Social Control of Hypothalamus-Mediated Male Aggression

Abstract

How environmental and physiological signals interact to influence neural circuits underlying developmentally programmed social interactions such as male territorial aggression is poorly understood. We have tested the influence of sensory cues, social context, and sex hormones on progesterone receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) that are critical for male territorial aggression. We find that these neurons can drive aggressive displays in solitary males independent of pheromonal input, gonadal hormones, opponents, or social context. By contrast, these neurons cannot elicit aggression in socially housed males that intrude in another male's territory unless their pheromone-sensing is disabled. This modulation of aggression cannot be accounted for by linear integration of environmental and physiological signals. Together, our studies suggest that fundamentally non-linear computations enable social context to exert a dominant influence on developmentally hard-wired hypothalamus-mediated male territorial aggression.

Keywords: VMH; aggression; castration; emotion; pheromone; progesterone receptor; sex hormones; sexual dimorphism; territorial behavior; ventromedial hypothalamus.

Figure 1. Chemogenetic activation of PR+ VMHvl neurons triggers aggression

All mice were injected with AAV encoding Cre-dependent hM3DGq-mCherry (A–G) or mCherry (D) into VMHvl of adult PR^Cre males. (A) Schematic showing bilateral stereotaxic delivery of AAV to the VMHvl of PR^Cre mice. Inset shows mCherry+ cells in VMHvl 10 days following injection. (B) Top: mCherry+ (PR+, hM3DGq+) neuron in VMHvl targeted for patch-clamp recording using a 1040 nm 2-photon excitation source. Bottom: target neuron filled with Alexa 594 was visualized with 810 nm 2-photon excitation to confirm neuronal identity. Scanning differential interference contrast was visualized in both imaging configurations to guide recording. (C) Example of such a patched mCherry+ neuron where 5 min CNO (1 μM, gray shading) depolarized the neuron and increased spiking activity. Numbers (1–3) highlight spiking during baseline, immediately after CNO, and 25 min following washout. (D) Summary of electrophysiological studies on PR+ VMHvl neurons expressing hM3DGq-mCherry (left) or control mCherry (right). Each dot represents a single neuron, and lines connect baseline and CNO conditions within recordings. Data from 6 hM3DGq-mCherry and 4 mCherry injected males. (E) CNO injection increases Fos expression in PR+ VMHvl neurons expressing hM3DGq-mCherry. Upper and lower histological panel images captured at 10X and 63X objectives, respectively. Quantification shows % hM3DGq-mCherry VMHvl neurons that express Fos following CNO or saline (control) injection. n = 3/condition. (F) Resident solitary PR^Cre males attack WT socially housed intruder males with increased probability and intensity when injected with CNO. There is no difference in percent resident males tail rattling to an intruder when given CNO or saline. n = 25/condition. (G) Resident solitary PR^Cre males tail rattle to and attack WT socially housed intruder females primed to be in estrus only when given CNO. n = 28/condition. na (not applicable): Only mice showing attack behavior were included in analysis of non-categorical data such as number of attacks and latency to first attack in this and all other figures. If no or too few males given saline displayed attacks, it precluded statistical analysis of these attack parameters. See also Table S1 for this and all other Figures, where we have analyzed these non-categorical data following inclusion of all animals, regardless of whether or not they displayed attacks. ns (not significant). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars = 100 μm and 500 μm for upper and lower insets, respectively (A), 10 μm (B), 100 μm and 20 μm for upper and lower insets, respectively (E).

Figure 2. PR+ VMHvl neurons elicit aggressive displays independent of chemosensory signaling

(A) Schematic showing that chemosensory cues detected by MOE or VNO neurons may elicit aggression by functioning in pathways upstream, downstream, or parallel to PR+ VMHvl neurons. AAV encoding Cre-dependent hM3DGq-mCherry was delivered to VMHvl of PR^Cre;Trpc2^−/− (B) or PR^Cre;Cnga2^−/Y (B) or PR^Cre (C, D) adult males. (B) Solitary male residents null for Trpc2 or Cnga2 tail rattled to and attacked socially housed female intruders only after being administered CNO. n = 12/condition (Trpc2^−/−) and 8/condition (Cnga2^−/Y). (C) Solitary male residents tail rattled to and attacked socially housed P. eremicus female intruders only after CNO injection. n = 10/condition. (D) Solitary male residents tail rattled more to inanimate objects after CNO administration. They also tail rattled more to the reflective side of the mirror compared to its non-reflective side or to a partially inflated glove. n = 13/condition (No mirror; Mirror); 12/condition (Flipped mirror); 13 (saline) and 9 (CNO) (Glove). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3. PR+ VMHvl neurons elicit aggressive displays independent of male gonadal hormones

(A) Schematic showing that testicular hormones may influence aggression by activating physiological function of PR+ VMHvl neurons directly or that of neural pathways upstream, downstream, or parallel to PR+ VMHvl neurons. (B) AAV encoding Cre-dependent hM3DGq-mCherry was delivered to VMHvl of PR^Cre adult males who were subsequently castrated or subjected to sham castration surgery. No differences between castrate and sham control males tail rattling to and attacking WT socially housed female intruders when injected with CNO. Mean ± SEM. n = 7/condition (Sham castrated) and 13/condition (Castrated). *p < 0.05, ***p < 0.001.

Figure 4. PR+ VMHvl neuron elicited aggression is dependent on social context

AAV encoding Cre-dependent hM3DGq-mCherry was delivered to VMHvl of PR^Cre adult males who were socially housed with other males (A) or singly housed (B) and used as intruders. Singly, but not socially, housed male intruders given CNO tail rattled to and initiated attacks toward a WT solitary male resident. Too few socially housed (n = 1) or solitary (n = 2) male intruders given saline attacked the resident, thereby precluding meaningful statistical analysis for non-categorical data. Mean ± SEM. n = 19 (saline) and 21 (CNO) (A); n = 15 (saline) and 14 (CNO) (B). ***p < 0.001.

Figure 5. PR+ VMHvl neurons can trigger aggression in socially housed males

AAV encoding Cre-dependent hM3DGq-mCherry was delivered to VMHvl of PR^Cre adult males who were socially housed with other males and used as residents (A) or intruders (B). (A) Socially housed resident males were injected with CNO or saline, their cage mates were removed from the homecage, and a WT intruder male was inserted into the cage. Such socially housed residents tail rattled to and attacked intruders significantly more with CNO compared to saline. n = 12/condition. (B) Socially housed males injected with CNO or saline were inserted into a clean cage (neutral arena) along with a WT socially housed male. CNO-administered males tail rattled to and attacked the WT male more than males injected with saline. n = 8/condition. Too few socially housed males given saline initiated attacks in their homecage or a neutral arena (n = 1 each), thereby precluding meaningful statistical analysis for non-categorical data. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. Male pheromones inhibit aggression elicited by PR+ VMHvl neurons

AAV encoding Cre-dependent hM3DGq-mCherry was delivered to VMHvl of PR^Cre adult males who were socially housed with other males and used as intruders. (A) Castrate male intruders injected with CNO did not tail rattle to or initiate attacks toward WT solitary resident males significantly more than when they were injected with saline. n = 15/condition. (B) Male intruders genetically disabled for chemosensory signaling via the VNO or MOE (PR^Cre; Trpc2^−/− or PR^Cre;Cnga2^−/Y) injected with CNO but not saline tail rattled to and attacked WT solitary resident males. n = 15/condition (Trpc2^−/−) and 8/condition (Cnga2^−/Y). (C) Male intruders injected with CNO but not saline tail rattled to and attacked WT solitary resident females. n = 13/condition. Mean ± SEM. **p < 0.01, ****p < 0.0001.

Figure 7. Modeling male aggression with logic circuits and neural networks

(A) Schematic of a logic circuit that summarizes results in which the output variable is attack and the opponent is a lab mouse. (B) A two layer but not a single layer network adequately captures the findings summarized in the logic circuit outlined in panel A and Table 1. We determined the fewest experiments that need to be excluded in order for the logic circuit to be implementable by a single layer network. This search showed that removal of only one experiment, outlined in rows 14, 15 (Table 1), is sufficient for the logic circuit to be realized by a linear classifier. The experiment outlined in these two rows demonstrates that socially housed intruder males do not initiate attacks toward resident males even upon stimulation of PR+ VMHvl neurons. Green bar indicates findings from rows in Table 1 that can be implemented by the neural network whereas findings from numbered rows (red) cannot be implemented by the network.