Loss of Ephaptic Contacts in the Murine Thalamus during Osmotic Demyelination Syndrome.

BACKGROUND AND AIM: A murine model mimicking osmotic demyelination syndrome (ODS) revealed with histology in the relay posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei adjoined nerve cell bodies in chronic hyponatremia, amongst the damaged 12 h and 48 h after reinstatement of osmolality. This report aims to verify and complement with ultrastructure other neurophysiology, immunohistochemistry, and molecular biochemistry data to assess the connexin-36 protein, as part of those hinted close contacts.This ODS investigation included four groups of mice: Sham (NN; n = 13), hyponatremic (HN; n = 11), those sacrificed 12 h after a fast restoration of normal natremia (ODS12h; n = 6) and mice sacrificed 48 h afterward, or ODS48 h (n = 9). Out of these, thalamic zones samples included NN (n = 2), HN (n = 2), ODS12h (n = 3) and ODS48h (n = 3). RESULTS: Ultrastructure illustrated junctions between nerve cell bodies that were immunolabeled with connexin36 (Cx36) with light microscopy and Western blots. These cell's junctions were reminiscent of low resistance junctions characterized in other regions of the CNS with electrophysiology. Contiguous neurons showed neurolemma contacts in intact and damaged tissues according to their location in the ODS zones, at 12 h and 48 h post correction along with other demyelinating alterations. Neurons and ephaptic contact measurements indicated the highest alterations, including nerve cell necrosis in the ODS epicenter and damages decreased toward the outskirts of the demyelinated zone. CONCLUSION: Ephapses contained C × 36between intact or ODS injured neurons in the thalamus appeared to be resilient beyond the core degraded tissue injuries. These could maintain intercellular ionic and metabolite exchanges between these lesser injured regions and, thus, would partake to some brain plasticity repairs.

Use of Active Salmon-Lecithin Nanoliposomes to Increase Polyunsaturated Fatty Acid Bioavailability in Cortical Neurons and Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) play an important role in the development, maintenance, and function of the brain. Dietary supplementation of n-3 PUFAs in neurological diseases has been a subject of particular interest in preventing cognitive deficits, and particularly in age-related neurodegeneration. Developing strategies for the efficient delivery of these lipids to the brain has presented a challenge in recent years. We recently reported the preparation of n-3 PUFA-rich nanoliposomes (NLs) from salmon lecithin, and demonstrated their neurotrophic effects in rat embryo cortical neurons. The objective of this study was to assess the ability of these NLs to deliver PUFAs in cellulo and in vivo (in mice). NLs were prepared using salmon lecithin rich in n-3 PUFAs (29.13%), and characterized with an average size of 107.90 ± 0.35 nm, a polydispersity index of 0.25 ± 0.01, and a negative particle-surface electrical charge (-50.4 ± 0.2 mV). Incubation of rat embryo cortical neurons with NLs led to a significant increase in docosahexaenoic acid (DHA) (51.5%, p < 0.01), as well as palmitic acid, and a small decrease in oleic acid after 72 h (12.2%, p < 0.05). Twenty mice on a standard diet received oral administration of NLs (12 mg/mouse/day; 5 days per week) for 8 weeks. Fatty acid profiles obtained via gas chromatography revealed significant increases in cortical levels of saturated, monounsaturated, and n-3 (docosahexaenoic acid,) and n-6 (docosapentaenoic acid and arachidonic acid) PUFAs. This was not the case for the hippocampus or in the liver. There were no effects on plasma lipid levels, and daily monitoring confirmed NL biocompatibility. These results demonstrate that NLs can be used for delivery of PUFAs to the brain. This study opens new research possibilities in the development of preventive as well as therapeutic strategies for age-related neurodegeneration.

Enhanced modulation of cell-type specific neuronal responses in mouse dorsal auditory field during locomotion.

As we move through the environment we experience constantly changing sensory input that must be merged with our ongoing motor behaviors - creating dynamic interactions between our sensory and motor systems. Active behaviors such as locomotion generally increase the sensory-evoked neuronal activity in visual and somatosensory cortices, but evidence suggests that locomotion largely suppresses neuronal responses in the auditory cortex. However, whether this effect is ubiquitous across different anatomical regions of the auditory cortex is largely unknown. In mice, auditory association fields such as the dorsal auditory cortex (AuD), have been shown to have different physiological response properties, protein expression patterns, and cortical as well as subcortical connections, in comparison to primary auditory regions (A1) - suggesting there may be important functional differences. Here we examined locomotion-related modulation of neuronal activity in cortical layers ⅔ of AuD and A1 using two-photon Ca2+ imaging in head-fixed behaving mice that are able to freely run on a spherical treadmill. We determined the proportion of neurons in these two auditory regions that show enhanced and suppressed sensory-evoked responses during locomotion and quantified the depth of modulation. We found that A1 shows more suppression and AuD more enhanced responses during locomotion periods. We further revealed differences in the circuitry between these auditory regions and motor cortex, and found that AuD is more highly connected to motor cortical regions. Finally, we compared the cell-type specific locomotion-evoked modulation of responses in AuD and found that, while subpopulations of PV-expressing interneurons showed heterogeneous responses, the population in general was largely suppressed during locomotion, while excitatory population responses were generally enhanced in AuD. Therefore, neurons in primary and dorsal auditory fields have distinct response properties, with dorsal regions exhibiting enhanced activity in response to movement. This functional distinction may be important for auditory processing during navigation and acoustically guided behavior.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Peculiar protrusions along tanycyte processes face diverse neural and nonneural cell types in the hypothalamic parenchyma.

Tanycytes are highly specialized ependymal cells that line the bottom and the lateral walls of the third ventricle. In contact with the cerebrospinal fluid through their cell bodies, they send processes into the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. In the present work, we combined transgenic and immunohistochemical approaches to investigate the neuroanatomical associations between tanycytes and neural cells present in the hypothalamic parenchyma, in particular in the arcuate nucleus. The specific expression of tdTomato in tanycytes first allowed the observation of peculiar subcellular protrusions along tanycyte processes and at their endfeet such as spines, swelling, en passant boutons, boutons, or claws. Interestingly, these protrusions contact different neural cells in the brain parenchyma including blood vessels and neurons, and in particular NPY and POMC neurons in the arcuate nucleus. Using both fluorescent and electron microscopy, we finally observed that these tanycyte protrusions contain ribosomes, mitochondria, diverse vesicles, and transporters, suggesting dense tanycyte/neuron and tanycyte/blood vessel communications. Altogether, our results lay the neuroanatomical basis for tanycyte/neural cell interactions, which will be useful to further understand cell-to-cell communications involved in the regulation of neuroendocrine functions.

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons.

BACKGROUND: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD-beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons. METHODS: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC. RESULTS: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls. CONCLUSION: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.

Cortical RORß is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy.

Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 µm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Ytterbium-doped fibre femtosecond laser offers robust operation with deep and precise microsurgery of C. elegans neurons.

Laser microsurgery is a powerful tool for neurobiology, used to ablate cells and sever neurites in-vivo. We compare a relatively new laser source to two well-established designs. Rare-earth-doped mode-locked fibre lasers that produce high power pulses recently gained popularity for industrial uses. Such systems are manufactured to high standards of robustness and low maintenance requirements typical of solid-state lasers. We demonstrate that an Ytterbium-doped fibre femtosecond laser is comparable in precision to a Ti:Sapphire femtosecond laser (1-2 micrometres), but with added operational reliability. Due to the lower pulse energy required to ablate, it is more precise than a solid-state nanosecond laser. Due to reduced scattering of near infrared light, it can lesion deeper (more than 100 micrometres) in tissue. These advantages are not specific to the model system ablated for our demonstration, namely neurites in the nematode C. elegans, but are applicable to other systems and transparent tissue where a precise micron-resolution dissection is required.

Connect-seq to superimpose molecular on anatomical neural circuit maps.

The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed "Connect-seq," which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.

A strategy for iron oxide nanoparticles to adhere to the neuronal membrane in the substantia nigra of mice.

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P-modulated growth hormone secretion.

Substance P is an undecapeptide affecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these effects, previous papers provided solid evidence that substance P exhibits regulatory effects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fibers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also affect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and somatostatinergic systems utilizing double label immunohistochemistry combined with high magnification light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fiber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addition, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also influences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts.

Gestational folic acid content alters the development and function of hypothalamic food intake regulating neurons in Wistar rat offspring post-weaning.

Background: Folic acid plays an important role in early brain development of offspring, including proliferation and differentiation of neural stem cells known to impact the function of food intake regulatory pathways. Excess (10-fold) intakes of folic acid in the gestational diet have been linked to increased food intake and obesity in male rat offspring post-weaning.Objective: The present study examined the effects of folic acid content in gestational diets on the development and function of two hypothalamic neuronal populations, neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), within food intake regulatory pathways of male Wistar rat offspring at birth and post-weaning.Results: Folic acid fed at 5.0-fold above recommended levels (5RF) to Wistar dams during pregnancy increased the number of mature NPY-positive neurons in the hypothalamus of male offspring, compared to control (RF), 0RF, 2.5RF, and 10RF at birth. Folic acid content had no effect on expression and maturation of POMC-positive neurons. Body weight and food intake were higher in all treatment groups (2.5-, 5.0-, and 10.0-fold folic acid) from birth to 9 weeks post-weaning compared to control. Increased body weight and food intake at 9-weeks post-weaning were accompanied by a reduced activation of POMC neurons in the arcuate nucleus (ARC).Conclusion: Gestational folic acid content modulates expression of mature hypothalamic NPY-positive neurons at birth and activation of POMC-positive neurons at 9-weeks post-weaning in the ARC of male Wistar rat offspring which may contribute to higher body weight and food intake later in life.

Isotopic variations of copper at the protein fraction level in neuronal human cells exposed in vitro to uranium.

The study of isotopic variations of endogenous and toxic metals in fluids and tissues is a recent research topic with an outstanding potential in biomedical and toxicological investigations. Most of the analyses have been performed so far in bulk samples, which can make the interpretation of results entangled, since different sources of stress or the alteration of different metabolic processes can lead to similar variations in the isotopic compositions of the elements in bulk samples. The downscaling of the isotopic analysis of elements at the sub-cellular level, is considered as a more promising alternative. Here we present for the first time the accurate determination of Cu isotopic ratios in four main protein fractions from lysates of neuron-like human cells exposed in vitro to 10 µM of natural uranium for seven days. These protein fractions were isolated by Size Exclusion Chromatography and analysed by Multi-Collector Inductively Coupled Plasma Mass Spectrometry to determine the Cu isotopic variations in each protein fraction with regard to the original cell lysate. Values obtained, expressed as δ65Cu, were -0.03 ± 0.14 ‰ (Uc, k = 2), -0.55 ± 0.20 ‰ (Uc, k = 2), -0.32 ± 0.21 ‰ (Uc, k = 2) and +0.84 ± 0.21 ‰ (Uc, k = 2) for the four fractions, satisfying the mass balance. The results obtained in this preliminary study pave the way for dedicated analytical developments to identify new specific disease biomarkers, to gain insight into stress-induced altered metabolic processes, as well as to decipher metabolic pathways of toxic elements.

Intracellular Recording of Cardiomyocyte Action Potentials with Nanopatterned Volcano-Shaped Microelectrode Arrays.

Micronanotechnology-based multielectrode arrays have led to remarkable progress in the field of transmembrane voltage recording of excitable cells. However, providing long-term optoporation- or electroporation-free intracellular access remains a considerable challenge. In this study, a novel type of nanopatterned volcano-shaped microelectrode (nanovolcano) is described that spontaneously fuses with the cell membrane and permits stable intracellular access. The complex nanostructure was manufactured following a simple and scalable fabrication process based on ion beam etching redeposition. The resulting ring-shaped structure provided passive intracellular access to neonatal rat cardiomyocytes. Intracellular action potentials were successfully recorded in vitro from different devices, and continuous recording for more than 1 h was achieved. By reporting transmembrane action potentials at potentially high spatial resolution without the need to apply physical triggers, the nanovolcanoes show distinct advantages over multielectrode arrays for the assessment of electrophysiological characteristics of cardiomyocyte networks at the transmembrane voltage level over time.

Genetic dissection of the different roles of hypothalamic kisspeptin neurons in regulating female reproduction.

The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedbacks are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest that activational effects of estradiol regulate surge generation and maintain cyclicity through AVPV and arcuate kisspeptin neurons, respectively, independent from its role in the development of hypothalamic kisspeptin neurons or puberty onset.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.

Pubertal development of estradiol-induced hypothalamic progesterone synthesis.

In females, a hallmark of puberty is the luteinizing hormone (LH) surge that triggers ovulation. Puberty initiates estrogen positive feedback onto hypothalamic circuits, which underlie the stimulation of gonadotropin releasing hormone (GnRH) neurons. In reproductively mature female rodents, both estradiol (E2) and progesterone (P4) signaling are necessary to stimulate the surge release of GnRH and LH. Estradiol membrane-initiated signaling facilitates progesterone (neuroP) synthesis in hypothalamic astrocytes, which act on E2-induced progesterone receptors (PGR) to stimulate kisspeptin release, thereby activating GnRH release. How the brain changes during puberty to allow estrogen positive feedback remains unknown. In the current study, we hypothesized that a critical step in estrogen positive feedback was the ability for estradiol-induced neuroP synthesis. To test this idea, hypothalamic neuroP levels were measured in groups of prepubertal, pubertal and young adult female Long Evans rats. Steroids were measured with liquid chromatography tandem mass spectrometry (LC-MS/MS). Hypothalamic neuroP increases from pre-puberty to young adulthood in both gonad-intact females and ovariectomized rats treated with E2. The pubertal development of hypothalamic E2-facilitated progesterone synthesis appears to be one of the neural switches facilitating reproductive maturation.