rAAV-VGAT1-CRE-EGFP-WPRE-hGH polyA mixed with AAV2/9-hSyn-FLEX-ChrimsonR-tdTomato-WPRE-pA was injected into LS of HDC; AI47 mouse brain. 5 ms, 1 Hz pulse of 635 nm red light was performed to evoke IPSCs on histaminergic neurons. f, g Representative images of clamped neuron (f) and all images of pulse laser evoked IPSCs (g), which still present after TTX and 4-AP administration but blocked by GABAA receptor antagonist bicuculline (n = 3). Traces coming from each cell are shown as same color. h Experimental scheme for viruses injection and electrophysiological test of histaminergic neurons. rAAV-VGLUT2-CRE-WPRE-hGH mixed with AAV2/9-hSyn-FLEX-ChrimsonR-tdTomato-WPRE-pA was injected into PVT of HDC; AI47 mouse brain. 5 ms, 1 Hz pulse of 635 nm red light was performed to evoke EPSCs on histaminergic neurons. i, j Representative images of clamped neuron (i) and all images of pulse laser evoked EPSCs (j), which still present after TTX and 4-AP administration but blocked by NMDA receptor antagonist APV and AMPA receptor antagonist CNQX (n = 3). Traces coming from each cell are shown as same color. APV 2-amino-5-phosphonovaleric, CNQX 6-cyano-7-nitroquinoxaline-2,3-dione, LS Lateral septal nucleus, PVT Paraventricular nucleus of the thalamus, TMN Tuberomammillary nucleus, TTX Tetrodotoxin, 4-AP: 4-aminopyridine.
