Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell–cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release.

Tuberoinfundibular dopamine (TIDA) neurons are the central regulators of prolactin (PRL) secretion. Their extensive functional plasticity allows a change from low PRL secretion in the non-pregnant state to the condition of hyperprolactinemia that characterizes lactation. To allow this rise in PRL, TIDA neurons are thought to become unresponsive to PRL at lactation and functionally silenced. Here we show that, contrary to expectations, the electrical properties of the system were not modified during lactation and that the neurons remained electrically responsive to a PRL stimulus, with PRL inducing an acute increase in their firing rate during lactation that was identical to that seen in non-pregnant mice. Furthermore, we show a long-term organization of TIDA neuron electrical activity with an harmonization of their firing rates, which remains intact during lactation. However, PRL-induced secretion of dopamine (DA) at the median eminence was strongly blunted during lactation, at least in part attributable to lack of phosphorylation of tyrosine hydroxylase, the key enzyme involved in DA synthesis. We therefore conclude that lactation, rather than involving electrical silencing of TIDA neurons, represents a condition of decoupling between electrical activity at the cell body and DA secretion at the median eminence.

The discoveries of novel functional adaptations of the hypothalamus and anterior pituitary gland for physiological regulation have transformed our understanding of their interaction. The activity of a small proportion of hypothalamic neurons can

The MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinase) pathway is a cardinal regulator of synaptic plasticity, learning, and memory in the hippocampus. One of major endpoints of this signaling cascade is the 5′ mRNA cap binding protein eIF4E (eukaryotic Initiation Factor 4E), which is phosphorylated on Ser 209 by MNK (MAPK-interacting protein kinases) and controls mRNA translation. The precise role of phospho-eIF4E in the brain is yet to be determined. Herein, we demonstrate that ablation of eIF4E phosphorylation in male mice (4Eki mice) does not impair long-term spatial or contextual fear memory, or the late phase of LTP. Using unbiased translational profiling in mouse brain, we show that phospho-eIF4E differentially regulates the translation of a subset of mRNAs linked to inflammation, the extracellular matrix, pituitary hormones, and the serotonin pathway. Consequently, 4Eki male mice display exaggerated inflammatory responses and reduced levels of serotonin, concomitant with depression and anxiety-like behaviors. Remarkably, eIF4E phosphorylation is required for the chronic antidepressant action of the selective serotonin reuptake inhibitor fluoxetine. Finally, we propose a novel phospho-eIF4E-dependent translational control mechanism in the brain, via the GAIT complex (gamma IFN activated inhibitor of translation). In summary, our work proposes a novel translational control mechanism involved in the regulation of inflammation and depression, which could be exploited to design novel therapeutics.SIGNIFICANCE STATEMENT We demonstrate that downstream of the MAPK (mitogen-activated protein kinase) pathway, eukaryotic Initiation Factor 4E (eIF4E) Ser209 phosphorylation is not required for classical forms of hippocampal LTP and memory. We reveal a novel role for eIF4E phosphorylation in inflammatory responses and depression-like behaviors. eIF4E phosphorylation is required for the chronic action of antidepressants, such as fluoxetine in mice. These phenotypes are accompanied by selective translation of extracellular matrix, pituitary hormones, and serotonin pathway genes, in eIF4E phospho-mutant mice. We also describe a previously unidentified translational control mechanism in the brain, whereby eIF4E phosphorylation is required for inhibiting the translation of gamma IFN activated inhibitor of translation element-containing mRNAs. These findings can be used to design novel therapeutics for depression.

There is increasing recognition that estrogen exerts multifaceted regulatory effects on GnRH neurons. The acute effects of estrogen on calcium dynamics in these cells were examined using a transgenic mouse line that allows real-time measurement of intracellular calcium concentration ([Ca2+]i) in GnRH neurons in the acute brain slice preparation. 17-beta-Estradiol (E2) at 100 pm-100 nm was found to activate [Ca2+]i transients in approximately 40% of GnRH neurons with an approximate 15-min latency. This effect was not replicated by E2-BSA, which limits E2 action to the membrane, 17-alpha-estradiol, the inactive isomer at classical estrogen receptors (ERs), or G-1 the GPR30 agonist. E2 continued to activate [Ca2+]i transients when transcription was blocked. An ER alpha-selective agonist was equally potent in activating [Ca2+]i transients, and E2 remained effective in ERbeta knockout x GnRH-Pericam mice. E2's activation of [Ca2+]i transients continued in the presence of tetrodotoxin, which blocks action potential-dependent transmission, but was abolished completely by the further addition of a gamma-aminobutyric acid (GABA)A receptor antagonist. Exogenous GABA was found to initiate [Ca2+]i transients in GnRH neurons. Whole cell, voltage-clamp recordings of GnRH-green fluorescence protein neurons revealed that E2 generated discrete bursts of miniature inhibitory postsynaptic currents with a latency of approximately 15 min. These observations provide evidence for a new mechanism of nonclassical estrogen action within the brain. Estrogen interacts with the classical ERalpha at the level of the GABAergic nerve terminal to regulate action potential-independent GABA release that, in turn, controls postsynaptic calcium dynamics.

The pattern of prolactin (PRL) secretion depends on the physiological state. Due to insufficient detection sensitivity of existing assays, the precise description of these patterns in mice is lacking. We described an ultrasensitive ELISA assay that can detect mouse PRL in small fractions of whole blood, allowing longitudinal studies of PRL secretion profiles in freely moving mice. Over a 24-hour period, males displayed no oscillation in PRL levels, whereas virgin and lactating females showed large pulses. Peaks of PRL secretion reached 30-40 ng/mL in lactating female mice and rarely exceeded 10 ng/mL in virgin females. These pulses of PRL in lactating females were associated with suckling. The return of pups after an experimental 12-hour weaning induced a pulse of PRL release, reaching 100 ng/mL. This approach also enabled us to assess the inhibitory tone from hypothalamic dopamine neurons on PRL secretion. We used a dopamine D2 receptor antagonist to relieve pituitary lactotrophs from the tuberoinfundibular dopaminergic inhibitory tone and demonstrate a D2-induced PRL rise that can be used to evaluate both the secretory capacity of lactotrophs and the magnitude of the inhibitory tone on pituitary PRL release. We demonstrate that, although lactotroph function is altered to enhance chronic PRL output, their secretory response to acute stimulus is not modified during lactation and that chronic hyperprolactinemia is linked to a lower inhibitory tone. The combination of a sensitive PRL ELISA and administration of D2 receptor antagonist provide a unique opportunity to investigate the function and plasticity of the lactotroph axis in freely moving mice.

Haematopoietic stem and progenitor cells (HSPCs) develop through distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) is able to recapitulate some of these processes, however, it has proven difficult to generate functional haematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we exploited single cell RNA sequencing (scRNAseq) in combination with single cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we identified CD44, CD326, ICAM2/CD9 and CD18 as markers of these progenitors, respectively. Using an artificial neural network (ANN), that we trained on a scRNAseq derived from human fetal liver, we were able to identify a wide range of hPSCs-derived HPSC phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded and was completely missing in the dataset that had been generated using cells selected on the basis of CD43expression. By comparing the single cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver we identified transcription factors and molecular pathways that can be exploited in the future to improve the in vitro production of HSCs.

Highlights d In lactation, TIDA neurons synthesize and release enkephalin in place of dopamine d This switch in neurotransmitter is driven by neuronal prolactin receptors d This adaptation coincides with altered prolactin signaling within TIDA neurons d This promotes rather than inhibits prolactin secretion during lactation