Hierarchical Representations of Aggression in a Hypothalamic-Midbrain Circuit

Abstract

Although the ventromedial hypothalamus ventrolateral area (VMHvl) is now well established as a critical locus for the generation of conspecific aggression, its role is complex, with neurons responding during multiple phases of social interactions with both males and females. It has been previously unclear how the brain uses this complex multidimensional signal and coordinates a discrete action: the attack. Here, we find a hypothalamic-midbrain circuit that represents hierarchically organized social signals during aggression. Optogenetic-assisted circuit mapping reveals a preferential projection from VMHvl^vGlut2 to lPAG^vGlut2 cells, and inactivation of downstream lPAG^vGlut2 populations results in aggression-specific deficits. lPAG neurons are selective for attack action and exhibit short-latency, time-locked spiking relative to the activity of jaw muscles during biting. Last, we find that this projection conveys male-biased signals from the VMHvl to downstream lPAG^vGlut2 neurons that are sensitive to features of ongoing activity, suggesting that action selectivity is generated by a combination of pre- and postsynaptic mechanisms.

Keywords: aggression; circuit; hypothalamus; jaw muscle; periaqueductal gray; social behavior.

Figure 1.

An excitatory circuit connect the VMHvl to jaw-projecting neurons in the lPAG (A) Viral strategy for targeting excitatory projections from VMHvl to lPAG. Slices were made of VMHvl and lPAG and whole cell recordings were performed. (B) Representative infrared differential interface contrast image (IR-DIC) from a recorded slice containing VMHvl (top, left) and lPAG (bottom, left). Yellow arrows indicate locations of recording pipette tips. Scale bar 500 μm. (right) Coronal section (right) showing expression of Chrimson-tdTomato (red) from a vGlut2 x Ai6 mouse. Scale bar 1mm. (C) Example trace showing current clamp recording of a VMHvl^vGlut2 neuron expressing Chrimson-tdTomato. 605 nm light pulses (20 Hz, 20 ms for 500 ms, red ticks) reliably evoked time-locked spiking. Scale bars: 100 ms (horizontal) and 10 mV (vertical). (D) Histological image showing distribution of glutamatergic cells (green) and Chrimson-tdTomato expressing fibers from the VMHvl (red) in the PAG. Blue: Topro-3. Scale bar, 200 μm. (E) Enlarged views from (D) showing biocytin-filled vGlut2 negative (top row) and positive cells (bottom row) and their corresponding recording traces showing 1ms 605 nm light evoked EPSC (−70 mV) and IPSC (0 mV). Yellow arrows indicate the locations of the biocytin filled cells. Scale bar (left), 20 μm. Scale bars (right): 10 ms (horizontal) and 10 pA (vertical). (F) Stacked bar graphs showing the percentage of recorded PAG cells receiving EPSC during light stimulation. Two-tail Fisher’s test. *p = 0.0132. (G) Light-evoked EPSC amplitude (left) and latency (right) in PAG glutamatergic neurons (N=12). Error bars show means ± SEM. (H) Example traces showing 1ms 605 nm light evoked EPSC before (black) and after 1 μM TTX and 100 μM 4AP perfusion (red) in PAG glutamatergic neurons. Scale bars: 100 ms (horizontal) and 10 pA (vertical). (I) No change in light-evoked EPSC amplitude before and after 1 μM TTX and 100 μM 4AP perfusion in PAG glutamatergic neurons (n=6). Paired t-test. p > 0.05. (J) Schematic describing strategy for quantifying jaw-projecting lPAG^vGlut2+ neurons. (K) 297/327 (90.8%) of PRV-614 labeled neurons in lPAG were vGlut2-Ai6 positive. (Example overlap shown in K, bottom right). (L) Strategy for functional targeting of jaw-projecting lPAG neurons (M) Example traces showing 1ms 605 nm light evoked EPSC in one PAG^vGlut2 neuron. Scale bars: 10 ms (horizontal) and 10 pA (vertical). (N) Summary (left; n=27 cells, n=8 mice) and failure rate (right). Stacked bar graph showing 12 out of 23 PAG PRV+ vGlut2+ neurons received glutamatergic input from VMHvl. (O) Light-evoked EPSC amplitude and latency in PAG PRV+ vGlut2+ neurons (n=23). Error bars show means ± SEM.

Figure 2.

Chemogenetic inactivation of lPAG results in aggression-specific deficits. (A) Viral strategy for reversible inactivation of lPAG. vGlut2-ires-cre animals were injected bilaterally with DIO-hM4D(Gi)-mCherry (N=8 animals) or DIO-mCherry (N=6 animals). After injection with either saline or CNO, animals were tested during interactions with males, females, then given access to palatable food. (B) Example histology showing expression of hM4Di-mCherry in the PAG. Scale bar 500μm. (C-F) Following CNO inactivation (left panels), percent time spent attacking was reduced relative to saline (C, p=0.016). This effect was due entirely to reduced attack duration following inactivation (D, p=0.004), and not to changes in attack latency (E, p=0.784) or to the number of attack episodes (F, p=0.772). No effects on attack were observed in control animals (right panels, C-F, p=0.854, p=0.236, p=0.480, p=0.854). (G-J) In contrast to attack, no effects on other social or jaw-dependent behaviors were observed. CNO Inactivation did not affect the perfect time spent investigating males (G, hM4Di: p=0.751 mCherry: p=0.239), time spent investigating females (H, hM4Di: p=0.270 mCherry: p=0.584), mounting females (I, hM4Di: p=0.332 mCherry: p=0.567), or the latency to approach and consume a yogurt pellet (J, hM4Di: p=0.998 mCherry: p=0.998), paired t-tests. We tracked the locations of the saline and CNO injected animals (K) during male-male (L) and male-female (M) interactions. No significant differences in the distribution of inter-animal distance (L-M left, p=0.243, p=0.154) or velocity (L-M right, p=0.999, 0.999) were observed between saline and CNO injection.

Figure 3.

Activity in the lPAG is more attack selective than the VMHvl. We performed chronic single unit recordings in the lPAG during social behaviors and compared these responses to activity in the VMHvl. (A) Histology showing example placement of electrode bundles (and one group of tetrodes (PAG3) in the lPAG and electrode track locations for all recording animals (N=6). Scale bar 1mm. (B) Example raster plot (top) and PETH (bottom) for activity of an example lPAG units aligned to attack (red), investigation of a male (blue), and investigation of a female (green) sorted by behavior. (C-E) Normalized responses of population (top) of recorded neurons sorted by peak of response aligned to attack (C, N = 159), investigation of male (D, N = 158), and investigation of female (E, N = 151). Bottom histograms show number of units with response peak above 95% CI in each bin. Dotted black lines (C-E) represent chance levels for each behavior. (F) Normalized population response mean ±SEM of VMHvl (top) and lPAG (bottom) during onsets of key behaviors interactions with males: attack (red) and investigate male (blue), and with females: investigate female (green). (N = 166,156 for VMHvl male attack and investigate, N=212 for female investigate, N = 159 for lPAG male attack and investigate, N=151 for female investigate). Comparison of responsivity of individual VMHvl neurons (G-H top, light gray) and lPAG neurons (G-H bottom, dark gray). VMHvl population is nonselective between attack and investigate male (G, top, p=0.806, N = 157), and selective for investigation of male compared to female (H, top, p=0.005, N=147), while lPAG is selective for attack relative to investigate male (G, bottom, p=3.4×10^−7, N=152), and nonselective for investigation of males and females (H, bottom, p=0.415, N=152). Tests in G-H performed using Wilcoxon signed-rank test. Pie chart insets displaying percentages of individually significant neurons (Bonferroni-corrected t-test) in VMHvl and lPAG show an increasing number of purely attack selective neurons in the lPAG relative to VMHvl and a decrease of investigation selective neurons in the lPAG. (I) Selectivity of population to attack compared to selectivity to investigate male shows that attack-shifted peak for lPAG population (dark gray) relative to VMHvl (light gray) shown using RI value. P=0.0001, Kolmogorev Smirnov test. (J) Attack responsive neurons in the VMHvl (light gray) have significantly increased activity prior to attack onset relative to attack responsive lPAG neurons (dark gray). N= 44 neurons in VMHvl N=46 neurons, lPAG, p = 0.0005, Bonferroni corrected unpaired ttest across all bins.

Figure 4.

lPAG spiking has precise temporal alignment with jaw muscle activity during aggression. (A) Example simultaneous recordings of jaw EMG and lPAG spiking during attack episodes, shown in red. (B) Example EMG (top) and activity from simultaneously lPAG neuron (bottom) during interaction with a male (C) Mutual information (MI) of lPAG spiking and EMG activity comparing during interactions with males and females (N=64 neurons). MI of activity during male interaction compared to time shuffled control (p=0.0214, paired t-test), MI of activity during female interactions to time shuffled control (p=0.1551, paired t-test), MI of activity during male and female interactions (p=0.0053, paired t-test). Example STEMG (D) and attack-aligned PETH (E) with precise temporal alignment to EMG. (F-G) STEMG (F) and attack aligned activity (G) of neurons with significant STEMG (red, top trace), and significant attack responsive neurons that are do not have significant STEMG (black, bottom trace), show distinct dynamics.

Figure 5.

Activity in lPAG-projecting VMHvl neurons conveys preferentially male-specific information (A) Experimental configuration of bilateral injection of GCaMP6f into vGlut2-ires-Cre mice for simultaneous fiber photometry recordings of VMHvl and VMHvl-PAG projection neurons. Ipsilateral injection targets VMHvl^vGlut2 neurons and the contralateral injection targets VMHvl-PAG^vGlut2 projection neurons. (B) Example histology of GCaMP-labeled VMHvl^vGlut2 neurons (right) and VMHvl-lPAG^vGlut2 neurons (left) and placement of fiber tracks. Scale bar 300μm. (C) Example simultaneous recording of VMHvl ^vGlut2 (black) and VMHvl-PAG ^vGlut2 (magenta) projection neurons during alternating interactions with males (blue) and females (red). (D) Population activity (mean +SEM for each animal, N=6 animals) of comparison between activity during male interaction and female interaction for VMHvl neurons (D left, p=0.0062) and for VMHvl-PAG projection neurons (D right, p=0.0002), shows that both populations exhibit increased activity to males. (E) Comparison of simultaneously recorded activity VMHvl ^vGlut2 and VMHvl-PAG ^vGlut2 neurons is not significantly different during male interactions (E left, p=0.3676), but activity during female interaction is reduced in VMHvl-PAG neurons (E right, p=0.044). (F) Distributions of the Pearson correlation between behavior-aligned activity in VMHvl and VMHvl-PAG. Correlation distributions are not significantly different between attack and investigation of male (F, left, p=0.358, unpaired t-test, N=83 attack trials, N=85 investigate male trials), but correlations are higher in both attack and investigate male than during investigation of females (F, middle p=0.00002; F, right p=0.004, unpaired t-test, N=180 investigate female trials). Solid lines represent distribution medians. (G) Correlation of simultaneously recorded VMHvl^vGlut2 and VMHvl-PAG^vGlut2 neurons is higher in male interactions than female interactions (p=0.0238). All tests (D-E,G) using paired t-test.

Figure 6.

Simultaneous recordings of VMHvl^vGlut2 and lPAG^vGlut2 reveal lPAG activity is preferentially coupled to VMHvl during attack and is preferentially sensitive to VMHvl activity history during male interactions (A) Experimental configuration of simultaneous recordings during interactions with males (B) and females (C). (D) Distributions of the Pearson correlation between behavior-aligned activity in VMHvl^vGlut2 and lPAG^vGlut2. Correlation distributions are significantly higher for attack than investigation of males or females (D, left, p=0.00003, unpaired t-test, D, right, p=0.00006, N=112 attack trials, N=73 investigate male trials, N=174 investigate female trials), but correlation distributions are similarly low for investigation of either males or females (D, middle p=0.240). Solid lines represent distribution medians. (E) Cross correlation of simultaneously recorded signals during male interactions (blue), female interactions (red) and no-interaction baseline (black). (F) Comparison of summed cross correlation in pre epoch (−10s to 0s) and post epoch (0s to 10s) for male, female, and no-interaction baseline shows significant asymmetry only during male interaction (N=7 animals, male p=0.0174*, female p=0.9333, baseline p=0.6148). (G) PAG ^vGlut2 activity was fit with a linear model using a variable amount of preceding VMHvl^vGlut2 signal as the regressors. (H) Fit percent (middle) and time (right) associated with best fit models of cross-validated data using a time-varying input from VMHvl. Dotted lines represent data from time shuffled controls. (I) Controls for time-varying regression model. Comparison of forward (VMHvl → lPAG) and reverse (lPAG → VMHvl) regressive models. Model fit percentages for fits during male (I, left) and female (I, right) interactions. Black traces (I) show reverse models. During male interactions (blue), fits are significantly increased in forward model relative to reverse model (*p<0.05, paired t-test for each time bin). (J) Conceptual model of pathway selectivity of male-responsive information.