A line attractor encoding a persistent internal state requires neuropeptide signaling

Abstract

Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1⁺ neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.

Keywords: CRISPR-Cas9; attractor dynamics; microendoscope imaging; neuropeptides; persistent activity.

Figure 1.. VMHvl^Esr1 neurons co-express Oxytocin and Vasopressin receptors and respond to these peptides ex vivo

A) Schematic illustrating resident-intruder (RI) behavioral assay and anatomy of VMH, with dorsomedial (dm), central (c) and ventrolateral (vl) subdivisions indicated. Esr1⁺ neurons in VMHvl respond specifically to intruder males- (red) or females-(green), or to both (gray). Male- vs. female-tuned neurons control male- vs. female-directed aggression and mating behaviors, respectively^,,. B) Violin plots illustrating expression of Esr1, Avpr1a and Oxtr mRNAs in VMH single cell transcriptomic clusters (top, colored boxes) and super-types (shaded regions). CPM^M, maximum counts per million reads. C) t-SNE plots illustrating the expression of Avpr1a and Oxtr mRNAs in single VMH cells (dots). Inset, cluster of Esr1⁺ cells (see also Supplemental Figure S1C). D) Levels of Esr1, Avpr1a, and Oxtr mRNA expression in ~50 VMH cells. E) Venn diagram of Esr1, Avpr1a, and Oxtr mRNA-expressing VMHvl neurons. Circle sizes represent relative number of neurons. F) Illustration of two-photon ex vivo calcium imaging preparation showing (i) perfusion of brain slices with peptides (400nM each AVP plus OXT) and (ii) average calcium responses in Esr1⁺ cells (n=124 cells, 4=mice). Gray shaded box depicts peptide perfusion period. Statistics: Values plotted as mean ± S.E.M. ****p<0.0001 Mann-Whitney test

Figure 2.. CRISPR/Cas9 based co-perturbation of Oxtr and Avpr1a reduces territorial aggression in males

A) Schematic illustrating viral constructs for region-restricted multiplex CRISPR/Cas9 gene editing in vivo, using a gRNA-DsRed LV (i) and Cas9 AAV (See Methods) (ii). Inset, males were co-injected bilaterally in VMH with the OAR gRNAs+Cas9 (experimental) or Scr gRNA+Cas9 (control) viral mixtures. B) Two different gRNAs each target Oxtr and Avpr1a. C) Immunostaining for DsRED (red) and Cas9 (green) in coronal VMH sections from co-injected animals. Sections are counterstained with DAPI (blue). D) Fraction of Cas9⁺ cells expressing DsRed (gRNA virus); n= 3 mice. E) Fraction of Esr1⁺ neurons responding to 400nM AVP+ OXT in VMH slices from ESR1-2A-CRE mice co-injected with Cas9 AAV plus Cre-dependent OAR gRNAs-GCaMP8s or Scr-GCaMP8s AAVs (see Figure 3Ai). Datapoints represent brain slices. n=2 Scr RNA (control) and n=3 OARs (experimental) mice. B) Flow diagram illustrating experimental paradigm. G) Male-directed behaviors in experimental (red bars) and control (gray bars) males. H) Female-directed behaviors in experimental and control males. n= 11 mice per group. Statistics: Values plotted as mean ± S.E.M. **p≤0.01 Mann-Whitney test was performed in (E), (G) and (H).

Figure 3.. Co-disruption of OXT/AVP signaling alters VMHvl^Esr1 bulk calcium activity

A) Cre-dependent AAV constructs for combining multiplex CRISPR/Cas9 gene editing and calcium imaging in vivo. B) Left, schematic of experimental design combining bilateral gene editing of Oxtr/Avpr1a with unilateral fiber photometry. Right, GCaMP8s expression (green) in a coronal VMHvl section, counterstained with DAPI (blue). C) Male-directed behaviors in experimental and control males (n=16 control and n=13 experimental mice). D) Female-directed behaviors; (n=15 control and n=13 experimental mice). E) Example fiber photometry traces from control (gray trace) and experimental (red trace) mice during male-directed sniffing bouts (behavior raster at top). F) Z-scored behavior-triggered average (BTA) of bulk calcium activity in VMHvl^Esr1 neurons during male-directed sniffing, as a peri-event time histogram (PETH). (i) AUC (area under the curve); (ii) peak PETH signal (first 1 min). Each datapoint represents a sniff bout (n=5 mice per group). G) Examples fiber photometry traces from control and experimental mice during attack. H) Z-scored BTA of VMHvl^Esr1 activity; (i) AUC and (ii) peak PETH during attack. n=5 per group. Statistics: Values plotted as mean ± S.E.M. *p≤0.05 **p≤0.01 Mann-Whitney test was used in (C), (D), (F) and (H).

Figure 4.. Single-cell imaging of VMHvl^Esr1 neurons with co-disruption of Oxtr/Avpr1a

A) Schematic of “CRISPRoscopy.” Upper, unilateral viral injection and miniscope placement; lower, coronal VMHvl section illustrating GCaMP8s expression (green); blue, DAPI counterstain. B) Sample z-scored unit responses towards female and male intruders from control (i) and experimental (ii) male residents. C) VMHvl^Esr1 ensemble representations of responses to intruder male (magenta trace) or female (green trace) for a control (i, Scr gRNAs) and experimental (ii, OARs gRNAs) male resident, projected onto a PLS regression against intruder sex. Axes indicate the percentage of variance explained for each PLS dimension. D) Accuracy of frame-wise decoders of intruder sex in control (Scr) and experimental (OARs) animals, trained on VMHvl^Esr1 unit activity. E, F) Single unit VMHvl^Esr1 activity (E) and cumulative distribution of responses (F) in units of σ above pre-intruder baseline during 1 minute of interaction. G) Percentage of male- or female-selective or co-active VMHvl^Esr1 units (≥ 2σ above the pre-intruder baseline) in control (i) and experimental (ii) mice. n=5 control, n=7 Oxtr/Avp1ra targeted animals. Statistics: Values are means ± S.E.M. Mann-Whitney test was used in (D); Nested Mann-Whitney and Kolmogorov–Smirnov test was performed in (E) and (F), respectively. **p≤0.01 ***p≤0.001 ****p≤0.0001

Figure 5.. Activity and tuning of VMHvl^Esr1 neurons with co-disruption of Oxtr/Avpr1a during social behaviors

A) Cumulative distribution plots of VMHvl^Esr1activity (in units of σ relative to pre-intruder baseline) during male- (sniffing and attack) or female- (sniffing and mounting) directed behaviors in control (Scr gRNAs) and experimental (OARs gRNAs) mice. B) Scatter plots of single VMHvl^Esr1 unit activity (σ above the pre-intruder baseline) during male-directed sniffing or attack in control and experimental mice. Green datapoints indicate sniff male (M)-selective units (≥ 2σ activity during male-directed sniffing and <2σ activity during attack); red datapoints depict attack male-selective units (≥2σ activity during attack and <2σ activity during sniffing). C) Average activity (σ) of single VMHvl^Esr1 units during sniff or attack male (M), or during sniff or mount female (F). D) Percentage of VMHvl^Esr1 units active (defined as ≥2σ relative to pre-intruder baseline) during male- (i) or female- (ii) directed behaviors in control vs experimental mice. E) Choice probability histograms of VMHvl^Esr1 behavioral tuning during male- and female-directed behaviors in control (Scr gRNAs) and experimental (OARs gRNAs) mice. Statistics: Values plotted as means ± S.E.M. Nested Kolmogorov–Smirnov test was used in (A), Nested Mann-Whitney test in (C). **p≤0.01 ***p≤0.001 ****p≤0.0001

Figure 6.. VMHvl^Esr1 line attractor dynamics require Oxtr/Avpr1a-mediated signaling

A, C) Time constants (τ) of all rSLDS dimensions, arranged in decreasing order in control (A) and experimental (C) mice. B, D) Normalized weighted average activity projections onto the time axis of the 1^st dimension (integration dimension) for VMHvl^Esr1 units in control (B) and experimental (D) M1 mice. E) Mean normalized VMHvl^Esr1 1^st dimension activity during all behavioral bouts, or the post-intruder removal period, in control (i) and experimental (ii) mice. F) Line attractor scores for VMHvl^Esr1 population activity in control (n = 4) and experimental (n =7) mice. G) Inferred flow fields of fit rSLDS models for VMHvl^Esr1 neuronal activity, reduced to the first 2 principal components (PCs), from control (i) and experimental (ii) mice M1 with projected neural trajectories (traces) and behavior annotations (color labels). H) Inferred 3D dynamic landscape of fit rSLDS model in VMHvl control mouse M1 (i) and experimental mouse M1 (ii). Different views of line (i) and point attractors (ii) are shown. Red arrows (i) depict movement along line attractor. All sniffing, dominance mounting and attack bouts towards an intruder male are depicted in the behavioral rasters in (B) and (D). Statistics: Values plotted as means ± S.E.M. Kruskal-Wallis test was performed in (A) and (C). Paired t-test was performed in (E) and Mann-Whitney test in (F) *p≤0.05 **p≤0.01

Figure 7.. Oxtr/Avp1ra-mediating signaling modulates VMHvl ^Esr1 persistent neural activity

A) (i) Behavioral raster plots and the corresponding neural activity rasters (z-scored) of individual VMHvl^Esr1 units weighted on the 1^st dimension in control (left) and experimental (right) mice. (ii) Absolute rSLDS weights of neurons contributing to the 1st dimension are shown as stem plots. B) Average single unit and cumulative distribution of neuronal persistence measured by ACHW of individual VMHvl^Esr1 units weighted on the 1^st dimension (i) and the 2^st dimension (ii) during the entire duration of male-male interactions. n=4 control, n=7 experimental animals. C) As in B), except data from all Esr1⁺ units during the first 1–2 minutes of male-male interactions. n=5 control, n=7 experimental animals. D) Schematic of working model. Oxtr and Avpr1a-mediated signaling controls aggression escalation by regulating VMHvl^Esr1 single cell dynamics (transient and persistent activity) and line attractor dynamics (left) during male-male interactions. Co-disruption of Oxtr and Avpr1a causes reduced persistent and increased transient responses, the absence of the line attractor (replaced by a trivial point attractor), and reduced aggression (right). Statistics: Values plotted as mean ± S.E.M. Nested Mann-Whitney test was performed for (Bi, Ci, Aii inset). Kolmogorov test was performed (Aii, Bii, Cii) *p≤0.05 **p≤0.01 ****p≤0.0001