Abstract
Compared to males, aggression is less frequently noticed in females. Fierce maternal-aggression to thwart the attack/threat of a male conspecific/intruder is transiently expressed as she defends her pups. The odor cues emanated by the intruder provoke aggressive behavior by robustly activating the ventral-premammillary nucleus (PMv) in the hypothalamic-attack area (HAA). But, how PMv activation triggers aggression is unclear. In view of neuropeptide cocaine- and amphetamine-regulated transcript (CART)'s potential to reconfigure neural circuits for behavioral demands, occurrence throughout aggression circuitry, and abundance particularly in PMv, we test the role of PMvCART in maternal and inter-male aggression in rats. Males/dams actively attacked the intruder; virgin females did not. The dams/males without intruder showed isolated c-Fos cells in PMv, but the intruder's presence triggered c-Fos-activation in different PMv-subdivisions in dams/males. Compared to dams without intruder, confrontation with intruder robustly activated PMvCART-neurons and augmented CART-ir in ventral-PMv and cart-mRNA in PMv-containing tissues in dams. Conversely, in males, the intruder's presence activated lateral-PMvCART neurons, but CART-levels remained unaltered. Intra-PMv CART-siRNA administration suppressed maternal-aggression, but male aggression was unaffected. Since PMv is strongly connected with the ventrolateral–ventromedial hypothalamus (VMHvl) and medial preoptic nucleus (MPN), we test whether CART signaling to these nuclei triggers maternal aggression. While VMHvl showed stronger CARTergic-axonal input than MPN, immunoneutralization of CART in VMHvl, but not MPN, blocked maternal-aggression. CART may drive the circuit beyond HAA since VMHvl neurons contacted by CART-axons project to periaqueductal gray. We identify the engagement of vPMv and lPMv during maternal and inter-male aggression, respectively, and CART as a key mediator in the PMv–VMHvl pathway to express maternal-aggression in rats.
- aggression circuitry
- CART peptide
- c-Fos
- inter-male aggression
- maternal aggression
- ventral premammillary nucleus
Significance Statement
A pregnant/lactating rat transiently becomes fiercely aggressive to protect her pups when challenged by an intruder. The neural mechanism underlying this transitory expression of aggressive behavior is not clear. We identify the role of neuropeptide cocaine- and amphetamine-regulated transcript (CART)-containing neurons in the hypothalamic ventral premammillary nucleus (PMv) in dams that give them the behavioral flexibility to display maternal-aggression. A subset of PMvCART neurons in dams shows dramatic activation when provoked by an intruder while silencing of these neurons suppressed maternal but not inter-male aggression. Furthermore, CART signals the ventrolateral part of the ventromedial hypothalamus to trigger aggression in dams. The study shows CART as a novel messenger in aggression circuitry and PMvCART as a key regulator of maternal aggression.
Introduction
Although inter-male interaction is fraught with aggression among conspecifics, the behavior is rarely seen in females (Stagkourakis et al., 2018). Pregnancy/lactation profoundly reconfigures neural pathways to elicit specific behavioral expression in the mother (Hasen and Gammie, 2005). The maternal aggression against the intruder is one such extraordinary behavioral flexibility transiently expressed by the mother. This response helps protect her offspring from the impending threats/attack from an intruder (Hahn-Holbrook et al., 2011; de Almeida et al., 2014). The ability to display aggression is acquired a day prior to parturition (Caughey et al., 2011), the intensity of the behavior increases at postpartum days 3–12 (Consiglio and Lucion, 1996), and this maternal prowess completely disappears with weaning of pups (Caughey et al., 2011). The neural mechanism underlying a transient shift in the behavior of females to express maternal aggression is unclear.
Extensive neural circuitry detects the intruder, interprets the threat component, and evokes aggression. The hypothalamic attack area (HAA) comprising the medial preoptic nucleus (MPN), tuberal region of the lateral hypothalamic area/ventrolateral part of the ventromedial hypothalamus (VMHvl), and ventral premammillary nucleus (PMv) is known to mediate aggression (Hasen and Gammie, 2005; Motta et al., 2013). The PMv is heavily interconnected with other components of the HAA and plays a crucial role in precipitating maternal aggression (Canteras et al., 1992; Motta et al., 2013). While the optogenetic stimulation of PMv or its activation following exposure to the intruder triggered maternal aggression, lesion or inhibition of PMv reduced the intensity of behavior (Motta et al., 2013; Stagkourakis et al., 2024). In addition, PMv is known to be involved in inter-male aggression (Stagkourakis et al., 2018). However, little is known about the signaling agents in the PMv that mediate aggression. The PMv is known to be composed of distinct subdivisions (Cavalcante et al., 2006); however, the functional significance of each, with reference to inter-male and maternal aggression, has not been defined.
Neuropeptide cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the brain and regulates a range of functions, including energy balance, reward, reproduction, fear, and emotions (Lau and Herzog, 2014; Subhedar et al., 2014). CART is also known to profoundly reconfigure the circuits for behavioral demands (Bodas et al., 2023). Although the abundance of CART throughout the aggression circuitry hints at a novel role of this neuropeptide in the brain, experimental evidence is lacking.
We set out to examine the role of PMvCART in aggression since these neurons are CART-enriched, activated by opposite-sex odor, and control neuroendocrine output in response to olfactory cues (Elias et al., 2001; Cavalcante et al., 2006; Vrang, 2006; Donato et al., 2010; Donato and Elias, 2011). We find that the neurons in the PMv across males, virgin females (VF), and dams display a subject-specific pattern of activation following exposure to an intruder. Interestingly, a selective subpopulation of CART neurons in PMv shows dramatic activation only in dams provoked by an intruder. The siRNA knockdown experiment targeting PMvCART attenuated maternal aggression, but not inter-male aggression. Furthermore, we identify VMHvl as a critical site for CART signaling to trigger aggression in dams. We propose that the circuit consisting of the PMvCART neurons and the CARTergic inputs converging on the VMHvl neurons, which further convey the output information to the lateral periaqueductal gray (lPAG), may play a key role in mediating aggressive behavior expressed by the dam.
Materials and Methods
Animals
Adult, male and female Sprague Dawley rats (approximately 3 months old) were used in the study. The animals were maintained at standard conditions of the animal facility (12 h light/dark photoperiod, temperature, and relative humidity) and provided rat chow and water ad libitum. Naïve, adult male Wistar rats housed individually in a separate room of the animal house were used as intruders (I) only once on the day of the experiment. All the experimental procedures were performed in compliance with the guidelines of the Institutional Animal Ethics Committee (IAEC), National Institute of Science Education and Research (Approved Protocol #AH212), and Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur (Approved Protocol #IAEC/UDPS/2022/02/28), under the Committee for the Control and Supervision of Experiments on Animals (CCSEA), New Delhi, India.
Maternal aggression behavior
The experiments were performed as described earlier (Hasen and Gammie, 2005; Motta et al., 2013). The female rats were randomly allocated to two groups, viz., (1) virgin (VF) and (2) lactating (dams, Dm). While the rats in the virgin group were individually kept in each cage, those in the lactating group were housed with a breeder male Sprague Dawley rat. The pregnant females were separated from the male a week before labor and housed individually. Following parturition, the litters were culled to eight pups (four males, four females) maximum on the postpartum (PP) day 1, considering the day pups were born as PP day 0. The behavior of the Dm with the pups was monitored from the PP days 1–4. The cages were changed once a week for the rats in the VF group, whereas those for Dm were not disturbed until the completion of the experiment. The VF and Dm were randomly divided into four groups (n = 6–8/group), viz., (1) VF without an intruder (VF − I), (2) VF with an intruder (VF + I), (3) Dm without an intruder (Dm − I), and (4) Dm with an intruder (Dm + I). The experimental plan is shown in Figure 1A. As described (Motta et al., 2013), the aggressive behavior was tested on PP day 5 or 6. The intruder was introduced individually into the cage housing the VF or Dm and the latency to the first attack, and the total number and duration of attacks on the intruder were monitored for 5 min (Gammie and Nelson, 2001; Gammie et al., 2004; Motta et al., 2013). Additionally, the sniffing and exploratory behavior of the VF and Dm toward the intruder and the maternal behavior of Dm were monitored during the experimentation.
A, Schematics of the experimental design. The virgin females (VF) and dams (Dm) were divided into four groups. The groups of VF and postnatal day 5 (PD5) Dm not exposed to an intruder (I) are identified as VF − I and Dm − I, respectively. The VF and Dm in the other two groups exposed to intruders are designated as VF + I and Dm + I, respectively. The behavior including the time sniffing intruder (B), number of attacks on an intruder (C), and duration of attack (D) were recorded for 5 min. After the experiment, brains were processed for immunofluorescence. Note that the VF spent more time sniffing the intruders as compared to the Dm. The Dm + I displayed maternal aggression with a higher number and duration of attacks over the 5 min testing period. X, absence of the specific behavior in VF + I. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by the Student's t test (***p < 0.0001).
Intermale aggression behavior
The male aggression protocol as described previously (Soden et al., 2016) was employed. Single-housed, sexually experienced resident male Sprague Dawley rats (M) were divided into two groups. Adult, male Wistar rats younger than the resident rats were used as intruders (I). In the first group, the intruder was placed into the resident male's cage (M + I), and behavior including the number and duration of attacks, exploration, and defense were analyzed as previously described (Van Berg et al., 1983). The rats in the second group (M − I) kept in their home cage but not exposed to the intruder served as controls.
After completion of the experiments, all the females in maternal aggression and the resident males in intermale aggression groups were perfused as described below.
Tissue processing and immunofluorescence
The rats were deeply anesthetized with an intraperitoneal injection of ketamine [90 mg/kg body weight (BW)] and xylazine (10 mg/kg BW) and transcardially perfused with the phosphate-buffered saline (PBS) followed by 100 ml of 4% formaldehyde prepared from paraformaldehyde in phosphate buffer (pH 7.4) as fixative. The brains were dissected out, postfixed in the same fixative, and cryoprotected in 25% sucrose solution in PBS for 24 h. The brains were sectioned on a cryostat (CM3050 S, Leica Microsystems), and 25 μm thick, coronal sections of the posterior hypothalamus encompassing the rostrocaudal extent of the PMv [coordinates: anterior–posterior (AP), −3.6 to −3.96 mm (Paxinos and Watson, 2007)] were collected in PBS and stored in the antifreeze solution (Basu et al., 2022) at −20°C until further processing.
The immunofluorescence method was employed as described previously (Basu et al., 2022). The sections were rinsed in PBS, treated with 0.5% Triton X-100 in PBS, and blocked in 3% normal horse serum in PBS for 30 min. One set of sections from animals in each group was incubated in either the rabbit polyclonal anti-CART antiserum (1:5,000, catalog #H-003-62, Phoenix Pharmaceuticals, RRID: AB_2313614) or a mixture of anti-CART (1:5,000) and sheep polyclonal anti-c-Fos (1:2,000, catalog #sc-52-G, Santa Cruz Biotechnology, RRID: AB_2629503) antisera for 24 h at 4°C. The sections were rinsed in PBS and incubated in either Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1,000, Life Technologies) or a mixture of diluted secondary antibodies [Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-sheep IgG (1:1,000 each, Life Technologies)] for 4 h at room temperature.
The sections encompassing the rostrocaudal extent of the PMv from the VF rats (n = 8) were processed for either Nissl staining (Mitra et al., 2021) or CART immunofluorescence as described above. In addition, the PMv sections were incubated as described above in a mixture of either guinea pig polyclonal anti-vGluT2 (1:500, catalog #AB22511, EMD Millipore, RRID: AB_2251-1) and rabbit polyclonal anti-CART (1:5,000) antisera or mouse monoclonal anti-neuronal nitric oxide synthase (nNOS) antibodies (1:1,000, catalog #37-2800, Thermo Fisher Scientific, RRID: AB_2533308) and rabbit polyclonal anti-CART antiserum (1:5,000). After rinsing in PBS, the sections were incubated in a cocktail of secondary antibodies [Alexa Fluor 594-conjugated donkey anti-rabbit IgG and either Alexa Fluor 488-conjugated donkey anti-mouse IgG or DyLight488-conjugated anti-guinea pig IgG (1:1,000 each, Life Technologies)] for 4 h at room temperature.
Sections were rinsed in Tris buffer, mounted on the glass slides, and coverslipped using the mounting medium (Vector Laboratories). The nomenclature of PMv subdivisions in the rats was adapted as described earlier (Cavalcante et al., 2006), with minor modifications.
Retrograde neuronal tracing
A retrograde neuronal tracer, FluoroGold (FG; Fluorochrome) was iontophoresed into the lPAG. The method described earlier (Singh et al., 2016) was employed with minor modifications. Briefly, the VF (n = 3) were anesthetized, the head was positioned into the stereotaxic frame, the hairs on the head were removed, and the skull surface was cleaned with betadine solution. Previously described stereotaxic coordinates for the lPAG [anterior–posterior (AP), −7.4 mm; mediolateral (ML), −0.7 mm from the bregma; and dorsoventral (DV), −5.7 mm from the skull surface; Lonstein and Stern, 1998; Calizo and Flanagan-Cato, 2003] were used. Craniotomy was performed, a glass capillary with a tip diameter of 15 μm (P97, Sutter Instruments) loaded with 2% solution of FG was lowered in the brain, and FG was iontophoresed using 6 µA positive current (Midgard Precision Current Source, Stoelting) for 15 min. After 15 d, the rats were anesthetized and perfused transcardially, and brains were isolated. The midbrain and hypothalamic tissues were sectioned coronally (25 µm thickness) as described above. The sections containing the VMHvl were processed for immunofluorescence using rabbit polyclonal anti-CART antiserum, as described above.
qRT-PCR analysis for cart mRNA expression
The qRT-PCR method as previously described (Basu et al., 2022) was employed. The female rats were divided into four groups (n = 5/group), viz., VF + I, VF − I, Dm + I, and Dm − I. For the inter-male aggression, the males were divided into two groups (n = 5/group), viz., the resident male with (M + I) or without (M − I) an intruder. In addition, a group of dams exposed to the intruder was also included to compare the response of the cart mRNA. After the experiment, the rats were decapitated, and brains were isolated and snap-frozen. Brains were coronally sectioned until the bregma level of −3.6 (Paxinos and Watson, 2007), and a 200-µm-thick hypothalamic section was obtained. The region containing the PMv was isolated, and the total RNA was extracted and purified using Qiagen's minikit and quantified in the NanoDrop One spectrophotometer (Thermo Fisher Scientific). The cDNA was synthesized from the RNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific). qRT-PCR was performed in QuantStudio 5 RT-PCR system (Applied Biosystems) with the primers for the target [CART (forward) 5′-AAGAAGTACGGCCAAGTCCC-3′; (reverse) 5′-GAAGTGCTTGTGAAGGGGTG-3′; amplicon length, 139 bp] and house-keeping gene [β-actin (forward) 5′-TCTTCCAGCCTTCCTTCCTG-3′; (reverse) 5′-CACCATGTACCCAGGCATTG-3′; amplicon length, 147 bp]. The samples were analyzed in biological and technical replicates to obtain the ct value. The relative cart mRNA expression was determined to that of the β-actin using the 2−ΔΔCT method. The changes in cart mRNA levels in the PMv-containing tissues in the different groups of rats were compared. In all comparisons, p < 0.05 was considered statistically significant.
Intra-PMv cannulation and CART siRNA administration
The experimental plan is shown in Figure 9A. The adult female rats, on Day 15 of the pregnancy, were used (Storlien et al., 2016). The surgery and the postsurgical care conform to the standardized procedures in our laboratory (Awathale et al., 2021). Briefly, the anesthetized rats were placed in the stereotaxic frame (Stoelting), bilateral craniotomy was performed, and the stainless steel guide cannulae were implanted in PMv [Fig. 9B; coordinates: AP, −3.8 mm; ML, ±1.0 mm; DV, −9.4 mm from bregma (Paxinos and Watson, 2007)]. A stylet projecting 0.5 mm below the guide cannula was inserted after the surgery. The rats were housed individually for postsurgical recovery and habituation. Superior bedding was provided to the female rats for the birth of litters. Similarly, to test the significance of CART in intermale aggression, the cannulae were placed bilaterally in the PMv (Fig. 9B) of the resident male rats as described above.
The rat CART siRNA comprising a pool of three target-specific 19 nt siRNA (NM_017110.1, AGCUCAAGAGUAAACGCAU, AGUCCUGAAGAAGCUCAAG, ACGCAUUCCGAUCUAUGAG; catalog #SR-NP001-001) and the control (scrambled siRNA, antisense-dTdT 3′ overhang and sense-dTdT 3′ overhang; catalog #SR-CL000-005) procured from Eurogentec, Belgium, were used in the study. The control and CART siRNAs were resuspended in RNase-free water, diluted (1:1) with lipofectamine messenger max solution (Invitrogen), and stored at 2–8°C. At the end of Day 4 before the dark period, the control or CART siRNA (1 µl/hemisphere/rat; n = 5/group) was injected intra-PMv of the dams or males using an internal cannula attached to microsyringe drive (Bioanalytical Systems). After 24 h (on Day 5), an intruder male was placed into the cage of either the dam with pups or the resident male, and maternal aggression in dams and intermale aggression in the resident male were analyzed, as described above. After completion of the experiment, the rats were killed, and brains were isolated, fixed, sectioned, and stained with cresyl violet to verify the placement of cannulae.
Intra-MPN or intra-VMHvl cannulation and CART immunoneutralization
The schematic of the experimental design is shown in Figure 10P1. The rats (n = 6/group) were anesthetized on Day 15 of their pregnancy. As described above, stereotaxic surgery was performed. The stainless steel guide cannulae were implanted either in the MPN (Fig. 10P2; coordinates: AP, −0.24 mm; ML, ±0.5 mm; DV, −8.0 mm) or VMHvl [Fig. 10P2; coordinates: AP, −2.52 mm; ML, ±1.0 mm; DV, −9.2 mm (Paxinos and Watson, 2007)]. The rats were subjected to postsurgical care and recovery and kept in the home cage till the birth of the litters. On PP day 5, dams were administered either aCSF containing the nonimmune serum (NIS; 0.25 μl/hemisphere/rat) or anti-CART antiserum (1:500; 0.25 μl/hemisphere/rat) either intra-MPN or intra-VMHvl, 15 min prior to the behavioral assessment. An intruder was introduced into the cage (Fig. 10P3) and the maternal aggression was analyzed as described above.
Image and statistical analyses
The sections were observed under an Axio Imager M2 fluorescence microscope equipped with an Apotome and a high-resolution camera (Carl Zeiss). The Nissl-stained sections were observed under an AxioLab A1 microscope with a camera attached (Carl Zeiss). The photomicrographs were captured and adjusted uniformly for size, brightness, and contrast in the Adobe Photoshop CS6 software (Adobe). The figure panels were prepared and labeled in the same software.
To determine whether the interaction with an intruder activates the PMvCART neurons, the changes in the (1) number of c-Fos-expressing neurons, (2) percentage of CART neurons coexpressing c-Fos, (3) percent fluorescent area of CART-ir, (4) CART-ir/neuron, and (5) percentage of weak and intense CART-ir neurons were analyzed in each PMv subdivision. Five coronal sections encompassing the rostrocaudal extent of the hypothalamus containing the PMv from each animal in different groups were analyzed. To determine the relative levels of CART-ir in each PMv subdivision of the VF, the percent fluorescent area of CART-ir and the density of CART-ir neurons were analyzed. The image analysis system consisted of a fluorescence microscope with a camera attached (Carl Zeiss). The photomicrographs of the rostrocaudal extent of the sections containing the PMv were captured. The total number of c-Fos and CART neurons and the CART neurons coexpressing c-Fos in each PMv subdivision were counted on either side of the brain of each animal, and the mean ± SEM was determined. The percentages of CART neurons coexpressing c-Fos were calculated, the data from each rat in the group were pooled separately, and the mean ± SEM was calculated for each group. The percentages of CART + vGluT2 or CART + nNOS were similarly determined. In addition, the changes in the density of c-Fos-expressing neurons in each PMv subdivision of VF + I and Dm + I were determined, as previously described (Csikós et al., 2020). Three c-Fos-labeled coronal sections containing the PMv were used for each animal. The photomicrographs of either side of the brain containing the PMv were captured and analyzed in ImageJ (NIH). In a predetermined square of 100 µm2, the total number of c-Fos cells in each PMv subdivision on either side of the brain of each animal in the group was counted, the data from animals in each group were pooled, and mean ± SEM was calculated. The data were expressed as c-Fos cell number/mm2. Similarly, the density of CART neurons in each PMv subdivision of the virgin females was analyzed.
The percent fluorescent area of CART-ir was analyzed using the previously described method (Mitra et al., 2021; Basu et al., 2022; Singh et al., 2023). The photomicrographs of the six rostrocaudal hypothalamic sections containing the PMv on either side of the brain of each animal were captured by keeping the microscope settings, like the exposure time, intensity, objective, and adjustments in the image acquisition software unchanged. In ImageJ, the photomicrographs were converted to 8 bit and thresholded, the immunoreactivity was selected, background was subtracted, and the percent fluorescent area was determined. The percent fluorescent area of CART-ir in each PMv subdivision of the animals in each group was pooled, and the mean ± SEM was calculated for each group.
The integrated intensity of CART immunofluorescence in each neuron residing in all four PMv subdivisions of the VF and Dm with or without an intruder was analyzed as previously described (Zahola et al., 2019; Mitra et al., 2022). High-magnification fluorescence photomicrographs of the six coronal sections containing the PMv were captured, the boundaries of the neuronal cell body and nucleus were determined, and the integrated fluorescence intensity of CART-ir in the cytoplasm was obtained, as previously described (Bhardwaj et al., 2018; Raudenska et al., 2019; Mitra et al., 2022). The background intensity of the nonimmunoreactive area was subtracted, and the neurons with distinct nuclei were counted and scored to avoid the recounting error. The data from animals in each group were pooled separately, and mean ± SEM was calculated. Based upon the intensity values of CART-ir, the PMvCART neurons were categorized into two types, viz., weak (intensity of CART-ir up to 299 au) and intense (intensity of CART-ir between 300 and 650 au). The number of weak and intense neurons was counted in each PMv subdivision of VF and Dm with or without the intruder, and the relative percentages of CART-ir neurons in each PMv subdivision were determined. For the number or intensity analysis of each neuron, the cell numbers were corrected with Abercrombie's method (Abercrombie, 1946).
In six preoptic area (POA)/hypothalamic sections, the percent fluorescent area of the CART-ir in the VMHvl and MPN, as well as the CART-ir in the close vicinity of the c-Fos cells in these nuclei of Dm with intruder were analyzed. The percent fluorescent area was calculated as described above. To determine the CART fiber innervation of the c-Fos cells in the MPN and VMHvl, the cellular boundary around the c-Fos cells was demarcated, and the percent fluorescent area of CART-ir around the cell was analyzed. The data from each animal were pooled, and the mean ± SEM was calculated for each region.
CART axonal innervation of the c-Fos cells in the VMHvl was analyzed under the super-resolution microscope (Elyra 7, Carl Zeiss). Using the lasers for CART (Alexa Fluor 488) and c-Fos (Alexa Fluor 594), a series of optical slices through the VMHvl were captured with a 63× oil objective, and the stacks were processed for SIM2 analysis in the ZEN black software. The maximum intensity projection file was generated from SIM2, and the CART fibers around the periphery of c-Fos cells were represented.
Student's t test was performed to analyze the significance between two groups whereas the differences between more than two groups were tested with one-way analysis of variance (ANOVA) followed by post hoc analysis using Tukey's multiple-comparisons test. The statistical analyses of the number and density of CART and c-Fos cells, the percentage of CART/c-Fos double-labeled neurons, the intensity of CART-ir neurons, percent fluorescent area of CART-ir, cart mRNA expression, and behavioral data were performed in Prism (GraphPad Software). The data of the number and density of c-Fos-ir neurons, percentage of CART/c-Fos double-labeled neurons, percent fluorescent area of CART-ir, cart mRNA levels, and the integrated fluorescence intensity of CART-ir/neuron in PMv were analyzed using one-way ANOVA followed by Tukey's multiple-comparisons test. The behavioral data were analyzed using Student's two-tailed unpaired t test. For all the statistical analyses, p < 0.05 was considered significant.
Results
Maternal aggression behavior
Compared to the virgin females (VF), dams (Dm) spent less time sniffing the intruder (I; Fig. 1A,B; p < 0.0001). However, the Dm exposed to the intruder (Dm + I) displayed distinct maternal aggression (Fig. 1A) with a higher number (Fig. 1C; Dm) and duration (Fig. 1D) of attacks on the intruder. The aggression behavior was not apparent in the VF exposed to the intruder (VF + I; Fig. 1C,D).
Organization of CART-ir in the ventral premammillary nucleus (PMv) of rats
The neuroanatomical organization of PMv and its subdivisions in the rats are shown in Figure 2, A and B. Using Nissl staining, four PMv subdivisions, viz., dorsal (dPMv), ventral (vPMv), caudal (cPMv), and lateral PMv (lPMv), were identified. While the neurons in the vPMv and cPMv were aggregated, those in the dPMv and lPMv were scattered (Fig. 2A,B). The CART neurons and fibers were observed in all the PMv subdivisions (Fig. 2C–H). The CART neurons in the vPMv (Fig. 2C,F) and cPMv (Fig. 2D,H) were compact, but those in the dPMv (Fig. 2C,E) and lPMv (Fig. 2C,G) were sparsely distributed. Semiquantitative image analysis of the CART-ir in each PMv subdivision showed a significantly higher density of CART neurons (Fig. 2I; vPMv, 214 ± 15.36; dPMv, 48 ± 5.8; cPMv, 98.0 ± 16.85; lPMv, 28.0 ± 5.83; p < 0.0001 vs dPMv, cPMv, and lPMv) and percent fluorescent area of CART-ir (Fig. 2J; vPMv, 9.81 ± 1.37; dPMv, 2.11 ± 0.22; cPMv, 5.52 ± 0.8; lPMv, 2.11 ± 0.4; p < 0.0001 vs dPMv and lPMv; p < 0.05 vs cPMv) in the vPMv as compared with those in other PMv subdivisions. In addition to CART, vGluT2, and nNOS/NADPHd are also expressed in the PMv (Donato et al., 2010, 2011; Donato and Elias, 2011). Application of double immunofluorescence showed 91 ± 2.3% CART neurons coexpressed nNOS (Fig. 2M,N; Na–Nc) whereas 33 ± 1.2% CART neurons contained vGluT2 (Fig. 2K,L; La–Lc). The colocalization of CART/nNOS in PMv neurons is comparable to a previous study showing NADPHd activity in PMvCART neurons (Donato et al., 2010).
Photomicrographs of the Nissl-stained coronal sections through the mediobasal hypothalamus of the virgin female rats showing the organization of the ventral premammillary nucleus (PMv; A,B). The PMv consists of four subdivisions, viz., dorsal (dPMv), ventral (vPMv), lateral (lPMv), and caudal (cPMv). Low-magnification (C,D) and high-magnification (E–H) fluorescence photomicrographs showing the distribution of CART cells and fibers in each PMv subdivision (E, dPMv; F, vPMv; G, lPMv; H, cPMv). The locations of high-magnification photomicrographs (E–H) are shown in C and D with respective labels. Semiquantitative image analysis of the (I) density of CART cells/mm2 and (J) percent fluorescent area of CART immunoreactivity in each PMv subdivision. Double immunofluorescence photomicrographs of the PMv of rat showing the coexpression of either (K,L) vGluT2 or (M,N) nNOS (pseudocolored red) in CART neurons (pseudocolored green). Magnified view of PMvCART neuron coexpressing (La–Lc) vGluT2 or (Na–Nc) nNOS. The magnified views La–Lc are of neurons (arrowheads) in K and L, whereas the neuron in Na–Nc is from M and N (arrows). Values (n = 5 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (I, F(3,28) = 47.28; J, F(3,28) = 19.57). *p < 0.05 and ***p < 0.0001 versus vPMv. Bregma level as per Paxinos and Watson (2007): −3.7 mm for A and C and −3.96 mm for B and D. A, rostral level; B, caudal level; ARC, arcuate nucleus; f, fornix; III, third ventricle. Scale bars: A–D, 100 µm; E–H, K–N, 25 µm; La–Lc and Na–Nc, 10 µm.
Exposure to the intruder differentially activates neurons in the PMv of the virgin females and dams
In addition to aggression (Motta et al., 2013), the PMv neurons respond to opposite-sex odor (Donato et al., 2010) and play a role in the regulation of GnRH neurons, luteinizing hormone (LH) secretion, and reproductive behavior (Swanson, 2000; Donato et al., 2010; Mei et al., 2023). However, the parcellation of the PMv into the subgroups that process the reproduction or aggression-related information has not been undertaken. To that end, we studied the c-Fos activation in each PMv subdivision [vPMv (Fig. 3), dPMv (Fig. 4), cPMv (Fig. 5), and lPMv (Fig. 6)] of the VF and Dm with and without the intruder. Isolated c-Fos-containing neurons were seen in each PMv subdivision of the VF − I (Figs. 3–6A) or Dm − I (Figs. 3–6G). However, a significant increase (p < 0.0001) in the number of c-Fos-containing neurons was seen in the PMv subdivisions of the VF + I (Figs. 3–6D,M). The Dm + I showed a robust induction in c-Fos in the PMv (Fig. 3N). A significantly higher (p < 0.0001) number and density of c-Fos cells were observed in each PMv subdivision of the Dm + I (Figs. 3–6J,M, 3N) as compared with those in the Dm − I (Figs. 3–6G,M), VF + I (Fig. 3–6D,M, 3N), and VF − I (Figs. 3–6A,M). An approximately threefold increase in the number of c-Fos and the density of c-Fos cells was observed in the PMv of Dm + I than those in the VF + I (p < 0.001). A gradient in the density of c-Fos cells was observed within the PMv of the Dm + I with the highest density in the vPMv followed by the cPMV, dPMv, and lPMv (p < 0.0001 between vPMv and other subdivisions and p < 0.001 between cPMv, dPMv, and lPMv; Fig. 7).
Fluorescence photomicrographs showing c-Fos-ir (red, A,D,G,J) and CART-ir (green, B,E,H,K) in the neurons of the ventral part of the ventral premammillary nucleus (vPMv) of the virgin females without (VF − I, A–C) or with (VF + I, D–F) an intruder (I) and the dams without (Dm − I, G–I) or with (Dm + I, J–L) an intruder. CART neurons are shown with arrows and the c-Fos-expressing CART neurons with arrowheads. Graphical representation of the semiquantitative analysis of the number (M) and density (N) of c-Fos-containing neurons, the percentage of CART neurons coexpressing c-Fos (O), percent fluorescence area of CART-ir (P), integrated fluorescence intensity of CART-ir/neuron (Q), and the relative percentages of weak and intense CART-ir neurons (R) in the vPMv. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (M,O–Q) and Student's t test (N). M, F(3,28) = 39.15, p < 0.0001. O, F(3,28) = 299.6, p < 0.0001. P, F(3,28) = 12.57, p < 0.0001. Q, F(3,42) = 10.60, p < 0.0001. *p < 0.05, **p < 0.001; ***p < 0.0001 in M and O–Q and ***p = 0.0001 in N. All photomicrographs are of the same magnification. Scale bar = 25 µm in A also applies uniformly to all the photomicrographs.
Fluorescence photomicrographs showing c-Fos-ir (red, A,D,G,J) and CART-ir (green, B,E,H,K) in the neurons of the dorsal part of the ventral premammillary nucleus (dPMv) of virgin females without (VF − I, A–C) or with (VF + I, D–F) an intruder (I) and the dams without (Dm − I, G–I) or with (Dm + I, J–L) an intruder. CART neurons are shown with arrows and the c-Fos-expressing CART neurons with arrowheads. Graphical representation of the number of c-Fos-ir neurons (M), percentage of CART-ir neurons coexpressing c-Fos (N), percent fluorescence area of CART-ir (O), integrated fluorescence intensity of CART-ir/neuron (P), and the relative percentages of weak and intense CART-ir neurons (Q) in the dPMv of different experimental groups. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (M) and Student's t test (N). M, F(3,28) = 181. ns, nonsignificant; ***p < 0.0001. X, no labeled neurons found. All photomicrographs are of the same magnification. Scale bar = 25 µm in A also applies uniformly to all the photomicrographs.
Fluorescence photomicrographs showing c-Fos-ir (red, A,D,G,J) and CART-ir (green, B,E,H,K) in the neurons in the caudal part of the ventral premammillary nucleus (cPMv) of virgin females without (VF − I, A–C) or with (VF + I, D–F) an intruder (I) and the dams without (Dm − I, G–I) or with (Dm + I, J–L) an intruder. CART neurons are shown with arrows and the c-Fos-expressing CART neurons with arrowheads. Graphical representation of the number of c-Fos-ir neurons (M), percentage of CART-ir neurons coexpressing c-Fos (N), percent fluorescence area of CART-ir (O), integrated fluorescence intensity of CART-ir/neuron (P), and the relative percentages of weak and intense CART-ir neurons (Q) in the cPMv of different experimental groups. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (M,N). M, F(3,28) = 492.8, (N) F(3,28) = 328.3. ns, nonsignificant; ***p < 0.0001. All photomicrographs are of the same magnification. Scale bar = 25 µm in A also applies uniformly to all the photomicrographs.
Fluorescence photomicrographs showing c-Fos-ir (red, A,D,G,J) and CART-ir (green, B,E,H,K) in the neurons of the lateral part of the ventral premammillary nucleus (lPMv) of virgin females without (VF − I, A–C) or with (VF + I, D–F) an intruder (I) and the dams without (Dm − I, G–I) or with (Dm + I, J–L) an intruder. c-Fos expressing CART neurons are shown with arrows in L. Graphical representation of the number of c-Fos-ir neurons (M), percentage of CART-ir neurons coexpressing c-Fos (N), percent fluorescence area of CART-ir (O), integrated fluorescence intensity of CART-ir/neuron (P), and the relative percentages of weak and intense CART-ir neurons (Q) in the lPMv of different experimental groups. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (M) and Student's t test (N). M, F(3,28) = 160. ns, nonsignificant; X, no labeled neurons found; ***p < 0.0001. All photomicrographs are of the same magnification. Scale bar = 25 µm in A also applies uniformly to all the photomicrographs.
Density of c-Fos-ir neurons in the ventral premammillary nucleus (PMv) subdivisions of the virgin females without (VF − I) or with (VF + I) and the dams without (Dm − I) or with (Dm + I) an intruder (I). dPMv, dorsal PMv; cPMv, caudal PMv; lPMv, lateral PMv; vPMv, ventral PMv. Values (n = 8 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (F(3,28) = 56.73). **p < 0.001 and ***p < 0.0001.
Maternal aggression is associated with a profound activation of PMvCART neurons
Next, we probed the response of the PMvCART neurons in VF and Dm exposed to the intruder. Application of double immunofluorescence showed c-Fos in isolated CART neurons in the vPMv (Fig. 3A–C,G–I,O) and cPMv (Fig. 5A–C,G–I,N) of the VF − I (Figs. 3, 5A–C) as well as Dm − I (Figs. 3, 5G–I). The CART/c-Fos double-labeled neurons were not detected in the dPMv (Fig. 4A–C,G–I,N) and lPMv (Fig. 6A–C,G–I,N) of these rats. Compared to the VF − I, a significant increase (p < 0.0001) in the percentage of CART neurons coexpressing c-Fos was observed in the vPMv [VF − I, 2.01 ± 0.32; VF + I, 48.38 ± 3.0 (Fig. 3D–F,O)] and cPMv [VF − I, 1.36 ± 0.24; VF + I, 54.35 ± 3.93 (Fig. 5D–F,N)] of the VF + I. The vPMv (86.94 ± 3.59; Fig. 3J–L,O), dPMv (75.82 ± 3.58; Fig. 4J–L,N), cPMv (87.22 ± 2.46; Fig. 5J–L,N), and lPMv (41.77 ± 3.11; Fig. 6J–L,N) in the Dm + I showed a significantly higher (p < 0.0001) percentage of CART neurons coexpressing c-Fos as compared with those in the VF + I.
The presence of intruder activated only a subpopulation of PMvCART neurons in VF whereas the confrontation with intruder robustly activated these neurons in Dm. The results suggest that the reproductive state of the females may influence the scaling up of the PMvCART neuronal activation when exposed to intruder.
The PMvCART level is elevated during maternal aggression in dams
We determined whether the activation of PMvCART neurons in VF and Dm exposed to the intruder altered expression of CART in the PMv. Compared to the VF + I, VF − I, or Dm − I, a significant increase (p < 0.05 compared with Dm − I and p < 0.001 compared with VF − I and VF + I) in cart mRNA level was observed in the PMv-containing hypothalamic tissue of Dm + I (Fig. 8A,B).
A, The schematic of the coronal section of the hypothalamus of rats showing the location of the ventral premammillary nucleus (PMv). The tissue containing the PMv (asterisk) was isolated for cart mRNA analysis. B, Graphical representation of the relative cart mRNA levels in the PMv of the virgin females without (VF − I) or with (VF + I) and dam without (Dm − I) or with (Dm + I) an intruder (I). Values (n = 5 rats/group) are represented as means (±SEM). The data were analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons test (F(3,16) = 13.65, p = 0.0001). f, fornix; v, ventricle. *p < 0.05 and **p < 0.001.
A significant increase in the percent fluorescent area (p < 0.0001 compared with VF − I and p < 0.001 compared with Dm − I and VF + I; Fig. 3P) as well as the intensity of CART-ir/neuron (p < 0.05 compared with Dm − I, p < 0.0001 compared with VF + I, and p < 0.001 compared with VF − I; Fig. 3Q) was observed in the vPMv of Dm + I. The percent fluorescent area of CART-ir and the intensity of CART-ir/neuron were comparable across the vPMv of VF − I, VF + I, and Dm − I (p > 0.05; Fig. 3B,E,H,K,P,Q). However, no significant difference was observed in the percent fluorescent area of CART-ir and the intensity of CART-ir/neuron in the dPMv (Fig. 4B,E,H,K,O,P), cPMv (Fig. 5B,E,H,K,O,P), and lPMv (Fig. 6B,E,H,K,O,P) of the VF and Dm with or without the intruder.
Although intense and weak CART-ir neurons were interspersed in the PMv, their relative percentage varied across different experimental groups (Figs. 3–6B,E,H,K, 3R, 4–6Q). While the vPMv in the VF − I, VF + I, and Dm − I showed several weakly labeled and only a few intensely labeled (approximately 20%) CART neurons, that in the Dm + I was largely occupied with intensely labeled CART neurons (approximately 80%; Fig. 3B,E,H,K,R). The dPMv in the VF − I, VF + I, and Dm − I contained only weak CART neurons whereas approximately 40% of that in the Dm + I was occupied with intensely labeled CART neurons (Fig. 4B,E,H,K,Q). The percentage of weak and intensely labeled CART neurons in the cPMv of the VF and Dm was comparable and not subjected to the presence or absence of the intruder (Fig. 5B,E,H,K,Q). In all four groups of rats, the lPMv showed the presence of only the weak CART neurons (Fig. 6B,E,H,K,Q). The results demonstrate that the confrontation with intruder triggered the synthesis of CART in the PMv neurons in Dm.
Intra-PMv CART siRNA suppressed maternal aggression in dams
We examined whether the enhanced CART levels in the PMv of dams confronting the intruder contribute to maternal aggression. The intra-PMv CART siRNA-treated dams were screened for aggressive behavior against the intruder (Fig. 9A,B). Compared to the control siRNA-treated dams, a significant delay in the latency to the first attack on the intruder was noticed in dams treated with CART siRNA (p < 0.0001, Fig. 9C). The control siRNA-treated Dm were actively engaged in attacking the intruder with a higher number (9.6 ± 0.51; Fig. 9D) and duration of attacks (58.0 ± 5.12 s; Fig. 9E). As compared with controls, the intra-PMv CART siRNA-treated Dm showed reduced (p < 0.0001) number (1.6 ± 0.4; Fig. 9D) and duration (16.0 ± 2.91; Fig. 9E) of attacks on the intruder. The data suggest a correlation between PMvCART signaling and expression of maternal aggression.
Effect of intra-ventral premammillary nucleus (intra-PMv) CART siRNA administration on maternal aggression in dams. A, Experimental design and (B) schematic of the coronal section [adapted from Paxinos and Watson (2007); bregma, −3.8; A] of the rat brain showing the placement of cannulae in the PMv. The red arrow indicates the time point when stereotaxic surgery to implant the cannulae in the PMv was performed. The green arrow indicates the timing of administering the control or CART siRNA intra-PMv. The red block differentiates the prenatal and postnatal period. The effect of siRNA treatment on maternal aggression including the latency to first attack (C), number of attacks (D), and attack duration (E). Values (n = 5 rats/group) are represented as means (±SEM). The data were analyzed by Student's t test. ***p < 0.0001 versus control siRNA.
Role of CART signaling in the VMHvl in maternal aggression
Next, we try to find out the potential target of PMvCART neurons that may mediate maternal aggression. The VMHvl and MPN serve as the two crucial nodes of the aggression circuitry (Canteras et al., 1992; Lonstein and Gammie, 2002; Motta et al., 2013; Hashikawa et al., 2017). The PMv neurons project to the MPN and VMHvl (Canteras et al., 1992) and control neuronal activation in these nuclei during maternal aggression (Motta et al., 2013). Particularly, the activation of the PMv inputs to the VMHvl elicited attacks in mice (Stagkourakis et al., 2018). While the VMHvl, as well as MPN, are innervated by CART axons, those in the MPN originate from PMv (Rondini et al., 2004). This underscores the importance of CART as a signaling agent within the HAA that may mediate the attack behavior. As previously reported (Motta et al., 2013), c-Fos induction was observed in the MPN (Fig. 10A) and VMHvl (Fig. 10D) of Dm confronting the intruder. However, a sharp contrast in the pattern of CART fibers was seen in the MPN and VMHvl of the Dm. While scattered CART fibers were observed in the MPN (Fig. 10B), a dense network of CART fibers was seen in the VMHvl (Fig. 10C,E). Compared to the VMHvl, other VMH subdivisions contained only isolated fibers (Fig. 10C). Semiquantitative analysis of the percent fluorescent area showed a significantly higher (p < 0.0001) CART-ir in the VMHvl than the MPN (Fig. 10F). Using double immunolabeling, we determined whether the CART axons contact c-Fos cells in the MPN and VMHvl of Dm exposed to the intruder. Only a few CART fibers/terminals were seen around the c-Fos-containing cell bodies in the MPN (Fig. 10B,I). Compared to the MPN, a higher percent fluorescent area of CART-ir and the number of CART-ir axonal contacts were observed around the c-Fos cells in the VMHvl (p < 0.0001; Fig. 10E,G–I). The stronger CART innervation suggests VMHvl as a crucial target of CART signaling during maternal aggression.
A,B,D,E, Photomicrographs of the coronal sections of the hypothalamus of dams exposed to the intruder showing CART fibers (green) innervating the c-Fos cells (red) in the medial preoptic nucleus (MPN, A,B) and ventrolateral part of the ventromedial hypothalamus (VMHvl, D,E). Low-magnification photomicrograph of the hypothalamus showing a dense network of CART fibers in the VMHvl (C). Note isolated fibers in other parts of the VMH. The insets in B and E are magnified views of the cells shown with arrowheads in B and E, respectively. Semiquantitative analysis of the percent fluorescent area of the CART-ir in the MPN and VMHvl (F) and around the c-Fos cells in these nuclei (I). G,H, Super-resolution microscope photomicrographs showing the CART fibers contacting c-Fos-expressing neurons (arrows). J, Schematics of the coronal sections of the rat brain showing the connection between the VMHvl and lateral periaqueductal gray (lPAG). The neurons in the VMHvl (red) project to the lPAG. K, Schematic of the sagittal section of the rat brain showing the site of iontophoresis of FluoroGold (FG; pseudocolored red) in the lPAG and its retrograde accumulation in the cell bodies in VMHvl. Photomicrographs of the coronal sections of the hypothalamus of rats showing the FG-containing cells (L,N) and CART fibers (M,O) in the VMHvl. Arrows in O show the FG-labeled neurons densely innervated by CART axons. Schematic of the experimental design showing the timeline of stereotaxic surgery (P1), implantation of cannulae intra-MPN or intra-VMHvl in the pregnant rats for administration of nonimmune serum (NIS) or anti-CART antibodies in these nuclei of dams (P2), and behavioral assessment (P3). Effect of intra-MPN and intra-VMHvl administration of NIS or CART ab on the latency to first attack (Q) and the number (R) and duration (S) of attacks. Values (n = 8 rats/group in F and I and 6 rats/group in Q–S) are represented as means (±SEM). The data were analyzed by Student's t test (F, p < 0.0001; I, p < 0.0001; Q, p = 0.0078; R, p = 0.0028; S, p = 0.0009). **p = 0.0078, ***p < 0.0001, #p = 0.0028, $p = 0.0009. ns, non-significant. A,B, −0.24 mm and, C–E, L, M, −2.52 mm from the bregma (Paxinos and Watson, 2007). ARC, arcuate nucleus; VMHdm, dorsomedial part of the ventromedial hypothalamus; III, third ventricle. Scale bars: A–E, L, and M, 25 µm; N and O, 20 µm; G, H, and the insets in B and E, 10 µm.
CART-containing axons innervate the lateral periaqueductal gray (lPAG)-projecting neurons in the VMHvl
The VMHvl neurons projecting to lPAG (Shimogawa et al., 2015; Lo et al., 2019) facilitate the motor output of aggressive actions (Hashikawa et al., 2017; Falkner et al., 2020). We probed whether the VMHvl neurons projecting to the lPAG are innervated by CART axons. As described previously (Falkner et al., 2020; Ma et al., 2023), iontophoresis of FG in the PAG retrogradely accumulated the tracer in the cell bodies of the VMHvl (Fig. 10J,K,L,N). Application of the FG + CART double immunofluorescence showed CART axonal contacts on the FG-labeled neurons in the VMHvl (Fig. 10M,O). The data suggest that CART may regulate the VMHvl–lPAG pathway to control motor output during maternal aggression.
Effect of immunoneutralization of CART in the MPN or VMHvl on maternal aggression in dams
Administration of CART activates c-Fos in different brain regions (Vrang et al., 1999). In view of the CART fiber innervation of the c-Fos cells in the MPN and VMHvl of Dm confronting the intruder, we hypothesized that the release of the peptide in these regions triggers maternal aggressive behavior. Since CART receptors have not been identified and CART receptor antagonist is not available, we have used the CART immunoneutralization approach (Rale et al., 2017; Awathale et al., 2021) to test the role of CART in the MPN/VMHvl in maternal aggression. Compared to the control, intra-VMHvl (Fig. 10P2) administration of the anti-CART antiserum increased the latency of the first attack (p = 0.0078, Fig. 10Q) but reduced the number of attacks (p = 0.0028, Fig. 10R) and the attack duration (p = 0.0009, Fig. 10S). The maternal aggression response in the Dm treated with intra-MPN (Fig. 10P2) aCSF or anti-CART antiserum was comparable (latency of the first attack, p = 0.92; number of attacks, p = 0.24; duration of attack, p = 0.71; Fig. 10Q–S). The results suggest that the VMHvl may serve as a target of PMvCART signaling and the release of the peptide in the VMHvl during an encounter with the intruder may generate aggressive behavior in Dm.
Intermale versus maternal aggression
A schematic of the experimental design is shown in Figure 11, A and B. As reported earlier (Gammie et al., 2003), the introduction of a male intruder triggered aggression in the resident males and dams, but the degree of response varied. The latency to the first attack on the intruder was significantly less in Dm than in the males (p < 0.0001; Fig. 11C) whereas the number of attacks on the intruder (p = 0.0003; Fig. 11D) and the attack duration (p < 0.0001; Fig. 11E) were significantly more in Dm than in males.
A,B, Schematics of the experimental design. An intruder (I) was placed into the cage of a male rat (M) or postnatal day 5 dam (Dm). The maternal aggression method was employed as shown in Fig. 1A. The males and the postnatal day 5 Dm exposed to the intruders are designated as M + I and Dm + I, respectively. The group of male rats without an intruder is designated as M − I. The behaviors including latency to the first attack (C), number of attacks on an intruder (D), and attack duration (E) were recorded for 5 min for each rat. Fluorescence photomicrographs of the coronal section of the hypothalamus of M − I (F,J), M + I (G,K), Dm − I (H,L), and Dm + I (I,M) showing c-Fos (red, F–I) and c-Fos-coexpressing CART neurons (green; J–M) in the ventral premammillary nucleus (PMv). Graphical representation of the number of c-Fos neurons (N), percentage of CART neurons co-expressing c-Fos (O), and percent fluorescence area of CART-ir (P) in different PMv subdivisions. Q, qRT-PCR analysis of the cart mRNA levels in the hypothalamic tissues containing PMv of M − I, M + I, and Dm + I. Note a significant increase in cart mRNA expression in the PMv of Dm + I as compared with M + I. Effect of intra-PMv administration of control or CART siRNA on the number (R) and duration (S) of attacks. Values (n = 5 rats/group) are represented as means (±SEM). The data were analyzed by Student's t test (C, p < 0.0001; D, p = 0.0003; E, p < 0.0001) or one-way ANOVA followed by Tukey's multiple-comparisons test (N, vPMv F(3,16) = 97.09, p < 0.0001; dPMv F(3,16) = 80.63, p < 0.0001; cPMv F(3,16) = 314.7, p < 0.0001; lPMv F(3,16) = 390.4, p < 0.0001; O, vPMv F(3,16) = 247.8, p < 0.0001; cPMv F(3,16) = 381, p < 0.0001; P, F(3,16) = 9.817, p = 0.0007; Q, F(2,12) = 11.96, p = 0.0014). O, Student's t test was used to analyze the data of dPMv (p < 0.0001) and lPMv (p = 0.0028). ns, non-significant; *p = 0.0028 in O and p < 0.05 in Q; **p = 0.0003 in D; ***p < 0.0001 in C, E, N, and O; #p < 0.05 in P. All fluorescence photomicrographs are of the same magnification. f, fornix; cPMv, caudal PMv; dPMv, dorsal PMv; lPMv, lateral PMv; vPMv, ventral PMv. Scale bar = 50 µm in F also applies uniformly to all the photomicrographs.
Confrontation with the intruder differentially activates the PMv subdivisions in males and dams
Since maternal and intermale aggression employs similar neuronal pathways to trigger the behavioral output, we compared the response of the PMv in the males and dams displaying aggression. Isolated c-Fos cells were seen in all the PMv subdivisions in the M − I (Fig. 11F); however, a robust c-Fos activation was observed in the PMv of the M + I (Fig. 11G; p < 0.0001). In Dm, the confrontation with intruder activated c-Fos in all the PMv subdivisions with a robust activation in the vPMv (Fig. 11I). In males confronting the intruder, the c-Fos induction was localized in the lPMv (Fig. 11G). As compared with the M − I and Dm − I, moderate c-Fos activation was seen in the lPMv of Dm + I (Fig. 11I). However, the number of c-Fos cells in the lPMv of M + I was significantly more as compared with that in Dm + I (p < 0.0001; Fig. 11G,I,N). While isolated c-Fos cells were found in the vPMv of M + I, a significantly higher (p < 0.0001) number of c-Fos cells was observed in this subdivision in Dm + I (Fig. 11G,I,N). The number of c-Fos cells across the PMv subdivisions in M − I and Dm − I were in a similar range (p > 0.05, Fig. 11F,H,N). As described above, a vast majority of CART neurons in each PMv subdivision of dams confronting the intruder coexpressed c-Fos (Fig. 11M,O). However, in M + I, a higher percentage of CART neurons coexpressing c-Fos were seen in the lPMv whereas only a few CART neurons in other PMv subdivisions of these rats coexpressed c-Fos (Fig. 11K,O). Compared to Dm + I, the lPMv in M + I contained a significantly higher percentage of CART neurons coexpressing c-Fos (Fig. 11K,O).
Effect of intermale aggression on CART expression in the PMv
Confrontation with intruder dramatically upregulates CART expression in the PMv of dams. We determined whether intermale aggression also has a similar effect. Compared to M − I, M + I, or Dm − I, a higher percent fluorescent area of CART-ir was observed in the vPMv of Dm + I (p < 0.05; Fig. 11M,P). The percent fluorescent area of CART-ir in the dPMv, cPMv, and lPMv of males and dams with or without the intruder was comparable (p > 0.05; Fig. 11J–L,P). Exposure to the intruder does not seem to influence the cart mRNA expression in the PMv in males (p > 0.05; Fig. 11Q). Compared to the M − I and M + I, significantly higher (p < 0.05) levels of cart mRNA were observed in the PMv of Dm + I (Fig. 11Q).
Effect of intra-PMv CART siRNA on intermale aggression
To test whether PMvCART mediates inter-male aggression, the aggressive behavior of the intra-PMv CART siRNA-treated resident males was analyzed. The intra-PMv control siRNA-treated males actively attacked the intruder with a higher number (Fig. 11R) and duration (Fig. 11S) of attacks. The number and duration of attacks on the intruder following the intra-PMv CART siRNA treatment were comparable to that observed in the male rats treated with control siRNA (p > 0.05; Fig. 11R,S).
The data suggest that different PMv subdivisions may be involved in triggering maternal and inter-male aggression, and unlike maternal aggression response, inter-male aggression is not mediated by PMvCART.
Discussion
Unlike inter-male aggression, a transient switch-on of the aggression circuitry during pregnancy/lactation provides behavioral flexibility to females to display fierce aggression toward an intruder. Although the importance of PMv as a critical site triggering aggression in both males and dams is well established (Motta et al., 2013; Stagkourakis et al., 2018; Chen et al., 2020; Stagkourakis et al., 2024), how its activation precipitates aggression is not well understood. In this study, we show that the rat PMv is highly compartmentalized and that exposure to intruder activated different subsets of neurons in the PMv in virgin females, dams, and males. Only a subset of vPMv neurons was activated in the virgin females with intruder whereas those in dams were robustly activated while confronting the intruder. In contrast, confrontation with the intruder activated lPMv but not other PMv subdivisions in males. Only the vPMv neurons showed induction in CART-ir during maternal aggression. We identify CART as a key regulator employed by the PMv neurons to control maternal but not intermale aggression. We show that the CART signaling in VMHvl mediates maternal aggression and provides neuroanatomical evidence implicating CART in driving the VMHvl–PAG pathway. The study contributes to the growing literature that identifies PMv as a region of paramount importance in precipitating aggression and demonstrates a key role for the neuropeptide CART in the neurocircuitry of aggression.
In addition to three PMv subdivisions (Cavalcante et al., 2006), we have identified the lPMv as the fourth PMv subdivision in rats. Lateral PMv was identifiable based on Nissl staining, distribution of CART neurons, and the nature of c-Fos activation after exposure to the intruder. While confrontation with intruder robustly activated vPMv in dams, the response in these neurons was subdued in virgin females. Conversely, the lPMv was activated in males exposed to the intruder. The data suggest that factors like the sex and reproductive state of the animal may engage PMv subdivisions and the extent of its neuronal activation following exposure to an intruder.
Exposure to intruder evoked aggression and a profound activation of PMvCART neurons in dams but not in virgin females. Particularly, the CART neurons in the vPMv of dams confronting the intruder were robustly activated. In parallel with the activation of the PMvCART, there was an increase in CART expression in the PMv of dams, suggesting active biosynthesis of PMvCART during maternal aggression. Although the mechanism controlling the activation of PMvCART neurons during aggression is not known, the neuronal inputs, sensitivity of neurons to hormones/intruder cues, and membrane potential may determine the engagement of these neurons to trigger aggression in dams. In addition, odor cues from the opposite sex have also been shown to activate PMvCART neurons (Cavalcante et al., 2006). A recent study showed the importance of priming with prolactin and oxytocin in changing the dormant nature of the PMvDAT neurons in virgin females to a depolarization state in dams (Stagkourakis et al., 2024). The hormonal changes during pregnancy/lactation may alter the properties of the PMvCART neurons in dams from a hyperpolarized to a readily excitable state enabling them to promptly respond to the intruder. In addition to aggression, prolactin regulates reproduction. It suppresses the kisspeptin neuronal system and reproductive behavior during lactation (Auriemma et al., 2020; Hackwell et al., 2024). The VMHvl, as well as PMv, are known to contain prolactin-responsive neurons (Furigo et al., 2014; Georgescu et al., 2022), and prolactin acting through its receptors in the VMHvl regulates the intensity of aggression during lactation (Georgescu et al., 2022). We suggest that pregnancy/lactation may suppress reproductive behavior and sensitize the PMvCART neurons and their target to drive maternal aggression. In addition, the PMv receives inputs from the medial amygdala (Canteras et al., 1995), which seems crucial to the activation of PMv during maternal aggression (Abellán-Álvaro et al., 2022). We observed reduced maternal aggression in dams following intra-PMv CART siRNA treatment. These results are comparable to a previous study showing suppressed maternal aggression in dams with PMv lesions (Motta et al., 2013). In contrast to dams, PMvCART levels remained unaltered in males confronting the intruder, and the intra-PMv CART siRNA treatment had no effect on intermale aggression. We suggest that PMvCART mediates maternal but not intermale aggression, and the reproductive state of females may regulate scaling up the activation of PMvCART neurons in response to an intruder.
The VMHvl plays a pivotal role in aggression (Yang et al., 2013, 2017, 2023), and our data identify the VMHvl as a crucial target for CART to express maternal aggression. This is based on (1) the presence of a dense network of CART fibers in the VMHvl, (2) stronger CART axonal innervation of the activated neurons in VMHvl in dams exposed to intruder, and (3) suppression of maternal aggression following immunoneutralization of CART in the VMHvl but not MPN. The CART fibers in the MPN/VMHvl seem to originate from PMv since the application of retrograde neuronal tracer in MPN resulted in labeling of the PMvCART neurons (Rondini et al., 2004), whereas anterograde neuronal tracing showed projections of PMv neurons to the VMHvl (Canteras et al., 1992). In view of stronger CART fiber innervation of the VMHvl, and the role of PMv in the activation of VMHvl during maternal aggression (Motta et al., 2013), we suggest that the PMvCART may signal VMHvl to trigger maternal aggression. However, the role of CART inputs to VMHvl from neuronal groups other than the PMv cannot be ruled out. The lPAG-projecting neurons in the VMHvl were densely innervated by CART axons. The VMHvl neurons are known to project to lPAG, and this pathway plays an important role in aggression (Falkner et al., 2020; Ma et al., 2023). The stimulation of PAG triggered (Fernandez de Molina and Hunsperger, 1962; Mos et al., 1982; Gregg and Siegel, 2001), whereas its destruction reduced (Gregg and Siegel, 2003; Zalcman and Siegel, 2006) the aggressive actions. Since the glutamatergic VMHvl neurons strongly influence the neurons in PAG (Shimogawa et al., 2015; Falkner et al., 2020) and CART is known to modulate the glutamatergic pathways in the brain (Meng et al., 2018), the role of this peptide in glutamatergic neurotransmission in the VMHvl–PAG pathway mediating maternal aggression is suggested.
Only a subset of PMvCART neurons in virgin females were activated following exposure to the intruder. Besides aggression, the PMv also plays a role in reproductive regulation (Donato et al., 2010; Donato and Elias, 2011; Mei et al., 2023). It integrates the sensory and gonadal cues (Donato et al., 2011; Sáenz de Miera et al., 2024), responds to opposite-sex odor (Yokosuka et al., 1999; Cavalcante et al., 2006; Leshan et al., 2009; Donato et al., 2010), projects to the POA (Canteras et al., 1992), and controls the kisspeptin/GnRH neurons (Rondini et al., 2004; Donato et al., 2010; Donato et al., 2013). Furthermore, the PMv-lesioned females displayed blunted reproductive function (Donato et al., 2010, 2013). We speculate that the chemosensory inputs may activate a subset of POA-projecting PMvCART neurons in virgin females which may regulate reproduction.
This study identifies CART as a key messenger in aggression circuitry and a novel mediator of maternal aggression in rats. The vPMv and lPMv in dams and males, respectively are activated during the confrontation with the intruder. Activation of a subset of PMvCART neurons in virgin females interacting with intruder may have relevance to the regulation of reproduction. The hormones of pregnancy/lactation may prime the PMvCART system in dams to respond robustly to the intruder's cues. We propose that the vPMvCART neurons and the CART signaling modulating the VMHvl–PAG pathway may fuel the aggressive outburst allowing dams to turn violent toward the intruder. PMvCART does not seem to mediate aggression in male rats. Aggression circuitry and the role of PMvCART in maternal aggression are summarized in Figure 12.
Schematic of the neural circuitry of aggression and proposed role of CART in the regulation of aggressive behavior. A, The circuitry of aggression consists of the accessory olfactory system, bed nucleus of stria terminalis (BNST), medial amygdala (MeA), hypothalamus, and periaqueductal gray (PAG). Three hypothalamic nuclei, viz., the ventral premammillary nucleus (PMv), ventrolateral part of the ventromedial hypothalamus (VMHvl), and medial preoptic nucleus (MPN), are critical components of the hypothalamic attack area. The PMv is strongly connected with the VMHvl and MPN. The intruder's odor cue detected by the vomeronasal organ travels through the accessory olfactory bulb and is transmitted to the MeA and BNST. The MeA sends the signals to the VMHvl directly or indirectly via PMv. The PMv plays an important role in the sensory detection of the intruder and signals the network for triggering aggression. B, The PMv consists of four subdivisions, viz., ventral (vPMv), dorsal (dPMv), caudal (cPMv), and lateral (lPMv). All four PMv subdivisions are schematically shown in one plane for comparison and labeled in one of the schematics. A compartment-specific activation is seen in the PMv in virgin females, dams, and males exposed to intruder. A robust neuronal activation is seen in the PMv of males and dams whereas only moderate activation of neurons is observed in the PMv of virgin females with intruder. Note that the confrontation with intruder robustly activates CART neurons in the vPMv whereas in males the CART neurons in the lPMv show c-Fos activation. While the upregulation of CART in the PMv of dams may trigger maternal aggression, the peptide does not seem to play a role in inter-male aggression. The VMHvl seems more densely innervated by CART axons (green arrow) than MPN (dashed green arrow). PMvCART may activate the neurons in the VMHvl which then project to the PAG for execution of the motor component of the attack. The activation of CART neurons in the PMv of the virgin females when exposed to intruder may regulate reproduction. AOB, accessory olfactory bulb; MOB, medial olfactory bulb; VNO, vomeronasal organ. The schematic is created in CorelDRAW and BioRender software.
Footnotes
This work was supported by funding from the National Institute of Science Education and Research (NISER)-Bhubaneswar, the Department of Atomic Energy (Grant No. RIN4002 XIII Plan), and the Science and Engineering Research Board (SERB, Grant No. CRG/2021/007466 to P.S.S. and CRG/2020/004971 to D.M.K.), Government of India. The Axio Imager M2 fluorescence microscope used in this study was generously provided by the SERB through Grant No. SR/SO/AS-83/2010. We thank the National Center for Animal Research and Experimentation (NCARE) at NISER for helping in conducting experiments on animals.
The authors declare no competing financial interests.
- Correspondence should be addressed to Praful S. Singru at pssingru{at}niser.ac.in.
