Esr1+ hypothalamic-habenula neurons shape aversive states.

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used patch-sequencing (Patch-seq) guided multimodal classification to define the structural and functional heterogeneity of the LHA-LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles and projection patterns. We found that genetically defined LHA-LHb neurons signal distinct aspects of emotional or naturalistic behaviors, such as estrogen receptor 1-expressing (Esr1+) LHA-LHb neurons induce aversion, whereas neuropeptide Y-expressing (Npy+) LHA-LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA-LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive signals in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA-LHb neurons. In summary, we describe the diversity of LHA-LHb neuron types and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.

Complete representation of action space and value in all dorsal striatal pathways.

The dorsal striatum plays a central role in the selection, execution, and evaluation of actions. An emerging model attributes action selection to the matrix and evaluation to the striosome compartment. Here, we use large-scale cell-type-specific calcium imaging to determine the activity of striatal projection neurons (SPNs) during motor and decision behaviors in the three major outputs of the dorsomedial striatum: Oprm1+ striosome versus D1+ direct and A2A+ indirect pathway SPNs. We find that Oprm1+ SPNs show complex tunings to simple movements and value-guided actions, which are conserved across many sessions in a single task but remap between contexts. During decision making, the SPN tuning profiles form a complete representation in which sequential SPN activity jointly encodes task progress and value. We propose that the three major output pathways in the dorsomedial striatum share a similarly complete representation of the entire action space, including task- and phase-specific signals of action value and choice.

A hypothalamus-habenula circuit controls aversion.

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

An interactive framework for whole-brain maps at cellular resolution.

To deconstruct the architecture and function of brain circuits, it is necessary to generate maps of neuronal connectivity and activity on a whole-brain scale. New methods now enable large-scale mapping of the mouse brain at cellular and subcellular resolution. We developed a framework to automatically annotate, analyze, visualize and easily share whole-brain data at cellular resolution, based on a scale-invariant, interactive mouse brain atlas. This framework enables connectivity and mapping projects in individual laboratories and across imaging platforms, as well as multiplexed quantitative information on the molecular identity of single neurons. As a proof of concept, we generated a comparative connectivity map of five major neuron types in the corticostriatal circuit, as well as an activity-based map to identify hubs mediating the behavioral effects of cocaine. Thus, this computational framework provides the necessary tools to generate brain maps that integrate data from connectivity, neuron identity and function.

Publisher Correction: An interactive framework for whole-brain maps at cellular resolution.

In the version of this article initially published online, Daniel Fürth was not listed as a corresponding author. The error has been corrected in the print, PDF and HTML versions of this article.

Application of multispectral imaging detects areas with neuronal myelin loss, without tissue labelling.

The application of multispectral imaging to discriminate myelinated and demyelinated areas of neural tissue is herein presented. The method is applied through a custom-made, multispectral imaging monochromator, coupled to a commercially available microscope. In the present work, a series of spinal cord sections were analysed derived from mice with experimental autoimmune encephalomyelitis (EAE), an experimental model widely used to study multiple sclerosis (MS). The multispectral microscope allows imaging of local areas with loss of myelin without the need of tissue labelling. Imaging with the aforementioned method and system is compared in a parallel way with conventional methods (wide-field and confocal fluorescence microscopies). The diagnostic sensitivity of our method is 90.4% relative to the 'gold standard' method of immunofluorescence microscopy. The presented method offers a new platform for the possible future development of an in vivo, real-time, non-invasive, rapid imaging diagnostic tool of spinal cord myelin loss-derived pathologies.

Crucial role of granulocytic myeloid-derived suppressor cells in the regulation of central nervous system autoimmune disease.

There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC-mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4(+) T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.

A novel role of peripheral corticotropin-releasing hormone (CRH) on dermal fibroblasts.

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh-/-) had higher proliferation and migration rates and compromised production of IL-6 and TGF-ß1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF(1) antagonist antalarmin recapitulated the findings in the Crh-/- cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis.

Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis.

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

Novel dehydroepiandrosterone derivatives with antiapoptotic, neuroprotective activity.

DHEA analogues with modifications at positions C3 or C17 were synthesized and evaluated for neuroprotective activity against the neural-crest-derived PC12 cell model of serum deprivation-induced apoptosis. The most potent compounds were the spiro-epoxy derivatives 17beta-spiro[5-androstene-17,2'-oxiran]-3beta-ol (20), (20S)-3beta,21-dihydroxy-17beta,20-epoxy-5-pregnene (23), and (20R)-3beta,21-dihydroxy-17alpha,20-epoxy-5-pregnene (27) with IC(50) values of 0.19 +/- 0.01, 99.0 +/- 4.6, and 6.4 +/- 0.3 nM, respectively. Analogues 20, 23, and 27, up to the micromolar range of concentrations, were unable to activate estrogen receptor alpha and beta (ERalpha and ERbeta) or to interfere with ER-dependent gene expression significantly. In addition, they were unable to stimulate the growth of Ishikawa, MCF-7, and LNCaP cells. Our results suggest that the spiro-epoxyneurosteroid derivatives 20, 23, and 27 may prove to be lead molecules for the synthesis of novel neuroprotective agents.

Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding.

Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.

G protein-associated, specific membrane binding sites mediate the neuroprotective effect of dehydroepiandrosterone.

The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects NMDA-/GABAA-receptor negative neural crest-derived PC12 cells from apoptosis. We now report that membrane-impermeable DHEA-BSA conjugate replaces unconjugated DHEA in protecting serum-deprived PC12 cells from apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the prosurvival factor Src and induction of the anti-apoptotic protein Bcl-2 and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated PC12 cell membranes revealed saturation within 30 min and binding of DHEA with a Kd of 0.9 nM. A similar binding activity was detectable in isolated membranes from rat hippocampus and from normal human adrenal chromaffin cells. The presence of DHEA-specific membrane binding sites was confirmed by flow cytometry and confocal laser microscopy of DHEA-BSA-FITC stained cells. In contrast to estrogens and progestins, glucocorticoids and androgens displaced DHEA from its membrane binding sites but with a 10-fold lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents acted as pure antagonists, blocking the antiapoptotic effect of DHEA as well as the induction of Bcl-2 proteins and Src kinase activation. In conclusion, our findings suggest that neural crest-derived cells possess specific DHEA membrane binding sites coupled to G proteins. Binding to these sites confers neuroprotection.