Figure 3. SPZ^→VMH neurons are more active at ZT1 than ZT13, and chemogenetic inhibition of SPZ^GABA transmission increases aggression at ZT1 but not ZT13.

(a) Retrograde tracer CTb injected into the VMH (right) labeled neurons (red, left and center) in the dorsal SPZ (dSPZ) that also show Fos expression (a marker of neuronal activation, turquoise) at ZT1. Representative of 4 mice. (b) dSPZ neurons are significantly more active at ZT1 (n=9) compared to ZT13 (n=9) (unpaired t test, two-tailed, t₍₁₆₎=3.903: *P=0.0013). (c) Sections depicting differences in dSPZ cFos expression at ZT1 (left, representative of 9 mice) and ZT13 (right, representative of 9 mice). (d) Construct of AAV-FLEX-hGlyR-mCherry inhibitory vector, containing a FLEX cassette in reverse orientation and flanked by loxP sites. Cre-recombinase excises these sites and the cassette flips and locks into the correct orientation, permitting expression of hGLYR-mCherry in cre+ cells. (e) At ZT1, Vgat-IRES-Cre mice (n=8) injected with AAV-FLEX-hGlyR-mCherry into the SPZ show increased total time attacking (left; paired t tests, two-tailed, t₍₇₎=4.404, *P=0.0031), increased number of attacks (center; paired t test, two-tailed, t₍₇₎=4.615, *P=0.0024), and decreased attack latency (right; paired t tests, two-tailed, t₍₇₎=5.686, **Ps=0.0007), following administration of IVM compared to VEH. Means ± s.e.m. (f) In additional Vgat-IRES-Cre mice (n=8) injected with AAV-FLEX-hGlyR-mCherry into the SPZ, IVM administration did not significantly increase aggression propensity compared to VEH at ZT13 (paired t tests, two-tailed; time attacking, t₍₇₎=0.5467, ^nsP=0.6016; number of attacks, t₍₇₎=1.93, ^nsP=0.0949; attack latency, t₍₇₎=1.118, ^nsP=0.3005). Means ± s.e.m. (g) Heat map depicting overlapping injection sites within the SPZ, each weighted according to the magnitude of difference in total time attacking for the IVM condition compared to the mean VEH response. Arbitrary units. (h) Vgat-IRES-Cre mice injected with AAV-FLEX-hGlyR-mCherry into the SPZ were injected with IVM (n=3) or VEH (n=3) at ZT1 and then perfused 90 min after ZT1 on the following day. Mice receiving IVM showed significantly fewer dsRed-containing (marker for neurons injected with AAV-FLEX-hGlyR-mCherry, brown) neurons that expressed Fos (black) compared to mice receiving VEH (unpaired t test, two-tailed, t₍₄₎=3.497, ^†P=0.025). Means ± s.e.m. (i) Representative sections depicting decrease in dsRed/Fos double labeling at ZT1 in the dSPZ (center boxes) following IVM (right, representative of 3 mice) compared to VEH (left, representative of 3 mice) administration.