Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks

Acknowledgments: Work funded by a Marie Curie Intra-European Fellowship FP7-PEOPLE-2009-IEF Project number 255559 to J.A.G.). The authors declare no conflicts of interest.Peer reviewedPublisher PD

Inhibitory Interplay between Orexin Neurons and Eating

We thank Drs. Cornelia Scho¨ ne, Rui Costa, and Guohong Cui for their assistance with fiber photometry. This work was funded by The Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust. L.F. is supported by the MRC and the Wellcome Trust.Peer reviewedPublisher PD

Optogenetic evidence for inhibitory signaling from orexin to MCH neurons via local microcircuits.

The lateral hypothalamus (LH) is a key regulator of multiple vital behaviors. The firing of brain-wide-projecting LH neurons releases neuropeptides promoting wakefulness (orexin/hypocretin; OH), or sleep (melanin-concentrating hormone; MCH). OH neurons, which coexpress glutamate and dynorphin, have been proposed to excite their neighbors, including MCH neurons, suggesting that LH may sometimes coengage its antagonistic outputs. However, it remains unclear if, when, and how OH actions promote temporal separation of the sleep and wake signals, a process that fails in narcolepsy caused by OH loss. To explore this directly, we paired optogenetic stimulation of OH cells (at rates that promoted awakening in vivo) with electrical monitoring of MCH cells in mouse brain slices. Membrane potential recordings showed that OH cell firing inhibited action potential firing in most MCH neurons, an effect that required GABAA but not dynorphin receptors. Membrane current analysis showed that OH cell firing increased the frequency of fast GABAergic currents in MCH cells, an effect blocked by antagonists of OH but not dynorphin or glutamate receptors, and mimicked by bath-applied OH peptide. In turn, neural network imaging with a calcium indicator genetically targeted to MCH neurons showed that excitation by bath-applied OH peptides occurs in a minority of MCH cells. Collectively, our data provide functional microcircuit evidence that intra-LH feedforward loops may facilitate appropriate switching between sleep and wake signals, potentially preventing sleep disorders.This work was supported by the Royal Society Dorothy Hodgkin Fellowship (JAS) and HFSP 37 Young Investigator Award (DB and AA, award ref. RGY0076/2012).This is the final version. It first appeared at http://www.jneurosci.org/content/35/14/5435.abstract

Role of the structure of transcription factor HNF-4a in apolipoprotein gene regulation

Τhe present study aimed at investigating amino acid residues in the LBD region of nuclear receptor HNF-4α that are critical for its activity. For this purpose selected point mutations were introduced into residues of the LBD region that were previously shown to be important for interactions with the ligand in other nuclear receptor molecules. Receptor activity depends on the presence of a ligand in the majority of nuclear receptors examined. However, HNF-4α is not known to alter its activity in response to an exogenous ligand. It rather seems to act as a constitutive activator of transcription, independently of any added molecules. The rationale was to test whether amino acids homologous to those of other nuclear receptors would participate in formation of the ligand binding pocket, in case there was an endogenous ligand for HNF-4α. The results of this study confirm the functional importance of residues leucine (L)220, arginine (R)226, isoleucine (I)338 and, to a lesser extent, isoleucine (I)346 of the LBD region in HNF-4α mediated activation of transcription. Furthermore, they help discriminate between residues of the LBD region that are critical for HNF-4α activity in the absence or presence of coactivators. In this context, mutation of isoleucine (I)338 results in complete loss of function by the receptor, whereas the observed loss of activity in the case of mutant R226G can be reversed by the action of coactivators. While the present analysis was carried out the HNF-4α LBD was crystallized, offering a structural context for the functional observations emerging from this study, by revealing that HNF-4α is endogenously complexed with fatty acid molecules, which act as natural ligands for the receptor. However, fatty acids were not shown to have the same structural consequences for HNF-4α as exogenous ligands do for their cognate receptors, that is, they were not shown to stabilize the LBD in the energetically favorable, “closed” state. On the contrary, the HNF-4α LBD was found to contain endogenous fatty acids both in the “open” and “closed” conformations of the receptor, which led to the proposal that the key molecule stabilizing HNF-4α in its closed and active state may be the coactivator rather than the ligand, in contrast to what has been observed for other nuclear receptors. Our results act in support of this hypothesis, by showing that coactivators are capable of discriminating between different residues and “rescuing” inactive mutants, thereby controling the molecule’s transcriptional activity. In this way, the present study offers functional evidence and expands predictions of the crystallographic model concerning the functional significance of amino acid residues, which participate in the HNF-4α binding pocket of the endogenous ligand. The significance of broader HNF-4α regions, such as the D domain, for transcriptional activation by the receptor was also examined. The data emerging from the study confirm the significance of the D region for HNF-4α transcriptional activity and interaction with coactivators, in agreement to what was found for other nuclear receptors.Η ανάλυση που διεξήχθη είχε ως πρώτο στόχο τη διερεύνηση των αμινοξέων, που είναι καθοριστικά για την ενεργότητα της LBD περιοχής του HNF-4α. Για το σκοπό αυτό εισήχθησαν επιλεγμένες σημειακές μεταλλάξεις σε αμινοξέα της LBD περιοχής, που είχε δειχθεί ότι είναι κρίσιμα για την αλληλεπίδραση με το συνδέτη σε άλλους πυρηνικούς υποδοχείς. Η ενεργότητα της LBD περιοχής εξαρτάται από την πρόσδεση του συνδέτη στην πλειοψηφία των πυρηνικών υποδοχέων, που έχουν εξεταστεί. Ωστόσο, ο HNF-4α δε διαφοροποιεί τη δραστηριότητά του ως απόκριση στην παρουσία εξωγενών μορίων-συνδετών. Αντίθετα, φαίνεται ότι δρα ως «ιδιοσυστατικός» ενεργοποιητής της μεταγραφής (constitutive activator) ανεξαρτήτως παρουσίας οποιουδήποτε εξωγενούς μορίου. Το σκεπτικό ήταν να διερευνηθεί κατά πόσο ομόλογα αμινοξέα με αυτά άλλων πυρηνικών υποδοχέων θα συμμετείχαν στην κοιλότητα πρόσδεσης του συνδέτη, σε περίπτωση που υπήρχε κάποιος ενδογενής συνδέτης για τον HNF-4α. Τα αποτελέσματα της παρούσας εργασίας αποδεικνύουν τη λειτουργική σημασία των αμινοξέων λευκίνη (L)220, αργινίνη (R)226, ισολευκίνη (I)338 και, σε μικρότερο βαθμό, ισολευκίνη (I)346 της LBD περιοχής για την ενεργοποίηση της μεταγραφής από τον HNF-4α. Επιπλέον, βοηθούν στη διάκριση αμινοξέων, που είναι καθοριστικά για την ενεργότητα της LBD περιοχής κάτω από διαφορετικές συνθήκες. Έτσι, μετάλλαξη της ισολευκίνης (I)338 οδηγεί σε ανεπανόρθωτη απώλεια της ενεργότητας του μεταγραφικού παράγοντα, ενώ η απώλεια ενεργότητας που παρατηρείται στην περίπτωση της μετάλλαξης R226G είναι δυνατόν να αντιστραφεί παρουσία συνενεργοποιητών. Η κρυστάλλωση της LBD περιοχής του HNF-4α κατά τη διάρκεια διεξαγωγής της μελέτης πρόσφερε το δομικό πλαίσιο για την ένταξη των λειτουργικών παρατηρήσεων, που προέκυψαν από τη μελέτη. Ειδικότερα, φανέρωσε ότι ο HNF-4α είναι ενδογενώς συνδεδεμένος με μόρια λιπαρών οξέων, τα οποία διαδραματίζουν το ρόλο φυσικών του συνδετών. Ωστόσο, τα μόρια αυτά δεν έχουν για τον HNF-4α τη δομική σημασία, που έχουν οι εξωγενείς συνδέτες για άλλους πυρηνικούς υποδοχείς, δηλαδή τη σταθεροποίηση της LBD περιοχής στην ενεργειακά ευνοϊκότερη, «κλειστή» διαμόρφωση. Αντίθετα, στον HNF-4α η LBD περιοχή περιέχει δεσμευμένα λιπαρά οξέα ανεξαρτήτως του αν βρίσκεται στην «ανοιχτή» ή «κλειστή» διαμόρφωση, κάτι που οδήγησε στη διατύπωση της πρότασης ότι το ρόλο σταθεροποιητή στην περίπτωση του HNF-4α μπορεί να παίζουν μόρια διαφορετικά από τους συνδέτες, όπως οι συνενεργοποιητές. Τα αποτελέσματά μας έρχονται να ενισχύσουν την υπόθεση της σπουδαιότητας των συνενεργοποιητών στη ρύθμιση της δράσης του HNF-4α, καθώς δείχνουν ότι τα συγκεκριμένα μόρια είναι σε θέση να διακρίνουν μεταξύ διαφορετικών αμινοξέων και να «διασώζουν» λειτουργικά ανενεργές μεταλλάξεις, έχοντας τον «τελικό λόγο» στη δραστικότητα του μεταγραφικού παράγοντα. Κατ’ αυτόν τον τρόπο η παρούσα μελέτη συμπληρώνει με λειτουργικά στοιχεία και επεκτείνει τις προβλέψεις του κρυσταλλογραφικού μοντέλου σχετικά με τη λειτουργική σημασία αμινοξέων, που συμμετέχουν στην κοιλότητα πρόσδεσης του ενδογενούς συνδέτη του HNF-4α. Ο δεύτερος στόχος της εργασίας ήταν η διερεύνηση της σημασίας ευρύτερων περιοχών του LBD του HNF-4α, όπως η περιοχή D, για τη μεταγραφική δραστηριότητα του υποδοχέα και την ενίσχυση από τους συνενεργοποιητές. Η παρούσα μελέτη επιβεβαίωσε τη σημασία της D περιοχής για την ενεργότητα του HNF-4α και την αλληλεπίδρασή του με τους συνενεργοποιητές, σε συμφωνία με ό,τι έχει δειχθεί ότι συμβαίνει και σε άλλους πυρηνικούς υποδοχείς

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion.

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion

A cortico-collicular circuit for accurate orientation to shelter during escape

When faced with predatorial threats, escaping towards shelter is an adaptive action that offers long-term protection against the attacker. From crustaceans to mammals, animals rely on knowledge of safe locations in the environment to rapidly execute shelter-directed escape actions1–3. While previous work has identified neural mechanisms of instinctive escape4–9, it is not known how the escape circuit incorporates spatial information to execute rapid and accurate flights to safety. Here we show that mouse retrosplenial cortex (RSP) and superior colliculus (SC) form a monosynaptic circuit that continuously encodes the shelter direction. Inactivation of SC-projecting RSP neurons decreases SC shelter-direction tuning while preserving SC motor function. Moreover, specific inactivation of RSP input onto SC neurons disrupts orientation and subsequent escapes to shelter, but not orientation accuracy to a sensory cue. We conclude that the RSC-SC circuit supports an egocentric representation of shelter direction and is necessary for optimal shelter-directed escapes. This cortical-subcortical interface may be a general blueprint for increasing the sophistication and flexibility of instinctive behaviours

A cortico-collicular circuit for orienting to shelter during escape

When faced with predatory threats, escape towards shelter is an adaptive action that offers long-term protection against the attacker. Animals rely on knowledge of safe locations in the environment to instinctively execute rapid shelter-directed escape actions1,2. Although previous work has identified neural mechanisms of escape initiation3,4, it is not known how the escape circuit incorporates spatial information to execute rapid flights along the most efficient route to shelter. Here we show that the mouse retrosplenial cortex (RSP) and superior colliculus (SC) form a circuit that encodes the shelter-direction vector and is specifically required for accurately orienting to shelter during escape. Shelter direction is encoded in RSP and SC neurons in egocentric coordinates and SC shelter-direction tuning depends on RSP activity. Inactivation of the RSP-SC pathway disrupts the orientation to shelter and causes escapes away from the optimal shelter-directed route, but does not lead to generic deficits in orientation or spatial navigation. We find that the RSP and SC are monosynaptically connected and form a feedforward lateral inhibition microcircuit that strongly drives the inhibitory collicular network because of higher RSP input convergence and synaptic integration efficiency in inhibitory SC neurons. This results in broad shelter-direction tuning in inhibitory SC neurons and sharply tuned excitatory SC neurons. These findings are recapitulated by a biologically constrained spiking network model in which RSP input to the local SC recurrent ring architecture generates a circular shelter-direction map. We propose that this RSP-SC circuit might be specialized for generating collicular representations of memorized spatial goals that are readily accessible to the motor system during escape, or more broadly, during navigation when the goal must be reached as fast as possible