Oxytocin and vasopressin within the ventral and dorsal lateral septum modulate aggression in female rats

Abstract

In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a combination of social isolation and aggression-training to specifically investigate the involvement of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the lateral septum (LS). Using neuropharmacological, optogenetic, chemogenetic as well as microdialysis approaches, we revealed that enhanced OXT release within the ventral LS (vLS), combined with reduced AVP release within the dorsal LS (dLS), is required for aggression in female rats. Accordingly, increased activity of putative OXT receptor-positive neurons in the vLS, and decreased activity of putative AVP receptor-positive neurons in the dLS, are likely to underly aggression in female rats. Finally, in vitro activation of OXT receptors in the vLS increased tonic GABAergic inhibition of dLS neurons. Overall, our data suggest a model showing that septal release of OXT and AVP differentially affects aggression in females by modulating the inhibitory tone within LS sub-networks.

Fig. 1. Social isolation and training reliably enhance aggression in female Wistar rats, independently of the estrous cycle.

a Scheme illustrating the animal groups and experimental timeline. Female Wistar rats were kept either group-housed (GH) or socially isolated (IS). After 5 days of social isolation, isolated and trained (IST) females were exposed to three consecutive female intruder tests (FIT1 to FIT3), i.e., to aggressive encounters with an unknown same-sex intruder. Black bars correspond to data from FIT4 (test). Partially drawn using https://biorender.com. b Both IS and IST rats showed increased total aggression (one-way ANOVA followed by Bonferroni F_(2,63) = 16.26, p < 0.0001), keep down (Kruskal–Wallis test followed by Dunn’s H₃ = 6.69, p = 0.035), threat (H₃ = 18.98, p < 0.0001) and offensive grooming (H₃ = 10.8, p = 0.005). c IS and IST females engaged more in attacks (Chi-squared test, X² = 40.81, p < 0.0001), and d spent a higher percentage of time attacking (H₃ = 8.8, p = 0.012) than GH females. e IS females in the proestrus and estrus (Pro-estrus) phase of the estrous cycle displayed less aggressive behavior than metestrus-diestrus (Met-diestrus) females (two-way ANOVA followed by Bonferroni; factor housing: F_{(2, 60)} = 12.7, p < 0.0001; estrous cycle: F_{(1, 60)} = 8.5, p = 0.005; housing × estrous cycle: F_{(2, 60)} = 1.54, p = 0.22). f IST and IS females compensated their increased aggression with decreased neutral behaviors (F_{(2, 63)} = 18.76, p < 0.0001), only IST females spent more time self-grooming (F_{(2, 63)} = 5.79, p = 0.005). All data are shown as mean+SEM. ^#p < 0.05, ^##p < 0.001, ^###p < 0.001 vs GH; ^ep < 0.05 vs met-diestrus. GH: n = 23; IS: n = 21; IST: n = 21.

Fig. 2. The high levels of aggression displayed by isolated and trained (IST) rats are accompanied by high OXT and low AVP concentrations in cerebrospinal fluid (CSF), and low OXT and V1a receptor binding in the lateral septum (LS).

a IS and IST females showed higher concentrations of OXT in CSF immediately after exposure to the female intruder test (FIT) compared with respective control rats (CTRL) (two-way ANOVA followed by Bonferroni: factor FIT: F_(1,44) = 3.91, p = 0.054; housing: F_(2,44) = 1.96, p = 0.152; FIT × housing: F_(2,44) = 4.68, p = 0.014). b IST females presented decreased OXT receptor (OXTR) binding, displayed as relative optical density (ROD), in the ventral portion of the LS (vLS) (Kruskal–Wallis test followed by Dunn’s: H₍₃₎ = 7.12, p = 0.02. c Scheme illustrating localization of OXTR in the vLS, and magnification of representative example autoradiograph (left: GH; right: IST). d FIT exposure decreased CSF AVP levels only in IST rats (two-way ANOVA, factor FIT: F_(1,50) = 15.98, p = 0.0002; housing: F_(2,50) = 2.13, p = 0.129; FIT × housing: F_(2,50) = 0.90, p = 0.41). e IST females presented decreased V1aR binding, shown as ROD, in the dorsal part of the LS (dLS) (H₍₃₎ = 8.72, p = 0.006). f Scheme illustrating localization of V1aR in the dLS and magnification of representative autoradiograph (left: GH, right: IST). All data are presented as mean+SEM. ^#p < 0.05, ^##p < 0.01 vs GH; ^*p < 0.05, ^**p < 0.01 vs control. Binding: GH^: n = 8; IS: n = 4; IST: n = 5; OXT: GH: n = 17; IS: n = 16; IST: n = 17; AVP: GH: n = 18; IS: n = 20; IST: n = 18.

Fig. 3. Endogenous OXT promotes, whereas synthetic AVP and OXT reduce aggression in females.

a Experimental design for pharmacological and chemogenetic experiments targeting the OXT and AVP systems in isolated and trained (IST) and group-housed (GH) rats. (AAV adeno-associated DREADD virus infusion into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei; AVP arginine vasopressin; arrow=drug infusions; FIT female intruder test, IS social isolation, OXT oxytocin, OXTR-A OXT receptor antagonist, SURG surgery, V1aR-A V1a receptor antagonist, WO wash-out). b i.c.v. infusion of OXT (50 ng/5 µl) increased aggression in GH (two-tailed Student’s t test t₍₁₉₎ = 2.46, p = 0.024), c but decreased aggression in IST females (t₍₈₎ = 2.33, p = 0.048, data corresponds to FIT4 and 6). d i.c.v. infusion of V1aR-A (750 ng/5 µl), but not OXTR-A (750 ng/5 µl), blocked the anti-aggressive effects of OXT in IST females (one-way ANOVA followed by Bonferroni F_(3,28) = 10.1, p = 0.001). Also, the V1aR-A alone did not affect aggression. Both, e i.c.v. infusion of OXTR-A (t₍₂₈₎ = 4.96, p < 0.0001) and f i.c.v. AVP (0.1 or 1 ng/5 µl) reduced total aggressive behavior (F_(3,54) = 7.48, p = 0.0003) in IST rats. g Chemogenetic activation of OXT neurons in the PVN and SON increased aggression only in metestrus-diestrus GH rats (two-way ANOVA, factor treatment: F_(1,19) = 3.342, p = 0.083; estrous cycle: F_(1,19) = 6.68, p = 0.018; treatment × estrous cycle: F_(1,19) = 6.45, p = 0.02). h Confirmation of virus infection in the PVN (right) and SON (left). OXT-neurophysin I staining: green; mCherry (virus): red. Scale bars 300 µm. Data are shown as mean+SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.0001 vs either vehicle or control; ^ep < 0.05 vs met-diestrus. OXT: GH: n = 9 and 12; IST: n = 9; AVP: n = 18, 9, and 9, respectively; OXTR-A: n = 14 and 15; combination OXT/OXTR-A/V1aR-A: n = 8, 6, 8, 9, and 7, respectively; chemogenetics: n = 6 and 15. Control group consisted of (i) rAAV1/2 OXTpr-mCherry+CNO, no virus infusion + (ii) saline, or (iii) CNO.

Fig. 4. The pro-aggressive effect of OXT is mediated in the vLS.

a Scheme illustrating the experimental design for pharmacological, microdialysis, and optogenetic experiments targeting the OXT system (AAV adeno-associated virus microinfusion into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei; arrow=drug infusions; FIT female intruder test; GH group-housed; IS social isolation; IST isolated and trained, MD microdialysis, SURG surgery, OXT oxytocin, OXTR-A OXT receptor antagonist, vLS ventral part of the lateral septum). b IST, but not GH females showed an increased rise in the percentage of OXT release (OXT content in microdialysates sampled during the FIT/OXT content in microdialysates sampled in the baseline × 100) in the vLS during the FIT (one sample Student’s t test IST: t₍₇₎ = 2.65, p = 0.033; GH: t₍₇₎ = 0.83, p = 0.43), thus OXT release during FIT tended to be higher in IST compared with GH rats (t₍₁₄₎ = 2.12, p = 0.053). Insert shows that absolute OXT content in microdialysates sampled under basal conditions did not differ between the groups (t₍₁₄₎ = 0.54, p = 0.60). c OXTR-A (100 ng/0.5 µl) infusion into the vLS reduced total aggression (two-tailed Student’s t test t₍₂₆₎ = 2.58, p = 0.016) in IST females. d–g Optogenetic stimulation (indicated by blue columns) of OXT axons in the vLS of GH females during the FIT. d Confirmation of virus infection in OXT neurons of the PVN (left) and SON (right). OXT-neurophysin I staining: green; mCherry (virus): red. Scale bars 300 µm. Blue-light stimulation of channelrhodopsin (ChR2)-OXT fibers enhanced aggressive behavior in e metestrus-diestrus (Met-diestrus) (two-way ANOVA followed by Bonferroni factor: time: F_(5,75) = 2.72, p = 0.026; virus: F_(5,75) = 20.03, p = 0.0004; time × virus: F_(5,75) = 1.056, p = 0.392), but not in f proestrus-estrus (Pro-estrus) females (time: F_(5,70) = 2.84, p = 0.02; factor virus: F_(1,14) = 2.73, p = 0.12; virus × time: F_(5,70) = 0.02, p = 0.97). g Cumulative analyses show that light stimulation enhanced aggression only in ChR2-OXT females in the metestrus-diestrus phase of the cycle (factor virus: F_(1,15) = 13.06, p = 0.0026; estrous cycle: F_(1,15) = 2.07, p = 0.17; virus × estrous cycle: F_(1,15) = 1.114, p = 0.31). Data are shown as mean+SEM. ^(#)p = 0.05 vs GH; ^*p < 0.05, ^**p < 0.01 vs either vehicle, baseline or ChR2 control; ⁺p < 0.05 vs 0–2 time point. Microdialysis: n = 8; OXTR-A: n = 13 and 15; optogenetics: n = 8 and 9. Control group consisted of rAAV1/2 OXTpr-mCherry + light stimulation.

Fig. 5. AVP exerts anti-aggressive effects within the dLS.

a Scheme illustrating the experimental design for pharmacology and microdialysis experiments targeting the AVP system (AVP arginine vasopressin, arrow=drug infusions, FIT female intruder test, GH group-housed, IS social isolation, IST isolated and trained; MD microdialysis, SURG surgery, dLS dorsal part of the lateral septum, OXT oxytocin, TGOT [Thr4,Gly]OXT, OXT receptor agonist, vLS ventral part of the lateral septum; V1aR-A V1aR receptor antagonist). b GH, but not IST females showed an increased rise in the percentage of AVP release (AVP content in microdialysates sampled during the FIT/AVP content in microdialysates sampled in the baseline × 100) in the dLS during the FIT (Wilcoxon signed-rank test GH: W₍₉₎ = 45, p = 0.0039; IST: W₍₇₎ = −4.00, p = 0.81), thus AVP release during the FIT was higher in GH than in IST rats (Mann–Whitney U test = 6.00, p = 0.0052). Insert shows that absolute AVP content in microdialysates sampled under basal conditions did not differ between the groups. c Infusion of AVP, but not OXT or TGOT (all at 0.1 ng/0.5 µl), into the dLS decreased total aggression in IST females (F_(3,30) = 7.292, p = 0.0008). d AVP infusion into the vLS did not change aggression in IST females (F_(3,25) = 0.11, p = 0.95). Local blockade of V1aR (100 ng/0.5 µl) prior to the FIT increased aggression in e GH (two-tailed Student’s t test t₍₁₃₎ = 3.31, p = 0.006) and f IST rats (t₍₂₀₎ = 2.14, p = 0.045). Data are shown as mean+SEM. ^#p < 0.05 vs GH; ^*p < 0.05, ^**p < 0.01 vs either vehicle or baseline. Microdialysis: n = 8 and 9; AVP, OXT, TGOT dLS: 12, 5, 5 and 12, respectively; vLS: 8, 8,7 and 6, respectively; V1aR-A: GH: n = 8 and 7, IST: n = 12 and 11.

Fig. 6. Spontaneous activity in neurons of the dLS and vLS is differentially modulated by activation of OXTRs.

a Scheme indicating the receptor binding-specific delimitation of the vLS (OXTR) and dLS (V1aR) used during voltage-clamp experiments, including two representative dLS and vLS cells. b Maximal z-projection of biocytin-filled vLS cell (streptavidin-CF633) overlaid with a single z-plane of VGAT and DAPI. c Magnification of cell body from the cell shown in b. d Neurite reconstruction of cell shown in b, and e of the cell shown in f; the gray-dotted line indicates the border of the lateral ventricle (LV). f Maximal z-projection of a biocytin-filled dLS (streptavidin-CF633) cell overlaid with a single z-plane of VGAT and DAPI. g Magnification of a single z-plane indicating the presence of somatostatin (SOM) cell bodies (arrowheads) only in the dLS. h Magnification of a single z-plane indicating the presence of ERα-positive cell bodies (arrows) only in the vLS. i Morphological characterization of LS neurons. dLS cells exhibited more neurite branches (two-tailed Student’s t test t₍₄₎ = 4.84, p = 0.0072) and branching points (t₍₄₎ = 4.85, p = 0.0072) longer neurite length (t₍₄₎ = 4.80, p = 0.0078), and wider soma areas (Mann–Whitney U test U = 1.0, p = 0.016) than vLS cells. Representative spontaneous current traces during TGOT (1 µM) and bicuculline (50 µM) bath application in dLS j and vLS l cells. k TGOT increased sIPSC frequencies in dLS cells (Wilcoxon signed-rank test W₍₉₎ = 55, p = 0.002) and m decreased sIPSC frequencies in vLS cells (W₍₇₎ = −36, p = 0.0078). TGOT had no effect whatsoever on the sIPSC amplitude independent of the subregion. Data are shown as mean+SEM. ^*p < 0.05, ^**p < 0.01, vs either dLS or aCSF. Morphology: n = 5; electrophysiology: dLS n = 10, vLS n = 8.

Fig. 7. An intrinsic GABAergic circuit within the subregions of the LS regulates aggression in female Wistar rats.

a–d Example average z-projects showing pERK (Alexa594) immunostaining in Venus-VGAT females after exposure to the female intruder test (FIT). a dorsal LS (dLS) of a group-housed (GH), i.e., low-aggressive female, b dLS of an isolated and trained (IST), i.e., high-aggressive female, c ventral LS (vLS) of a GH female, d vLS of an IST female (LV lateral ventricle, VGAT vesicular GABA transporter). e–h Neuronal activity in the LS reflected by pERK staining after FIT exposure: e In the dLS of IST females less pERK-positive cells (two-tailed Student’s t test t₍₉₎ = 4.20, p = 0.0023) and fewer pERK/VGAT co-localized cells were found (t₍₉₎ = 3.75, p = 0.004). f Aggression negatively correlated with the number of pERK/VGAT-positive cells in the dLS (Pearson’s correlation r = −0.746, p = 0.008). g In the vLS of IST females a tendency of more pERK-positive cells (t₍₉₎ = 2.07, p = 0.068) and a higher number of pERK/VGAT-positive cells was found (T₍₉₎ = 2.28, p = 0.049). h Aggression did not correlate with the number of pERK/VGAT-positive cells in the vLS (r = 0.4915, p = 0.1247). i Infusion of muscimol (10 ng/0.5 µl) into the dLS increased total aggression (t₍₁₂₎ = 2.52, p = 0.027) and threat (Mann–Whitney U test U = 5.00, p = 0.011) in GH rats. j Inhibition of the dLS also enhanced the percentage of rats showing attacks (Fisher exact test, p < 0.0001). k Muscimol in the vLS decreased total aggression (t₍₁₃₎ = 3.191, p = 0.0071) and tended to decrease threat behavior (t₍₁₃₎ = 1.832, p = 0.090). l Inhibition of the vLS also reduced the percentage of rats showing attacks (p < 0.0001). Data are shown as mean+SEM. ^#p < 0.05, ^##p < 0.01 vs GH^{; *}p < 0.05, ^**p < 0.01^{, ***}p < 0.0001 vs vehicle. Neural activity: GH n = 6 and IST n = 5; muscimol: dLS: n = 7 vLS: n = 8 and 7.

Fig. 8. The balance between OXT and AVP regulates the inhibitory tonus within the LS in order to control aggression in female rats.

The scheme depicts the main findings of this manuscript. Furthermore, it hypothesizes that subtle alterations in OXT and AVP release within the LS may impact GABAergic neurotransmission to generate aggressive behavior in female rats. On the left side is depicted, how the brain of a GH female responds to an intruder: a combination of low OXT release ventrally and high AVP release dorsally evokes an increased activity of the dLS, thereby reducing aggression. On the right side is depicted, how the brain of an IST female responds to an intruder: a combination of high OXT release ventrally and low AVP release dorsally culminates in increased tonic inhibition of dLS (dotted line indicates hypothetical pathway) and increased activation of the vLS, promoting aggression. Dorsal and ventral neurons are shown in proportion to their real size. AVP arginine vasopressin, ERα estrogen receptor α, IPSC inhibitory post-synaptic current, LV lateral ventricle, MS medial septum, OXT oxytocin, OXTR oxytocin receptor, SOM somatostatin, V1aR V1a receptor. Drawn using https://biorender.com.