‘Sundowning’ in dementia and Alzheimer’s disease is characterized by early-evening agitation and aggression. While such periodicity suggests a circadian origin, whether the circadian clock directly regulates aggressive behavior is unknown. We demonstrate that a daily rhythm in aggression propensity in male mice is gated by GABAergic subparaventricular zone (SPZGABA) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZGABA neurons receive input from vasoactive intestinal polypeptide suprachiasmatic nucleus neurons and innervate neurons in the ventrolateral part of the ventromedial hypothalamus (VMH), which is known to regulate aggression. Additionally, VMH-projecting dorsal SPZ neurons are more active during early day than early night, and acute chemogenetic inhibition of SPZGABA transmission phase-dependently increases aggression. Finally, SPZGABA-recipient central VMH neurons directly innervate ventrolateral VMH neurons, and activation of this intra-VMH circuit drove attack behavior. Altogether, we reveal a functional polysynaptic circuit by which the suprachiasmatic nucleus clock regulates aggression.
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We thank Q. Ha, M. Thompson, S. Bandaru, C. Friedman, R. Thomas, and M. Ha for excellent technical assistance and C. Dulac for helpful comments during the early stages of this project. We thank J. Lynch (University of Queensland) for the hGlyR construct; and D. Anderson, X. Burgos-Artizzu, and P. Dollár (California Institute of Technology) for the Behavior Annotator MatLab script and code. This work was supported by the G. Harold and Leila Y. Mathers Foundation and the US National Institutes of Health (NIH) grants NS072337, NS085477, AG09975, and HL095491 to C.B.S.; NS073613, NS092652, and NS103161 to P.M.F.; and DK111401, DK075632, DK096010, DK089044, DK046200, and DK057521 to B.B.L. W.D.T. was supported by Alzheimer’s Association grant AARF-16-443613 and NIH grants NS084582-01A1 and HL00701-15. N.L.M. was supported by CNPq (National Health Council for Scientific and Technological Development/Brazil, grant 200881/2014-0), CAPES (Coordination for the Improvement of Higher Education Personnel).
Integrated supplementary information
Supplementary Figure 1 Representative hypothalamic sections depicting Cre expression following AAV-iCre-2A-Venus in the SPZ.
Sections were immunohistochemically double-labeled for Cre (black), to reveal cells transfected by the AAV, and VIP (brown) to reveal fibers from the SCN that delineate the SPZ. ot, optic tract. f, fornix. Representative of 16 mice.
Representative section from a Vgat in situ hybridization demonstrating unilateral SPZ Vgat-deletion compared to an undeleted SPZ. White ovals depict the SPZ. Representative of 2 mice.
In constant darkness (free-running conditions), intact mice (C57Bl6/J, n=12) show differences in aggression propensity (total time attacking) between early subjective day [circadian time (CT)1] and early subjective night (CT13). Planned comparisons, paired t test between CT1 and CT13, two-tailed, t(11)=2.482, †P=0.0305. Means ± s.e.m.
(a) Mean (± s.e.m.) Tb per hour in SPZ Vgat-deleted mice (red, n=8) and intact GFP-injected littermate controls (blue, n=8) (two-way repeated measures ANOVA). (b) Double plotted actogram for Tb in LD (12:12 light-dark cycle) and constant darkness (DD). Arrow denotes the beginning of the DD period (from the end of the normal dark period).
Darkfield photomicrographs depicting hrGFP-labeled neurons (orange/brown) in the SPZ and fibers (yellow/gold) in the DMH and VMH. (a, b) One Vgat-IRES-Cre mouse with an AAV-FLEX-hrGFP injection site that transfected neurons in both the ventral SPZ (a) and dorsal SPZ (b). Images are from the same mouse, but from adjacent sections, as depicted in Fig. 2d-e. Representative of 2 mice. (c, d) Two additional Vgat-IRES-Cre mice with different AAV-FLEX-hrGFP injection sites transfecting all but the ventral SPZ (c), representative of 2 mice, or dorsal SPZ (d), representative of 4 mice, [see white ovals, compared to (a) and (b)]. (e-f) A lack of transfected cells in the ventral SPZ results in less hrGFP-labeled fibers and terminals in the DMH (e, same mouse as c), representative of 2 mice, while a lack of transfected cells in the dorsolateral SPZ results in less hrGFP labeling in the VMH (f, same mouse as d), representative of 4 mice. Using subtraction methods we can deduce that, in mice, the ventral SPZGABA population projects more heavily to the DMH while the dorsal SPZGABA population projects more heavily to the VMH (arrows).
Supplementary Figure 6 The dorsal SPZ shows high numbers of c-Fos+ neurons at ZT1 in intact, uninjected mice.
C57BL6/J mice (n=6) were maintained under the same lighting and housing conditions as Vgat-IRES-Cre mice prior to perfusion (Fig. 4f-g), except they did not undergo prior surgery or IP injections of IVM/VEH. When perfused 90 minutes after ZT1, these mice showed intense Fos immunolabeling (black) within the dorsal SPZ (blue ovals). This area densely projects to the VMH (Fig. 3 and Fig. S3) and closely mirrors the anatomical region of SPZGABA neurons that we identified as critical for mediating changes in aggressive behavior (Fig. 1g and 3b).
There were no significant differences between IVM and VEH in total time attacking [left; paired t tests, two-tailed: t(7)=0.44, nsP=0.67), number of attack bouts [center; paired t tests, two-tailed: t(7)=0.22, nsP=0.83), or attack latency [right; paired t tests, two-tailed: t(7)=0.22, nsP=0.83] at ZT1 in control Vgat-IRES-Cre mice (n=8) injected with ChR2 into the SPZ, which does not respond to IVM. Means ± s.e.m.