Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of Escherichia coli leads to bacterial replication within liver abscesses, while other organs such as the spleen clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Abscess susceptibility is heightened in the C57BL lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as 1 d post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4 (Toll-like receptor 4), are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Congenital ablation of Tacr2 reveals overlapping and redundant roles of NK2R signaling in the control of reproductive axis.

Tachykinin (TAC) signaling is an important element in the central control of reproduction. TAC family is mainly composed of substance P (SP), neurokinin A (NKA), and NKB, which bind preferentially to NK1, NK2, and NK3 receptors, respectively. While most studies have focused on the reproductive functions of NKB/NK3R, and to a lesser extent SP/NK1R, the relevance of NK2R, encoded by Tacr2, remains poorly characterized. Here, we address the physiological roles of NK2R in regulating the reproductive axis by characterizing a novel mouse line with congenital ablation of Tacr2. Activation of NK2R evoked acute luteinizing hormone (LH) responses in control mice, similar to those of agonists of NK1R and NK3R. Despite the absence of NK2R, Tacr2-/- mice displayed only partially reduced LH responses to an NK2R agonist, which, nonetheless, were abrogated after blockade of NK3R in Tacr2-/- males. While Tacr2-/- mice displayed normal pubertal timing, LH pulsatility was partially altered in Tacr2-/- females in adulthood, with suppression of basal LH levels, but no changes in the number of LH pulses. In addition, trends for increase in breeding intervals were detected in Tacr2-/- mice. However, null animals of both sexes were fertile, with no changes in estrous cyclicity or sex preference in social behavioral tests. In conclusion, stimulation of NK2R elicited LH responses in mice, while congenital ablation of Tacr2 partially suppressed basal and stimulated LH secretion, with moderate reproductive impact. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.NEW & NOTEWORTHY We have explored here the impact of congenital ablation of the gene (Tacr2) encoding the tachykinin receptor, NK2R, in terms of neuroendocrine control of the reproductive axis, using a novel Tacr2 KO mouse line. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.

Tachykinin Signaling Is Required for Induction of the Preovulatory Luteinizing Hormone Surge and Normal Luteinizing Hormone Pulses.

Tachykinins (neurokinin A [NKA], neurokinin B [NKB], and substance P [SP]) are important components of the neuroendocrine control of reproduction by direct stimulation of Kiss1 neurons to control GnRH pulsatility, which is essential for reproduction. Despite this role of tachykinins in successful reproduction, knockout (KO) mice for Tac1 (NKA/SP) and Tac2 (NKB) genes are fertile, resembling the phenotype of human patients bearing NKB signaling mutations, who often reverse their hypogonadal phenotype. This suggests the existence of compensatory mechanisms among the different tachykinin ligand-receptor systems to maintain reproduction in the absence of one of them. In order to test this hypothesis, we generated complete tachykinin-deficient mice (Tac1/Tac2KO). Male mice displayed delayed puberty onset and decreased luteinizing hormone (LH) pulsatility (frequency and amplitude of LH pulses) but preserved fertility. However, females did not show signs of puberty onset (first estrus) within 45 days after vaginal opening, they displayed a low frequency (but normal amplitude) of LH pulses, and 80% of them remained infertile. Further evaluation identified a complete absence of the preovulatory LH surge in Tac1/Tac2KO females as well as in wild-type females treated with NKB or SP receptor antagonists. These data confirmed a fundamental role of tachykinins in the timing of puberty onset and LH pulsatility and uncovered a role of tachykinin signaling in facilitation of the preovulatory LH surge. Overall, these findings indicate that tachykinin signaling plays a dominant role in the control of ovulation, with potential implications as a pathogenic mechanism and a therapeutic target to improve reproductive outcomes in women with ovulation impairments.

Bidirectional conditioning: Revisiting Asratyan's 'alternating' training technique.

The study of bidirectional conditioning began more than a century ago, yet it has failed to take strong root in psychology and neuroscience. We revisit this topic by exploiting E. A. Asratyan's alternating procedure of stimulus presentation, in which both forward (e.g., A â†’ B) and backward (e.g., B â†’ A) training trials are concurrently given, in order to analyze their potential interaction. Specifically, using a two-alternative, forced-choice task, we trained humans and pigeons to learn associations between stimuli depending on whether they were presented as sample stimuli or choice stimuli. Trials were selected from an associative network in which forward and backward associations between sample and choice stimuli were synergistic (bidirectional network) or from an associative network in which these associations were not synergistic (unidirectional network). Humans were faster to learn associations from the bidirectional network than from the unidirectional network; additionally, they performed poorly on unidirectional trials that allowed for the expression of (incorrect) bidirectional associations. Unlike humans, pigeons showed no evidence of bidirectional associations. The reasons for this species difference as well as future directions for research deploying Asratyan's two-way training technique are discussed.

Taking pigeons to heart: Birds proficiently diagnose human cardiac disease.

In two experiments, we trained pigeons (Columba livia) to sort visual images (obtained by clinical myocardial perfusion imaging techniques) depicting different degrees of human cardiac disfunction (myocardial hypoperfusion of the left ventricle) into normal and abnormal categories by providing food reward only after correct choice responses. Pigeons proved to be highly proficient at categorizing pseudo-colorized images as well as highly sensitive to the degree of the perfusion deficit depicted in the abnormal images. In later testing, the pigeons completely transferred discriminative responding to novel stimuli, demonstrating that they had fully learned the normal and abnormal categories. Yet, these pigeons failed to transfer discriminative responding to grayscale images containing no color information. We therefore trained a second cohort of pigeons to categorize grayscale image sets from the outset. These birds required substantially more training to achieve similar levels of performance. Yet, they too completely transferred discriminative responding to novel stimuli by relying on both global and local disparities in brightness between the normal and abnormal images. These results confirm that pseudo-colorization can enhance pigeons' categorization of human cardiac images, a result also found with human observers. Overall, our findings further document the potential of the pigeon as a useful aide in studies of medical image perception.

Clinical presentations and outcomes of bile duct loss caused by drugs and herbal and dietary supplements.

Bile duct loss during the course of drug-induced liver injury is uncommon, but can be an indication of vanishing bile duct syndrome (VBDS). In this work, we assess the frequency, causes, clinical features, and outcomes of cases of drug-induced liver injury with histologically proven bile duct loss. All cases of drug-induced liver injury enrolled into a prospective database over a 10-year period that had undergone liver biopsies (n = 363) were scored for the presence of bile duct loss and assessed for clinical and laboratory features, causes, and outcomes. Twenty-six of the 363 patients (7%) with drug-, herbal-, or dietary-supplement-associated liver injury had bile duct loss on liver biopsy, which was moderate to severe (<50% of portal areas with bile ducts) in 14 and mild (50%-75%) in 12. The presenting clinical features of the 26 cases varied, but the most common clinical pattern was a severe cholestatic hepatitis. The implicated agents included amoxicillin/clavulanate (n = 3), temozolomide (n = 3), various herbal products (n = 3), azithromycin (n = 2), and 15 other medications or dietary supplements. Compared to those without, those with bile duct loss were more likely to develop chronic liver injury (94% vs. 47%), which was usually cholestatic and sometimes severe. Five patients died and 2 others underwent liver transplantation for progressive cholestasis despite treatment with corticosteroids and ursodiol. The most predictive factor of poor outcome was the degree of bile duct loss on liver biopsy. CONCLUSION: Bile duct loss during acute cholestatic hepatitis is an ominous early indicator of possible VBDS, for which at present there are no known means of prevention or therapy. (Hepatology 2017;65:1267-1277).

Nonhormonal Therapies for Menopausal Vasomotor Symptoms.

This JAMA Insights in the Women's Health series describes the use of fezolinetant and neurokinin B antagonists as nonhormonal therapies for menopausal vasomotor symptoms.

GnRH Neuron-Specific Ablation of Gαq/11 Results in Only Partial Inactivation of the Neuroendocrine-Reproductive Axis in Both Male and Female Mice: In Vivo Evidence for Kiss1r-Coupled Gαq/11-Independent GnRH Secretion.

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

Central precocious puberty caused by mutations in the imprinted gene MKRN3.

BACKGROUND: The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified. METHODS: We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages. RESULTS: We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty. CONCLUSIONS: Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Expanding the Role of Tachykinins in the Neuroendocrine Control of Reproduction.

Reproductive function is driven by the hormonal interplay between the gonads and brain-pituitary axis. Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner, which is critical for the attainment and maintenance of fertility, however, GnRH neurons lack the ability to directly respond to most regulatory factors, and a hierarchical upstream neuronal network governs its secretion. We and others proposed a model in which Kiss1 neurons in the arcuate nucleus (ARC), so called KNDy neurons, release kisspeptin (a potent GnRH secretagogue) in a pulsatile manner to drive GnRH pulses under the coordinated autosynaptic action of its cotransmitters, the tachykinin neurokinin B (NKB, stimulatory) and dynorphin (inhibitory). Numerous genetic and pharmacological studies support this model; however, additional regulatory mechanisms (upstream of KNDy neurons) and alternative pathways of GnRH secretion (kisspeptin-independent) exist, but remain ill defined. In this aspect, attention to other members of the tachykinin family, namely substance P (SP) and neurokinin A (NKA), has recently been rekindled. Even though there are still major gaps in our knowledge about the functional significance of these systems, substantial evidence, as discussed below, is placing tachykinin signaling as an important pathway for the awakening of the reproductive axis and the onset of puberty to physiological GnRH secretion and maintenance of fertility in adulthood.

Leptin-responsive GABAergic neurons regulate fertility through pathways that result in reduced kisspeptinergic tone.

The adipocyte-derived hormone leptin plays a critical role in the central transmission of energy balance to modulate reproductive function. However, the neurocircuitry underlying this interaction remains elusive, in part due to incomplete knowledge of first-order leptin-responsive neurons. To address this gap, we explored the contribution of predominantly inhibitory (GABAergic) neurons versus excitatory (glutamatergic) neurons in the female mouse by selective ablation of the leptin receptor in each neuronal population: Vgat-Cre;Lepr(lox/lox) and Vglut2-Cre;Lepr(lox/lox) mice, respectively. Female Vgat-Cre;Lepr(lox/lox) but not Vglut2-Cre;Lepr(lox/lox) mice were obese. Vgat-Cre;Lepr(lox/lox) mice had delayed or absent vaginal opening, persistent diestrus, and atrophic reproductive tracts with absent corpora lutea. In contrast, Vglut2-Cre;Lepr(lox/lox) females exhibited reproductive maturation and function comparable to Lepr(lox/lox) control mice. Intracerebroventricular administration of kisspeptin-10 to Vgat-Cre;Lepr(lox/lox) female mice elicited robust gonadotropin responses, suggesting normal gonadotropin-releasing hormone neuronal and gonadotrope function. However, adult ovariectomized Vgat-Cre;Lepr(lox/lox) mice displayed significantly reduced levels of Kiss1 (but not Tac2) mRNA in the arcuate nucleus, and a reduced compensatory luteinizing hormone increase compared with control animals. Estradiol replacement after ovariectomy inhibited gonadotropin release to a similar extent in both groups. These animals also exhibited a compromised positive feedback response to sex steroids, as shown by significantly lower Kiss1 mRNA levels in the AVPV, compared with Lepr(lox/lox) mice. We conclude that leptin-responsive GABAergic neurons, but not glutamatergic neurons, act as metabolic sensors to regulate fertility, at least in part through modulatory effects on kisspeptin neurons.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

Enhanced attention in rats following blast-induced traumatic brain injury.

Purpose: To evaluate visuo-cognitive sequelae following blast-induced traumatic brain injury in a rat model. Methods: Rats were randomly assigned to one of four groups depending on the intensity/quantity of a blast received in a blast chamber: sham (no blast), low intensity (22 psi), medium intensity (26 psi), or three medium intensity blasts (26 psi × 3). After recovery, all subjects were given visual discrimination tasks of increasing complexity, until mastery. After behavioral training, visual function was assessed via spectral-domain optical coherence tomography and pattern electroretinogram, and the extent of retinal damage was quantified via immunohistochemistry of retinal ganglion cells. Results: None of the measures assessing visual function revealed significant differences as a function of blast intensity/quantity. Behavioral training did not disclose short-term effects of blast in general motivation or the development of anticipatory responding. No differences in general learning ability and the number of perseverative errors were observed. However, behavioral training found effects of blast in attentional function; relative to controls, subjects that received blasts were faster in learning to attend to informative (over non-informative) cues in the most difficult visual discrimination task. Conclusion: Blast exposure in rats resulted in increased attention following blast, with no appreciable deficits in visual function. These results are contrary to what is often reported for human clinical populations; as such, more research bridging methodological differences is necessary.

Metabolic control of puberty: 60 years in the footsteps of Kennedy and Mitra's seminal work.

An individual's nutritional status has a powerful effect on sexual maturation. Puberty onset is delayed in response to chronic energy insufficiency and is advanced under energy abundance. The consequences of altered pubertal timing for human health are profound. Late puberty increases the chances of cardiometabolic, musculoskeletal and neurocognitive disorders, whereas early puberty is associated with increased risks of adult obesity, type 2 diabetes mellitus, cardiovascular diseases and various cancers, such as breast, endometrial and prostate cancer. Kennedy and Mitra's trailblazing studies, published in 1963 and using experimental models, were the first to demonstrate that nutrition is a key factor in puberty onset. Building on this work, the field has advanced substantially in the past decade, which is largely due to the impressive development of molecular tools for experimentation and population genetics. In this Review, we discuss the latest advances in basic and translational sciences underlying the nutritional and metabolic control of pubertal development, with a focus on perspectives and future directions.

POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons.

Inactivating mutations in the melanocortin 4 receptor (MC4R) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females. In vivo, deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro, we dissect the specific action of MC4R on Kiss1ARH vs Kiss1AVPV/PeN neurons and show that MC4R activation excites Kiss1ARH neurons through direct synaptic actions. In contrast, Kiss1AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMCARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1ARH neurons and restricting the activation of Kiss1AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

Kisspeptin signaling in astrocytes modulates the reproductive axis.

Reproduction is safeguarded by multiple, often cooperative regulatory networks. Kisspeptin signaling, via KISS1R, plays a fundamental role in reproductive control, primarily by regulation of hypothalamic GnRH neurons. We disclose herein a pathway for direct kisspeptin actions in astrocytes that contributes to central reproductive modulation. Protein-protein-interaction and ontology analyses of hypothalamic proteomic profiles after kisspeptin stimulation revealed that glial/astrocyte markers are regulated by kisspeptin in mice. This glial-kisspeptin pathway was validated by the demonstrated expression of Kiss1r in mouse astrocytes in vivo and astrocyte cultures from humans, rats and mice, where kisspeptin activated canonical intracellular signaling-pathways. Cellular co-expression of Kiss1r with the astrocyte markers, GFAP and S100-ß, occurred in different brain regions, with higher percentage in Kiss1- and GnRH-enriched areas. Conditional ablation of Kiss1r in GFAP-positive cells, in the G-KiRKO mouse, altered gene expression of key factors in PGE2 synthesis in astrocytes, and perturbed astrocyte-GnRH neuronal appositions, as well as LH responses to kisspeptin and LH pulsatility, as surrogate marker of GnRH secretion. G-KiRKO mice also displayed changes in reproductive responses to metabolic stress induced by high-fat diet, affecting female pubertal onset, estrous cyclicity and LH-secretory profiles. Our data unveil a non-neuronal pathway for kisspeptin actions in astrocytes, which cooperates in fine-tuning the reproductive axis and its responses to metabolic stress.

Role of neurokinin B in the control of female puberty and its modulation by metabolic status.

Human genetic studies have revealed that neurokinin B (NKB) and its receptor, neurokinin-3 receptor (NK3R), are essential elements for normal reproduction; however, the precise role of NKB-NK3R signaling in the initiation of puberty remains unknown. We investigated here the regulation of Tac2 and Tacr3 mRNAs (encoding NKB and NK3R, respectively) in female rats and demonstrated that their hypothalamic expression is increased along postnatal maturation. At puberty, both genes were widely expressed throughout the brain, including the lateral hypothalamic area and the arcuate nucleus (ARC)/medial basal hypothalamus, where the expression of Tacr3 increased across pubertal transition. We showed that central administration of senktide (NK3R agonist) induced luteinizing hormone (LH) secretion in prepubertal and peripubertal females. Conversely, chronic infusion of an NK3R antagonist during puberty moderately delayed the timing of vaginal opening (VO) and tended to decrease LH levels. The expression of NKB and its receptor was sensitive to changes in metabolic status during puberty, as reflected by a reduction in Tacr3 (and, to a lesser extent, Tac2) expression in the ARC after a 48 h fast. Yet, acute LH responses to senktide in pubertal females were preserved, if not augmented, under fasting conditions, suggesting sensitization of the NKB-NK3R-gonadotropin-releasing hormone signaling pathway under metabolic distress. Moreover, repeated administration of senktide to female rats with pubertal arrest due to chronic undernutrition rescued VO (in ∼50% of animals) and potently elicited LH release. Altogether, our observations suggest that NKB-NK3R signaling plays a role in pubertal maturation and that its alterations may contribute to pubertal disorders linked to metabolic stress and negative energy balance.

Interactions between kisspeptins and neurokinin B.

Reproductive function is tightly regulated by an intricate network of central and peripheral factors; however, the precise mechanism triggering critical reproductive events, such as puberty onset, remains largely unknown. Recently, the neuropeptides kisspeptin (encoded by Kiss1) and neurokinin B (NKB, encoded by TAC3 in humans and Tac2 in rodents) have been placed as essential gatekeepers of puberty. Studies in humans and rodents have revealed that loss-of-function mutations in the genes encoding either kisspeptin and NKB or their receptors, Kiss1r and neurokinin 3 receptor (NK3R), lead to impaired sexual maturation and infertility. Kisspeptin, NKB, and dynorphin A are co-expressed in neurons of the arcuate nucleus (ARC), so-called Kisspeptin/NKB/Dyn (KNDy) neurons. Importantly, these neurons also co-express NK3R. Compelling evidence suggests a stimulatory role of NKB (or the NK3R agonist, senktide) on LH release in a number of species. This effect is likely mediated by autosynaptic inputs of NKB on KNDy neurons to induce the secretion of gonadotropin-releasing hormone (GnRH) in a kisspeptin--dependent manner, with the coordinated actions of other neuroendocrine factors, such as dynorphin, glutamate, or GABA. Thus, we have proposed a model in which NKB feeds back to the KNDy neuron to shape the pulsatile release of kisspeptin, and hence GnRH, in a mechanism also dependent on the sex steroid level. Additionally, NKB may contribute to the regulation of the reproductive function by metabolic cues. Investigating how NKB and kisspeptin interact to regulate the gonadotropic axis will offer new insights into the control of GnRH release during puberty onset and the maintenance of the reproductive function in adulthood, offering a platform for the understanding and treatment of a number of reproductive disorders.

Prediction error in models of adaptive behavior.

Pavlovian conditioning is evident in every species in which it has been assessed, and there is a consensus about its interpretation across behavioral,1,2 brain,3,4,5,6 and computational analyses7,8,9,10,11: conditioned behavior reflects the formation of a directional associative link from the memory of one stimulus (e.g., a visual stimulus) to another (e.g., food), with learning stopping when there is no error between the prediction generated by the visual stimulus and what happens next (e.g., food). This consensus fails to anticipate the results that we report here. In our experiments with rats, we find that arranging predictive (visual stimulus→food) and nonpredictive (food→visual stimulus) relationships produces marked and sustained changes in conditioned behaviors when the visual stimulus is presented alone. Moreover, the type of relationship affects (1) the distribution of conditioned behaviors related to the properties of both food (called goal-tracking) and the visual stimulus (called sign-tracking) and (2) when in the visual stimulus, these two behaviors are evident. These results represent an impetus for a fundamental shift in how Pavlovian conditioning is interpreted: animals learn about the relationship between two stimuli irrespective of the order in which they are presented, but they exhibit this knowledge in different ways. This interpretation and our new results are captured by a recent model of Pavlovian conditioning,12,13 HeiDI, and both are consistent with the need for animals to represent the fact that the impact of a cause (e.g., the ingestion of nutrients or the bite of a predator) can be felt before or after the cause has been perceived.

Differential modulation of gonadotropin responses to kisspeptin by aminoacidergic, peptidergic, and nitric oxide neurotransmission.

Kisspeptins (Kp), products of the Kiss1 gene, have emerged as essential elements in the control of GnRH neurons and gonadotropic secretion. However, despite considerable progress in the field, to date limited attention has been paid to elucidate the potential interactions of Kp with other neurotransmitters known to centrally regulate the gonadotropic axis. We characterize herein the impact of manipulations of key aminoacidergic (glutamate and GABA), peptidergic (NKB, Dyn, and MCH), and gaseous [nitric oxide (NO)] neurotransmission on gonadotropin responses to Kp-10 in male rats. Blockade of ionotropic glutamate receptors (of the NMDA and non-NMDA type) variably decreased LH responses to Kp-10, whereas activation of both ionotropic and metabotropic receptors, which enhanced LH and FSH release per se, failed to further increase gonadotropin responses to Kp-10. In fact, coactivation of metabotropic receptors attenuated LH and FSH responses to Kp-10. Selective activation of GABA(A) receptors decreased Kp-induced gonadotropin secretion, whereas their blockade elicited robust LH and FSH bursts and protracted responses to Kp-10 when combined with GABA(B) receptor inhibition. Blockade of Dyn signaling (at κ-opioid receptors) enhanced LH responses to Kp-10, whereas activation of Dyn and NKB signaling modestly reduced Kp-induced LH and FSH release. Finally, MCH decreased basal LH secretion and modestly reduced FSH responses to Kp-10, whereas LH responses to Kp-10 were protracted after inhibition of NO synthesis. In summary, we present herein evidence for the putative roles of glutamate, GABA, Dyn, NKB, MCH, and NO in modulating gonadotropic responses to Kp in male rats. Our pharmacological data will help to characterize the central interactions and putative hierarchy of key neuroendocrine pathways involved in the control of the gonadotropic axis.