Microinjector

For exposure of LPS by microinjection, 3 dpf larvae were collected and incubated with embryo media containing sterile distilled water (vehicle control) or 800 μg/mL spermidine (final concentration) at 28.5°C. After 18 and 20 h incubation, larvae were anesthetized and 1 mg/mL or 0.125 mg/mL LPS was injected into the yolk using a microinjector (Harvard Apparatus, Inc., Cambridge, MA, USA) for sudan black or neutral red staining, respectively, according to the methods used by Yang et al (2014) (link). Microinjection volume was 2 nL per larvae, and the control group was injected with an equal volume of PBS. The larvae from each group were further cultured for up to 4 dpf in the embryo media with or without 100 μg/mL spermidine for staining with sudan black and neutral red.

The anesthetized animals were fixed in a stereotaxic instrument (Narishige, Tokyo, Japan), and the scalp was incised at the midline of the skull. A hole was made in the skull based on a stereotaxic atlas [26 ]. A microtrocar was inserted into the left MVN and RVLM and fixed with dental cement. The microtrocar for the MVN was placed 11.6 mm posterior to the bregma, 1.5 mm left from the midline, and 6.4 mm depth from the dura mater. The microtrocar for the RVLM was located 12.7 mm posterior to the bregma, 2.4 mm left of the midline, and 7.3 mm depth from the dura mater [26 ]. 10 µl of either ACSF, NMDA, AMPA, MK801, or CNQX (all 1 mM) was injected into the MVN or RVLM using a microinjector (Harvard Apparatus, Holliston, MA, USA). All of the animals were dosed while maintaining consciousness. Hypotension was induced by an intravenous injection of SNP, 10 min after drug injection. After the end of the experiment, the MVN or RVLM were histologically confirmed by a stereotaxic atlas [26 ].