NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

Mapping of c-Fos expression in the medial amygdala following social buffering in male rats.

Social buffering is the phenomenon in which an affiliative conspecific (associate) ameliorates stress responses of a subject. We previously found that social buffering in Wistar subject rats is induced if the strain of the associate is Wistar or a strain derived from Wistar rats. In the present study, we assessed the possible role of medial amygdala (Me) in this strain-dependent induction of social buffering. The subjects were exposed to the conditioned stimulus (CS) that had been paired or unpaired with a foot shock either alone, with an unfamiliar Wistar associate, or with an unfamiliar Fischer 344 (F344) associate. We found that the Wistar associates, but not F344 associates, ameliorated increased freezing and Fos expression in the paraventricular nucleus of the hypothalamus and lateral amygdala caused by the CS. In addition, Fos expression in the posterior complex of the anterior olfactory nucleus and lateral intercalated cell mass of the amygdala was increased simultaneously. These results suggest that Wistar associates, but not F344 associates, induced social buffering. In the Me, we did not find any differences associated with stress responses or amelioration of stress responses. In contrast, a comparison among the unpaired subjects found that the Wistar associates, but not F344 associates, increased exploratory behavior and Fos expression in the posteroventral subdivision of the Me (MePV). Based on these results, we propose that the MePV is involved in the recognition of social similarity with the associates. Taken together, the present study provides information about the possible role of Me in social buffering.

Arginine vasopressin in the medial amygdala causes greater post-stress recruitment of hypothalamic vasopressin neurons.

Arginine vasopressin (AVP) is expressed in both hypothalamic and extra-hypothalamic neurons. The expression and role of AVP exhibit remarkable divergence between these two neuronal populations. Polysynaptic pathways enable these neuronal groups to regulate each other. AVP neurons in the paraventricular nucleus of the hypothalamus increase the production of adrenal stress hormones by stimulating the hypothalamic-pituitary-adrenal axis. Outside the hypothalamus, the medial amygdala also contains robust amounts of AVP. Contrary to the hypothalamic counterpart, the expression of extra-hypothalamic medial amygdala AVP is sexually dimorphic, in that it is preferentially transcribed in males in response to the continual presence of testosterone. Male gonadal hormones typically generate a negative feedback on the neuroendocrine stress axis. Here, we investigated whether testosterone-responsive medial amygdala AVP neurons provide negative feedback to hypothalamic AVP, thereby providing a feedback loop to suppress stress endocrine response during periods of high testosterone secretion. Contrary to our expectation, we found that AVP overexpression within the posterodorsal medial amygdala increased the recruitment of hypothalamic AVP neurons during stress, without affecting the total number of AVP neurons or the number of recently activated neurons following stress. These observations suggest that the effects of testosterone on extra-hypothalamic AVP facilitate stress responsiveness through permissive influence on the recruitment of hypothalamic AVP neurons.

Neurotrophic factors in the posterodorsal medial amygdala of male and cycling female rats.

The posterodorsal medial amygdala (MePD) has a high concentration of receptors for gonadal hormones, is a sexually dimorphic region and dynamically controls the reproductive behavior of both males and females. Neurotrophic factors can promote dendritic spine remodeling and change synaptic input strength in a region-specific manner. Here, we analyzed the gene and protein expression of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-1), polysialylated neural cell adhesion molecule (PSA-NCAM) and Ephrin-A4 in the MePD of adult males and females in diestrus, proestrus and estrus using real-time qPCR and fluorescent immunohistochemistry. The first approach showed their amplification except for Igf1 and the latter revealed that BDNF, IGF-1, PSA-NCAM and Ephrin-A4 are expressed in the MePD of the adult rats. Protein expression of these neurotrophic factors showed no differences between groups. However, proestrus females displayed a higher number of labelled puncta than males for BDNF expression and diestrus females for IGF-1 expression. In conjunction, results indicate that IGF-1 might be released rather than synthetized in the MePD, and the expression of specific neurotrophic factors varies specifically during proestrus. The dynamic modulation of BDNF and IGF-1 during this cyclic phase is coincident with synaptic changes and spine density remodeling in the MePD, the disinhibition of gonadotrophin secretion for ovulation and the display of sexual behavior.

Age-specific and context-specific responses of the medial extended amygdala in the developing prairie vole.

The social needs of organisms change as they mature. Yet, little is known about the mechanisms that subserve processing social interactions or how these systems develop. The medial extended amygdala (meEA) is comprised of the medial bed nucleus of the stria terminalis (BSTm) and the medial amygdala (MeA). This neural complex holds great promise for understanding how the social brain processes information. We assessed expression of the immediate early gene cFos and the enzyme tyrosine hydroxylase (TH) at three developmental time-points (postnatal day [PND] 2, 9, and 21) to determine how developing prairie voles process familial social contact, separation, and reunion. We demonstrate that (1) BSTm cFos responses were sensitive to separation from family units at PND 9 and PND 21, but not at PND 2; (2) MeA cFos responses were sensitive to reunion with the family, but only in PND 21 pups; (3) BSTm TH neurons did not exhibit differential responses to social condition at any age; and (4) MeA TH neurons responded strongly to social contact (remaining with family or following reunion), but only at PND 21. Our results suggest that the sub-units of the meEA become functionally responsive at different developmental time points, and are differentially activated in response to distinct social contexts. Overall, our results support the notion that interconnected regions of the meEA follow divergent developmental timelines and are sensitive to distinct properties of social contexts.

A combinatorial modulation of synaptic plasticity in the rat medial amygdala by oxytocin, urocortin3 and estrogen.

The medial nucleus of the amygdala (MeA) plays a pivotal role in a variety of mammalian social behaviors. Specifically, activity of the hypothalamic pro-social neuropeptide oxytocin in the MeA was shown to be crucial for social recognition memory. The MeA is also a hub of neuroendocrine activity and expresses a large number of receptors of neuropeptides and hormones. These include oxytocin receptor, estrogen receptor alpha and corticotropin-releasing factor (CRF) receptor type 2 (CRFR2). In a previous study we found that intracerebroventricular (ICV) oxytocin application to anesthetized rats promotes long-term depression (LTD) of the MeA response to electrical stimulation of its main sensory input, the accessory olfactory bulb (AOB). We also reported that this type of synaptic plasticity contributes to long-term social recognition memory. Here we used similar methodology to examine the possibility that various neuromodulators pose a combinatorial effect on synaptic plasticity in the MeA. We found that ICV administration of the CRF-related peptide urocortin3 fifteen minutes before oxytocin, caused long-term potentiation (LTP), via CRFR2 activation. Similarly, ICV administration of 17ß-estradiol forty-five minutes before oxytocin induced LTP, which was blocked by an antagonist of the estrogen receptors alpha and beta. Notably, none of these two neuromodulators had any effect on its own, suggesting that they both turn the oxytocin-mediated synaptic plasticity from LTD to LTP. Finally, we found that application of 17ß-estradiol, forty-five minutes before urocortin3 also caused LTP in the MeA response to AOB stimulation, even without oxytocin application. We suggest that the combinatorial modulation of the bidirectional synaptic plasticity in the AOB-MeA pathway by oxytocin, 17ß-estradiol and urocotin-3 serves to modify social information processing according to the animal's internal state.

Estrogens and their receptors in the medial amygdala rapidly facilitate social recognition in female mice.

Estrogens have been shown to rapidly (within 1 h) affect learning and memory processes, including social recognition. Both systemic and hippocampal administration of 17ß-estradiol facilitate social recognition in female mice within 40 min of administration. These effects were likely mediated by estrogen receptor (ER) α and the G-protein coupled estrogen receptor (GPER), as administration of the respective receptor agonists (PPT and G-1) also facilitated social recognition on a rapid time scale. The medial amygdala has been shown to be necessary for social recognition and long-term manipulations in rats have implicated medial amygdalar ERα. As such, our objective was to investigate whether estrogens and different ERs within the medial amygdala play a role in the rapid facilitation of social recognition in female mice. 17ß-estradiol, G-1, PPT, or ERß agonist DPN was infused directly into the medial amygdala of ovariectomized female mice. Mice were then tested in a social recognition paradigm, which was completed within 40 min, thus allowing the assessment of rapid effects of treatments. 17ß-estradiol (10, 25, 50, 100 nM), PPT (300 nM), DPN (150 nM), and G-1 (50 nM) each rapidly facilitated social recognition. Therefore, estrogens in the medial amygdala rapidly facilitate social recognition in female mice, and the three main estrogen receptors: ERα, ERß, and the GPER all are involved in these effects. This research adds to a network of brain regions, including the medial amygdala and the dorsal hippocampus, that are involved in mediating the rapid estrogenic facilitation of social recognition in female mice.

Place preferences associated with pups or cocaine change the expression of D2R, V1aR and OTR in the NAcc and MeA and the levels of plasma AVP, OT, T and E2 in mandarin vole fathers.

Drug abuse often has negative impacts on parenting behavior. The dopamine (DA), arginine vasopressin (AVP) and oxytocin (OT) systems are involved in paternal behavior and drug-induced behaviors. Mandarin voles (Microtus mandarinus) are socially monogamous rodents with high levels of paternal behavior. The aims of this study were to examine the protein expression levels of the DA 2-type receptor (D2R), AVP receptor 1A(V1aR) and OT receptor (OTR) in the nucleus accumbens (NAcc) and medial amygdala (MeA) as well as the plasma hormone responses after mandarin vole fathers were conditioned with their pups or cocaine. Our experimental models are based on the conditioned place preference (CPP) paradigm. We observed CPP in response to either pup- or cocaine-associated cues in the mandarin vole fathers. Fathers that were conditioned to either pups or cocaine had a lower expression of D2R and V1aR in the NAcc than did controls. Fathers that were conditioned to pups had higher levels of OTR expression in the MeA and higher plasma levels of AVP, OT, estradiol (E2), and lower plasma levels of testosterone (T) than did controls. Fathers that were conditioned to cocaine exhibited lower levels of plasma AVP and T. These results indicate that the reward effects of pup and cocaine are both mediated by D2R, V1aR and OTR in the NAcc and MeA and that there are subtle differences between the pup and cocaine reward mechanisms that are associated with altered plasma AVP, OT, T and E2.

Kisspeptin in the medial amygdala and sexual behavior in male rats.

The medial amygdala (MeA) is crucial for sexual behavior; kisspeptin (Kiss1) also plays a role in sexual function. Kisspeptin receptor (Kiss1r) knockout mice display no sexual behavior. Recently Kiss1 and Kiss1r have been discovered in the posterodorsal subnucleus of the medial amygdala (MePD). We hypothesised that Kiss1 in the MePD may have an influence on male sexual behavior. To test this we bilaterally cannulated the MePD and infused kisspeptin-10 in male rats. This caused the rats to have multiple erections, an effect specific to Kiss1 receptor activation, because Kiss1r antagonism blocked the erectile response. When Kiss1 was infused into the lateral cerebroventricle, there were no observed erections. We also measured the plasma levels of LH when Kiss1 is infused into the MePD or lateral cerebroventricle; Kiss1 increased plasma LH to comparable levels when infused into both sites. We conclude that Kiss1 has a role in male sexual behavior, which is specific to the MePD.