Molecular diversity of corticotropin-releasing hormone mRNA-containing neurons in the hypothalamus

in Journal of Endocrinology
View More View Less
  • 1 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria
  • | 2 MTA-SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Hungarian Academy of Sciences, Budapest, Hungary
  • | 3 Department of Anatomy, Semmelweis University, Budapest, Hungary
  • | 4 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Free access

Hormonal responses to acute stress rely on the rapid induction of corticotropin-releasing hormone (CRH) production in the mammalian hypothalamus, with subsequent instructive steps culminating in corticosterone release at the periphery. Hypothalamic CRH neurons in the paraventricular nucleus of the hypothalamus are therefore considered as ‘stress neurons’. However, significant morphological and functional diversity among neurons that can transiently produce CRH in other hypothalamic nuclei has been proposed, particularly as histochemical and molecular biology evidence associates CRH to both GABA and glutamate neurotransmission. Here, we review recent advances through single-cell RNA sequencing and circuit mapping to suggest that CRH production reflects a state switch in hypothalamic neurons and thus confers functional competence rather than being an identity mark of phenotypically segregated neurons. We show that CRH mRNA transcripts can therefore be seen in GABAergic, glutamatergic and dopaminergic neuronal contingents in the hypothalamus. We then distinguish ‘stress neurons’ of the paraventricular nucleus that constitutively express secretagogin, a Ca2+ sensor critical for the stimulus-driven assembly of the molecular machinery underpinning the fast regulated exocytosis of CRH at the median eminence. Cumulatively, we infer that CRH neurons are functionally and molecularly more diverse than previously thought.

Abstract

Hormonal responses to acute stress rely on the rapid induction of corticotropin-releasing hormone (CRH) production in the mammalian hypothalamus, with subsequent instructive steps culminating in corticosterone release at the periphery. Hypothalamic CRH neurons in the paraventricular nucleus of the hypothalamus are therefore considered as ‘stress neurons’. However, significant morphological and functional diversity among neurons that can transiently produce CRH in other hypothalamic nuclei has been proposed, particularly as histochemical and molecular biology evidence associates CRH to both GABA and glutamate neurotransmission. Here, we review recent advances through single-cell RNA sequencing and circuit mapping to suggest that CRH production reflects a state switch in hypothalamic neurons and thus confers functional competence rather than being an identity mark of phenotypically segregated neurons. We show that CRH mRNA transcripts can therefore be seen in GABAergic, glutamatergic and dopaminergic neuronal contingents in the hypothalamus. We then distinguish ‘stress neurons’ of the paraventricular nucleus that constitutively express secretagogin, a Ca2+ sensor critical for the stimulus-driven assembly of the molecular machinery underpinning the fast regulated exocytosis of CRH at the median eminence. Cumulatively, we infer that CRH neurons are functionally and molecularly more diverse than previously thought.

Introduction

The introduction of molecular methods in the neurobiologists’ tool kit has changed many long-held views on the structural and functional organization of the nervous system. One of the most recent additions is the use of single-cell RNA-sequencing (scRNA-seq), which can produce quantitative mRNA counts to predict the origin, lineage segregation, functional diversification and disease susceptibility of any cell type in the nervous system. Taking the power of ‘forward’ (or predictive) transcriptomics to the level of neuroanatomy, connectivity mapping by electrophysiology and genetic models, one can expect a sea-change of organizational rules that have hitherto been applied on neuronal identity and function (Shapiro et al. 2013, Romanov et al. 2015, Zeisel et al. 2015). Alternatively, the use of ‘reverse’ transcriptomics can validate many datasets that were generated using alternative tools. This is a particularly exciting prospect in neurobiology where sometimes mm-to-cm-scale distances exist between synaptic release sites and the somata of relevant projection neurons. Thus, functional transcriptomics at projection sites might be validated by scRNA-seq of somatic compartments (Romanov et al. 2015), which contain all mRNAs for the protein domain sustaining the regulated release of any neurotransmitter or neuroactive modulator. Here, we will review recent data on corticotropin-releasing hormone (CRH) mRNA-containing neurons in the mammalian hypothalamus to describe their unexpected molecular heterogeneity. By relying on a combination of novel experimental tools, we suggest that Crh expression can occur in glutamatergic, GABAergic and dopaminergic neurons and is a descriptor of specific ‘working modes’ and that the co-localization of CRH with secretagogin is a defining hallmark of ‘stress on’ CRH neurons in the paraventricular nucleus of the hypothalamus (PVN).

Organization of the hypothalamus–pituitary–adrenal axis: from systems to molecules

The body’s reaction to environmental stressors, including physical and psychosocial factors (McEwen 2007), is orchestrated by sequential hormone release in the hypothalamus–pituitary–adrenal (HPA) axis (Herman et al. 2003). HPA activation triggers corticosteroid release from the cortex of the adrenal gland to mobilize energy utilization, thus leading to integrative-protective responses to noxious conditions (Sapolsky et al. 2000, McEwen 2007). The first hierarchical level in the HPA axis is the release of CRH (Spiess et al. 1981, Vale et al. 1981) from the terminals of parvocellular hypothalamic neurons into hypophyseal portal vessels (Green & Harris 1947) at the median eminence (Swanson et al. 1983). According to the classical definition, the peripheral stress response then progresses through the secretion of adrenocorticotropic hormone (ACTH) (Mains et al. 1977, Roberts & Herbert 1977, Nakanishi et al. 1979) from the anterior lobe of the pituitary. Finally, ACTH induces steroidogenesis in, and corticosteroid release from, the adrenal cortex (Munck et al. 1984, McEwen & Stellar 1993). Corticosteroid action is then executed through glucocorticoid receptors (Gustafsson et al. 1987), encoded by the Nr3c1 gene (McEwen 2012), with ensuing changes in cellular states (‘cell-state switch’) tuned to overcome metabolic compromise.

Parvocellular neurons are broadly viewed as the origins of CRH for stress-induced, fast release and are situated in the PVN (Merchenthaler et al. 1983, Swanson et al. 1983, 1986, Alonso et al. 1986, Ceccatelli et al. 1989b, Swanson 1991). The PVN itself showcases the complexity of neuronal organization in the hypothalamus: it contains at least eight subdivisions (Swanson & Kuypers 1980, Simmons & Swanson 2009) with the tight spatial concentration of magnocellular and parvocellular neuroendocrine secretory motor neurons (that is, cells that release bioactive peptides into the portal circulation through release sites located outside the blood–brain barrier), neurons projecting to the brainstem and locally projecting hypothalamic neurons (Daftary et al. 1998, Krashes et al. 2014). Oxytocin and vasopressin (Du Vigneaud 1954) are produced in magnocellular neurons projecting directly to the posterior pituitary (Swaab et al. 1975, Rhodes et al. 1981). In contrast, parvocellular neurons are a kaleidoscope of neuroendocrine modalities, often co-releasing several neuropeptides and fast neurotransmitters in an ‘on-demand’ manner (Swanson et al. 1986, Ceccatelli et al. 1989a,b). Among parvocellular PVN neurons, CRH cells, besides projecting to the median eminence, also form axon collaterals toward other intrahypothalamic nuclei (e.g., lateral hypothalamus), thus increasing integrative capacity through the coincident control of neuronal circuitries and hormonal responses at the pituitary (Fuzesi et al. 2016).

Besides CRH at its ‘stress center’ (Alonso et al. 1986), parvocellular neuroendocrine cells can contain ‘releasing’ and ‘release-inhibiting’ hormones (e.g., growth hormone-releasing hormone and somatostatin (growth hormone-inhibiting hormone), thyrotropin-releasing hormone, gonadotropin-releasing hormone and dopamine (as prolactin-inhibiting hormone)) to regulate hormone release from the anterior pituitary (classed as growth hormone, thyroid-stimulated hormone, luteinizing and follicle-stimulating hormones and prolactin) (Guillemin 1978, Schally 1978, Ben-Jonathan & Hnasko 2001). Of particular interest in relation to stress, vasopressin potentiates the effect of CRH (Gillies et al. 1982, Turkelson et al. 1982, Rivier & Vale 1983) and can undergo induced expression in CRH neurons after adrenalectomy (Tramu et al. 1983, Kiss et al. 1984, Sawchenko et al. 1984). As such, these peptides co-exist in the same storage granules in boutons residing in the external layer of the median eminence (Whitnall et al. 1985).

The observation that basal ACTH and corticosterone levels can always be detected in blood (rather than being ‘on’/‘off’ signals) (Girotti et al. 2009, Romanov et al. 2015) indirectly suggests that CRH is constitutively released in minute amounts to continuously stimulate the production and release of downstream hormones. This concept has broad physiological significance as, for example, pancreatic β cells require glucocorticoids for their survival. Consequently, hypothalamic CRH hypofunction leads to Addison’s disease with diabetes being its primary comorbidity (Miller & O’Callaghan 2002, Napier & Pearce 2014, Kahaly & Hansen 2016). There also is a well-established relation of CRH (and corticosteroids) to mood disorders (Gold et al. 1995, Reul & Holsboer 2002, Lloyd & Nemeroff 2011, Waters et al. 2015): CRH release follows the diurnal cycle with autonomous peaks observed during the early morning hours and late afternoon/evening (Salata et al. 1988, Owens et al. 1990, Watts et al. 2004, Girotti et al. 2009). These data suggest that CRH might have more widespread biological roles than previously thought through cell- or tissue-specific expression of its receptors (see below). As such, and beyond regulating the stress response, CRH is increasingly implicated in the regulation of appetite (Drescher et al. 1994, Uehara et al. 1998).

CRH production in the brain

Beyond its canonical expression site identified as the parvocellular neurons of the PVN (Simmons & Swanson 2009), CRH production is not restricted to the PVN. In the hypothalamus, CRH is also detected in magnocellular cells of the lateral hypothalamus and preoptic area (Alon et al. 2009). A critical difficulty to identify the cellular sites of CRH production is its unusually low levels at rest and fast induction during stressful conditions or in particular metabolic states. Therefore, histochemical solutions so far relied on agents blocking the axonal transport machinery to elevate CRH levels in neuronal somata (Merchenthaler et al. 1982, Alonso et al. 1986) or by experimentally boosting its expression (Ceccatelli et al. 1991). In accord with the expansion of amenable molecular genetic tools, e.g. the Cre/lox system, we recently used Crh-IRES-Cre-driven green fluorescent protein (GFP) expression to allow the accumulation of GFP in response to episodic Crh expression during a cell’s life time (Fig. 1A). These results document many GFP+ neurons scattered within the hypothalamus, including its anterior and ventromedial areas. This genetic model suggests, in accord with earlier histochemical studies (Merchenthaler et al. 1982, Swanson et al. 1983, Keegan et al. 1994, Potter et al. 1994, Alon et al. 2009), that episodic Crh mRNA expression might be intrinsic to many hypothalamic neurons. Likewise, Crh gene transcription and protein expression were documented in many extrahypothalamic brain areas, including thalamic nuclei, bed nucleus of the stria terminalis, amygdala, medulla oblongata (especially inferior olive), piriform cortex and many neocortical and hippocampal subregions (Merchenthaler et al. 1982, Potter et al. 1994, Lantos et al. 1995, Alon et al. 2009, Zeisel et al. 2015, Hooper & Maguire 2016). Thus, an essential heterogeneity of CRH neurons emerges with CRH likely being a molecular determinant of functional cellular competence under particular behavioral and metabolic conditions.

Figure 1
Figure 1

Molecular heterogeneity of Crh+ neurons in the hypothalamus. (A) Distribution of GFP+ neurons outside the PVN in the hypothalamus of Crh-IRES-Cre::GFP mice. (B) Single-cell RNA-seq distinguishes 62 neuronal subclasses along the PVN–arcuate axis of the hypothalamus (Romanov et al. 2015). Expression levels (horizontal axis) were plotted as means of log2 transformed mRNA copy numbers ± s.e.m. Red and green colors identify GABAergic (#14, #15 and #24) and glutamatergic clusters (#44 and #45) of hypothalamic neurons, which express Crh mRNA at levels exceeding 2× the s.e.m. *q < 0.05 (Wilcoxon rank-sum test corrected for multiple testing). (C) Among both GABA and glutamate neurons, subsets are endowed with Crh mRNAs. (D) CRH co-exists in Lhx6-GFP neurons (asterisks) particularly in the bed nucleus of stria terminalis and preoptic area. (E) Neurotransmitter heterogeneity of Crh+ neurons. Venn diagrams demonstrate the proportion of dual and triple neuronal phenotypes in GABA/glutamate (top), GABA/dopamine (middle) and GABA/glutamate/dopamine (bottom) neurons. This analysis was performed using a threshold for mRNA expression at a level of ≥2 mRNA transcripts for each gene. Percentage values indicate the proportion of Crh+ neurons falling into groups and intersections (‘dual phenotype’ categories). Scale bars = 250 µm (A), 10 µm (D). Adapted, with permission, from Romanov et al. (2016).

Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0256

Exquisite co-localization studies suggest that CRH-positive(+) neurons could mediate either excitatory or inhibitory neurotransmission in a regionally defined manner. For excitatory CRH+ neurons, those of the bed nucleus of stria terminalis are considered as prime candidates (Lee & Davis 1997, McNally & Akil 2002, Hrabovszky et al. 2005). Likewise, CRH+ cells of the central nucleus of the amygdala participate in extended excitatory circuits in which these neurons might potentiate differential responses to tonic/unpredictable vs phasic/predictable stressors is currently under debate (Lee & Davis 1997, Palkovits 2000, McNally & Akil 2002), even including peripheral responses such as the regulation of heart rate (Nijsen et al. 2001). In turn, cortical and hippocampal interneurons might also express CRH, which use GABA as inhibitory neurotransmitter (Zeisel et al. 2015, Hooper & Maguire 2016). Nevertheless, CRH actions are chiefly diversified at the subcellular level as its two G-protein coupled receptors (with Gs/Gq coupling), CRHR1 (Crhr1) and CRHR2 (Crhr2) can differentially signal in response to this neuropeptide (Dautzenberg et al. 2001, Dautzenberg & Hauger 2002, Aguilera et al. 2004, Gutknecht et al. 2010).

ScRNA-seq-based subclassification of Crh mRNA+ hypothalamic neurons

The classical doctrine of hypothalamic stress induction posits CRH neurons being parvocellular neurosecretory cells situated in the PVN in mammals (Swanson et al. 1986, Simmons & Swanson 2009). However, ‘why’, ‘when’ and ‘for how long’ other neurons (GFP+; Fig. 1A) beyond those in the PVN produce CRH and at which biophysical thresholds and synaptic sites release their CRH contents are much less unequivocal. Among the many reasons for this ambiguity at the molecular regulatory levels, we emphasize the rapid induction of CRH production (Watts et al. 1999) under certain metabolic demands. This would allow for CRH being re-classified as a permissive cellular feature underscoring functional competence and circuit recruitment rather than a cell-identity mark (we define a cell-identity mark as a molecular feature that determines the cell’s function, sets it apart from neighboring cells and whose loss might render the cell functionally incompetent and misplaces it in the molecular taxonomy of cells in a given brain volume. In contrast, functional modality refers to a molecular feature (gene, gene set and combinatorial code) that is only present under certain (patho)physiological conditions yet does not define either its neurotransmitter phenotype or connectivity pattern over long time scales. To test if CRH is an identity mark or a cell-state modality, we analyzed the level of heterogeneity of gene expression defining CRH signaling (that is, Crh, Crhr1, Crhr2 and Crhbp) in hypothalamic neurons (Fig. 2A)). If so, a combination of novel methods including scRNA-seq and in situ gene expression (Stahl et al. 2016) profiling could decode the heterogeneity of hypothalamic CRH+ neurons and allow for their subclassification based on the correlated analysis of fast neurotransmitter contents. In addition, one would expect these data be supportive of more conventional approaches, such as the intracerebroventricular injection of colchicine, an axonal transport inhibitor (Ceccatelli et al. 1991), which is widely used for the improved detection of neuropeptide expression (and also represents a substantial stress stimulus).

Figure 2
Figure 2

Molecular determinants of CRH signaling in the mouse hypothalamus. (A) Neuronal subclusters expressing CRH receptors (Crhr1 and Crhr2) and CRH-binding protein (Crhbp). Note that some cells in clusters #14 (GABA) and #44 (glutamate) can express at least one receptor and binding protein for CRH signaling. Red and green colors identify respective GABAergic and glutamatergic clusters, which express Crh mRNA at levels exceeding 2× the s.e.m. Note that significant levels of gene expression were found only for Crhr2 and Crhbp but not for Crhr1 within the hypothalamus (*q < 0.05, Wilcoxon rank-sum test corrected for multiple testing). (B) Secretagogin expression among hypothalamic neurons. Cluster #44 (glutamate) coexpresses both Scgn and Crh genes, and this cluster localizes to the PVN (Romanov et al. 2015). (C) Secretagogin expression in the hypothalamus of laboratory rodents and humans. In mouse, secretagogin co-exists with neither oxytocin nor vasopressin. In contrast, a subset of vasopressin+ and oxytocin+ neurons can co-express secretagogin in rat and human, respectively. Color code: secretagogin (red), vasopressin (green) and oxytocin (blue). Scale bars = 20 µm. (A and B) adapted, with permission, from Romanov et al. (2016).

Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0256

As such, many neuropeptides (beyond CRH in the PVN) are used as specific hallmarks of neuronal identity in the hypothalamus (e.g. neuropeptide Y, oxytocin, agouti-related peptide (AgRP)) (Meister et al. 1990, Lantos et al. 1995, Aponte et al. 2011, Dietrich et al. 2015), asserting that expression of a single gene (or at most their circumscribed subset) typifies a neuronal subtype. This knowledge can thus be used to justify recent developments in quantitative mRNA profiling tools, as any single-cell analysis, if correct, would lean toward neuronal identity marks that are stably expressed in a given contingent of neurons. Accordingly, the recent development of mathematical algorithms (t-distributed stochastic neighbor embedding (tSNE) and BackSpin clustering (van der Maaten & Hinton 2008, Islam et al. 2014, Macosko et al. 2015, Zeisel et al. 2015)) allows for the refined analysis of scRNA-seq data on hypothalamic neurons (Romanov et al. 2015, 2016). This approach confirmed that many neuropeptide mRNAs (Hcrt, Agrp, Npy and Trh) are indeed neuronal identity marks (Romanov et al. 2016) and once ensuing peptides are produced, their ‘use-dependent’ release will impinge upon a neuronal circuit to determine metabolic output (e.g. AgRP invariably present in a neuronal subset in the arcuate nucleus that controls body weight and metabolic rate (Mizuno et al. 2003)). In contrast, Crh mRNA expression fails to meet these criteria: t-SNE projections demonstrate widespread Crh expression in hypothalamic neurons, which lack subtype definition. In accord, BackSpin clustering characterized several neuronal subtypes (including clusters #14, 15, 44 and 45 as per Romanov et al. (2016)) that contain fractions of Crh mRNA-expressing neurons (Fig. 1B and C). Thus, we conclude that a neuron’s ability to produce CRH is a functional modality rather than an identity-defining feature.

If CRH expression is a functionally induced modality, then the diversity of fast neurotransmitter contents in hypothalamic neurons expressing this neuropeptide can also be expected (Fig. 1C). Our detailed analysis of all hypothalamic Crh neurons showed their segregation into either GABAergic (that is, Gad1, Gad2 and Slc32a1 co-expression) or glutamatergic (Slc17a6) neurotransmitter phenotypes. As neurotransmitter coexistence appears as a unique and relatively common feature of hypothalamic neurons (in 10–20% of cells as coincidently revealed by both Fluidigm C1 and Drop-seq transcriptomics) (Romanov et al. 2016), dual GABA/glutamate, dopamine/GABA and dopamine/glutamate neurons can also contain Crh mRNA (Fig. 1E). In sum, Crh+ neurons encompass several subtypes of GABA and glutamate neurons, reinforcing the concept that episodic CRH production can be associated with multiple network-driven, metabolic or disease-related contexts rather than being a feature of a developmentally coded subset of hypothalamic neurons (that is, phylogeny defined at the transcriptional level).

ScRNA-seq uncovered that CRH neurons residing in the PVN are glutamatergic. This cell cluster can be distinguished by the co-existence of several mRNA marks, including Tmem59l, Fuca1, Npr3 and Arnt2, and whose existence in the PVN is validated by open-source histochemical databases (see in situ hybridization, Allen Brain Atlas/brain-map.org). Meanwhile, GABAergic neurons that can contain CRH co-express Pgr15l, a histochemical mark mapped onto the anterior part of the PVN, cells spread around the histochemical boundaries of the PVN and those residing in the dorsomedial hypothalamus (Fig. 1C). Alternatively, GABAergic CRH neurons can co-express LIM homeobox 6 (Lhx6) transcription factor and map onto the preoptic area (Fig. 1D). Moreover, a continuum of Lhx6+/Crh+ neurons extends toward the bed nucleus of stria terminalis, which is an extrahypothalamic area with innate projections to, among other, the PVN where it forms local circuits regulating the stress axis (Choi et al. 2007). Based on their transcriptional code, we hypothesize that GABAergic CRH+ neurons, despite their spatial heterogeneity, can originate from the same precursors, migrate toward different (extra)hypothalamic subregions and ultimately be a unified neuronal subtype. An outstanding question based on novel scRNA-seq and localization data is the determination of when (context and mode of operation) and for how long (induction) any neuronal subclass can express physiologically meaningful levels of CRH protein and if their postsynaptic targets are endowed with CRH receptors for the efficient translation of CRH signals into functional outcome.

CRH receptors in the brain

Two genes (Crhr1 and Crhr2) encode receptors to mediate CRH action with their mRNAs detected in many brain regions (Wynn et al. 1984, Aguilera et al. 2004). Crhr1 is widely expressed in the neocortex, piriform cortex, olfactory system and cerebellum (Van Pett et al. 2000). Most recently, cortical and hippocampal Crhr1 mRNA expression was also validated by scRNA-seq (Zeisel et al. 2015), showing pronounced Crhr1 gene expression in pyramidal cells in cortical layers 2/3, 4 and 5. These findings contrast those in interneurons, which generally lack Crhr1 mRNA transcripts. Given that interneurons can instead express CRH itself, the combination of cell-type-specific transcriptomics with circuit mapping allows predictions on local CRH signaling between CRH+ interneurons and Crhr1-laden pyramidal cells. In situ hybridization demonstrates a more restricted distribution pattern for Crhr2 mRNA, with its primary concentration in lateral and triangular nuclei of the septum and the amygdaloid complex. Thus, CRHR1 and CRHR2 functions might mediate vastly different biological responses and involve a combination of short- and long-range modes of intercellular signaling.

When analyzing CRH receptor distribution in the hypothalamus, expression levels are relatively low for both Crhr1 and Crhr2 with the former being present in arcuate, suprachiasmatic, anterior and dorsomedial hypothalamic nuclei (Van Pett et al. 2000), whereas the latter is distributed across anterior periventricular, ventromedial, arcuate and anterior hypothalamic nuclei (Van Pett et al. 2000). Notably, stress induces Crhr1 expression in the PVN, which is a significant feedback station for sustained CRH production and release (Makino et al. 1995, Aguilera et al. 2004).

Recently, we sought to address the precise cellular distribution of neurons that can respond to CRH as a neuromodulator locally in the hypothalamus (Romanov et al. 2016). Although ~10% of hypothalamic neurons express Crh mRNA (>1 mRNA copy), Crhr1 and Crhr2 were only sparsely present in hypothalamic neurons (4.5% and 2.6% of 898 neurons, respectively) at unexpectedly low copy numbers. The following steps of caution are warranted when considering these data: (i) low levels of Crhr1 mRNA detection (Hata et al. 1993, Sollner et al. 1993, Sutton et al. 1998, Pang & Sudhof 2010) might lead to false negatives (missed fraction of neurons), (ii) mRNA-to-protein translation and the stability of ensuing receptor complexes and their effectors (Fig. 2A) might diversify the efficacy of signal transduction and (iii) induced expression (Aguilera et al. 2004) could grossly affect CRH-responsive hypothalamic neuronal networks. Yet, our comparison of scRNA-seq data from hypothalamic and cortical datasets (Romanov et al. 2015, Zeisel et al. 2015) produced on identical platforms suggests that CRH+ efferents commonly form synapses outside the hypothalamus and/or CRH is mainly released into the ventricular and hypophyseal portal systems. Conversely, the likelihood of local-circuit neuronal ensembles using CRH as neuromodulator at rest is relatively modest. An exception to the above rules might be an autocrine role for CRH in the PVN as these neurons express both Crh and Crhbp (CRH binding protein) mRNAs with Crhbp inactivating CRH action (Dautzenberg & Hauger 2002, Seasholtz et al. 2002). Cumulatively, these data suggest a dichotomy of CRH content and CRH-responsive neuronal substrates within the hypothalamus and at extrahypothalamic sites.

Secretagogin controls CRH release at the median eminence

The regulated release of any bioactive peptide within the brain or at the median eminence relies on the coordinated assembly of a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) complex, allowing for the priming, docking and fusion of neuropeptide-filled large dense-core vesicles (LDCVs) (Hata et al. 1993, Sollner et al. 1993, Sutton et al. 1998, Pang & Sudhof 2010). In the hypothalamus, the exocytosis machinery includes many region- and cell-type-specific proteins, such as CAPS-1 and secretogranins (Miyazaki et al. 2011, Tobin et al. 2012). Until recently, our understanding of the molecular underpinnings of CRH release was limited. Recently, a focused scRNA-seq study (Romanov et al. 2015) in the mouse PVN identified the coincident presence of Crh, Nr3c1 (glucocorticoid receptor, subfamily 3, group C member 1) (van Rossum et al. 2004) and secretagogin (Scgn) (Romanov et al. 2015) (Fig. 2B and C). These data were validated by light and electron microscopy in both the PVN and the median eminence, localizing secretagogin to membrane surfaces at terminals releasing CRH into the hypophyseal portal system (Romanov et al. 2015) (Fig. 3B and C), anatomically tying secretagogin to the machinery priming CRH release.

Figure 3
Figure 3

Secretagogin affects hormone secretion. (A) Secretagogin knockdown in INS-1E insulinoma cells limits insulin release. (B and C) Secretagogin co-localizes with CRH at the median eminence (B) and accumulates as a membrane-associated synaptic component at the ultrastructural level (C). (D) Plasma ACTH levels 12 min after formalin (4%) injection into a paw of conscious adult mice. Red color: secretagogin knockdown in vivo. Note that secretagogin deletion occludes ACTH release. *, P < 0.05 vs. formalin-stressed controls. (E) The scheme showing involvement of secretagogin in hypothalamic CRH release, including potential protein-protein interactions. Scale bars = 10 µm (B) and 500 nm (C). Reproduced, with permission, from Romanov et al. (2015).

Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0256

Secretagogin is an appealing subject of study as it is a member of the EF-hand superfamily of Ca2+-binding proteins, expressed in all organ systems typically associated with neuroendocrine cells (Wagner et al. 2000, Adolf et al. 2007, Alpar et al. 2012), including the pituitary, adrenal gland, gut and pancreas (Wagner et al. 2000, Hasegawa et al. 2013). In the nervous system, secretagogin is localized to neurons populating the neocortex, hippocampus, cerebellum, basal ganglia, rostral migratory stream and olfactory bulb (Attems et al. 2008, Mulder et al. 2009, 2010, Gati et al. 2014). Secretagogin is a Ca2+ sensor protein ([Ca2+]0.5 is of ~25 µM for secretagogin in physiological salt buffers), which by definition induces protein–protein interactions through conformational change upon Ca2+ binding followed by downstream signaling to control discrete cellular functions (Schwaller 2010). Secretagogin’s in vitro interactome initially included synaptosomal-associated protein 25 kDa (SNAP-25) (Rogstam et al. 2007) with the more recent discovery of vesicle cargo, traffic and docking/release proteins (Bauer et al. 2011a,b, Romanov et al. 2015). Particularly, the 5th EF-hand domain can interact with cytoskeletal components (microtubules) (Maj et al. 2010, Yang et al. 2016). Thus, secretagogin possesses basic properties of an integrative sensor that can orchestrate the release of neuropeptide-containing LDCVs.

In the hypothalamus, secretagogin is chiefly expressed in the PVN and arcuate nuclei (Mulder et al. 2009, Romanov et al. 2015). Even though secretagogin can partially co-exist with vasopressin and oxytocin in some mammals (Fig. 2C), the majority of secretagogin+ neurons lacks any appreciable neuropeptide/hormone co-labeling in resting mice (Romanov et al. 2015). This constellation led to the detailed study of secretagogin+ neurons and their molecular interrogation by correlated transcriptomics and proteomics. Notably, converging genetic and histochemical evidence suggests that secretagogin is a constitutive mark of stress-activated CRH+ neurons that gate ACTH release from the pituitary (Romanov et al. 2015). Acute silencing of secretagogin expression (by siRNA) in vivo and in vitro provided critical insights into secretagogin’s function (Hasegawa et al. 2013, Romanov et al. 2015): (i) secretagogin knockdown induces CRH accumulation in the PVN. This suggests that secretagogin deficiency might limit the translocation of CRH to release sites and its Ca2+-dependent release into the portal circulation and (ii) concordantly, ACTH (Cam & Bassett 1983) and corticosterone production are blunted after secretagogin knockdown in animals exposed to acute formalin stress (Romanov et al. 2015) (Fig. 3D). These data also provide genetic evidence on a selective parvocellular Ca2+-dependent gatekeeper to the HPA axis whose function underpins a peripheral corticosterone surge from the adrenal cortex (Makara et al 1981).

Even though secretagogin is broadly recognized to scale hormone release (Wagner et al. 2000, Hasegawa et al. 2013, Romanov et al. 2015) (Fig. 3A), its exact interacting partners in neurons that might prime axonal transport and docking secretory vesicles remained unknown until recently. Using subtractive proteomics comparing Ca2+-saturated and Ca2+-free conditions and ‘reverse’ transcriptomics to verify the expression of co-purifying proteins in parvocellular neurons, we have defined protein subfamilies essential for the formation, intracellular traffic, priming and docking of release vesicles (examples: α-SNAP, Rab family members, syntaxins, synaptophysin, TMED10 and CAPS1), and some of them are specific to hypothalamic neurons (Fig. 3E) and release mechanisms (e.g. CAPS-1) (Tobin et al. 2012). In accord, ultrastructural data localized secretagogin to large axon terminals at the median eminence and substantiated its association to the readily releasable pool of vesicles, as well as plasmalemmal compartments. Because CRH, as many other peptide hormones released at the median eminence, is neither reused nor recycled after entering the blood stream, we hypothesize that impaired CRH secretion into the portal blood upon genetic secretagogin ablation impairs not one but many regulated Ca2+-sensitive intracellular checkpoints in parvocellular neurons (Fig. 3E). Thus, secretagogin qualifies as a molecular determinant rate-limiting CRH release.

Conclusions

Here, we collated recent molecular evidence highlighting the unprecedented molecular heterogeneity of hypothalamic neurons. The use of multiparametric experimental paradigms on the backdrop of array technologies provides unique precision and analytical power to distinguish neuronal modalities, let these be graded identity differences or temporal recruitment to neuronal circuits. Besides CRH neurons, novel levels of evidence emerge on the molecular heterogeneity of those affecting food intake (Henry et al. 2015), hedonism (amygdala), multisensory processing (e.g. association of vision and smell) (Macosko et al. 2015, Saraiva et al. 2015) and conscious representation of metabolic demands (cortex) (Zeisel et al. 2015). Thus, an unexpected and rapid expansion of concepts substantially refining and sometimes revising decade-long theories might be expected, allowing multimodal integration of information flow and refinement at extra- and intrahypothalamic circuits. The advent of deciphering the molecular heterogeneity of CRH neurons already produced novel insights into permissive checkpoints of the release of this hormone, as well as the parsing of probable subclasses of CRH neurons. Accordingly, CRH belongs primarily to ‘functional modality’ rather than ‘cell-identity’ marks. Future studies dissecting the upstream control of CRH neurons classically not considered ‘stress effectors’ in the hypothalamus, the metabolic contexts prompting their CRH production and if CRH receptors are coincidently expressed at synaptically wired circuit components and if receptor-level switches in decoding the physiological gain of CRH signaling exists will undoubtedly fuel the establishment of novel organizing principles of hypothalamic neuronal diversity.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This work was supported by the Swedish Research Council (T Ha, T Hö), Hjärnfonden (T Ha), the Novo Nordisk Foundation (T Ha, T Hö), the European Research Council (Secret-Cells; T Ha), the National Brain Research Program of Hungary (MTA-SE NAP B, KTIA_NAP_13-2014-0013; A A) and intramural funds of the Medical University of Vienna (T Ha). R A R was supported by the European Molecular Biology Organization (ALTF reference 596-2014) and EU FP7 program (Marie Curie Actions EMBOCOFUND2012, grant GA-2012-600394).

Acknowledgements

The authors thank Dr A Zeisel, Dr S Linnarsson (Karolinska Institutet), Dr M Farlik and Dr C Bock (CeMM, Austrian Academy of Sciences) for their initial contributions to single-cell RNA-sequencing and Dr J S Bains (The Hotchkiss Brain Institute, University of Calgary, Calgary, Canada) for CRH-Ires-Cre mice.

References

  • Adolf K, Wagner L, Bergh A, Stattin P, Ottosen P, Borre M, Birkenkamp-Demtroder K, Orntoft TF & Torring N 2007 Secretagogin is a new neuroendocrine marker in the human prostate. Prostate 67 472484. (doi:10.1002/pros.20523)

    • Search Google Scholar
    • Export Citation
  • Aguilera G, Nikodemova M, Wynn PC & Catt KJ 2004 Corticotropin releasing hormone receptors: two decades later. Peptides 25 319329. (doi:10.1016/j.peptides.2004.02.002)

    • Search Google Scholar
    • Export Citation
  • Alon T, Zhou L, Perez CA, Garfield AS, Friedman JM & Heisler LK 2009 Transgenic mice expressing green fluorescent protein under the control of the corticotropin-releasing hormone promoter. Endocrinology 150 56265632. (doi:10.1210/en.2009-0881)

    • Search Google Scholar
    • Export Citation
  • Alonso G, Szafarczyk A & Assenmacher I 1986 Immunoreactivity of hypothalamo-neurohypophysial neurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp): immunocytochemical evidence for a correlation with their functional state in colchicine-treated rats. Experimental Brain Research 61 497505. (doi:10.1007/bf00237574)

    • Search Google Scholar
    • Export Citation
  • Alpar A, Attems J, Mulder J, Hokfelt T & Harkany T 2012 The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage. Cellular Signalling 24 378387. (doi:10.1016/j.cellsig.2011.09.028)

    • Search Google Scholar
    • Export Citation
  • Aponte Y, Atasoy D & Sternson SM 2011 AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nature Neuroscience 14 351355. (doi:10.1038/nn.2739)

    • Search Google Scholar
    • Export Citation
  • Attems J, Preusser M, Grosinger-Quass M, Wagner L, Lintner F & Jellinger K 2008 Calcium-binding protein secretagogin-expressing neurones in the human hippocampus are largely resistant to neurodegeneration in Alzheimer’s disease. Neuropathology and Applied Neurobiology 34 2332. (doi:10.1111/j.1365-2990.2007.00854.x)

    • Search Google Scholar
    • Export Citation
  • Bauer M, Maj M, Wagner L, Cahill DJ, Linse S & O’Connell DJ 2011a Protein networks involved in vesicle fusion, transport, and storage revealed by array-based proteomics. Methods in Molecular Biology 781 4758. (doi:10.1007/978-1-61779-276-2_3)

    • Search Google Scholar
    • Export Citation
  • Bauer MC, O’Connell DJ, Maj M, Wagner L, Cahill DJ & Linse S 2011b Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion. Molecular BioSystems 7 21962204. (doi:10.1039/c0mb00349b)

    • Search Google Scholar
    • Export Citation
  • Ben-Jonathan N & Hnasko R 2001 Dopamine as a prolactin (PRL) inhibitor. Endocrine Reviews 22 724763. (doi:10.1210/edrv.22.6.0451)

  • Cam GR & Bassett JR 1983 The plasma levels of ACTH following exposure to stress or nicotine. Archives Internationales de Pharmacodynamie Et de Therapie 264 154167.

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Eriksson M & Hokfelt T 1989a Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus. Neuroendocrinology 49 309323. (doi:10.1159/000125133)

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Villar MJ, Goldstein M & Hokfelt T 1989b Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress. PNAS 86 95699573. (doi:10.1073/pnas.86.23.9569)

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Cortes R & Hokfelt T 1991 Effect of reserpine and colchicine on neuropeptide mRNA levels in the rat hypothalamic paraventricular nucleus. Brain Research: Molecular Brain Research 9 5769. (doi:10.1016/0169-328X(91)90130-P)

    • Search Google Scholar
    • Export Citation
  • Choi DC, Furay AR, Evanson NK, Ostrander MM, Ulrich-Lai YM & Herman JP 2007 Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic-pituitary-adrenal axis activity: implications for the integration of limbic inputs. Journal of Neuroscience 27 20252034. (doi:10.1523/JNEUROSCI.4301-06.2007)

    • Search Google Scholar
    • Export Citation
  • Daftary SS, Boudaba C, Szabo K & Tasker JG 1998 Noradrenergic excitation of magnocellular neurons in the rat hypothalamic paraventricular nucleus via intranuclear glutamatergic circuits. Journal of Neuroscience 18 1061910628.

    • Search Google Scholar
    • Export Citation
  • Dautzenberg FM & Hauger RL 2002 The CRF peptide family and their receptors: yet more partners discovered. Trends in Pharmacological Sciences 23 7177. (doi:10.1016/S0165-6147(02)01946-6)

    • Search Google Scholar
    • Export Citation
  • Dautzenberg FM, Py-Lang G, Higelin J, Fischer C, Wright MB & Huber G 2001 Different binding modes of amphibian and human corticotropin-releasing factor type 1 and type 2 receptors: evidence for evolutionary differences. Journal of Pharmacology and Experimental Therapeutics 296 113120.

    • Search Google Scholar
    • Export Citation
  • Dietrich MO, Zimmer MR, Bober J & Horvath TL 2015 Hypothalamic Agrp neurons drive stereotypic behaviors beyond feeding. Cell 160 12221232. (doi:10.1016/j.cell.2015.02.024)

    • Search Google Scholar
    • Export Citation
  • Drescher VS, Chen HL & Romsos DR 1994 Corticotropin-releasing hormone decreases feeding, oxygen consumption and activity of genetically obese (ob/ob) and lean mice. Journal of Nutrition 124 524530.

    • Search Google Scholar
    • Export Citation
  • Du Vigneaud V 1954 Hormones of the posterior pituitary gland: oxytocin and vasopressin. Harvey Lectures 50 126.

  • Fuzesi T, Daviu N, Wamsteeker Cusulin JI, Bonin RP & Bains JS 2016 Hypothalamic CRH neurons orchestrate complex behaviours after stress. Nature Communications 7 11937. (doi:10.1038/ncomms11937)

    • Search Google Scholar
    • Export Citation
  • Gati G, Lendvai D, Hokfelt T, Harkany T & Alpar A 2014 Revival of calcium-binding proteins for neuromorphology: secretagogin typifies distinct cell populations in the avian brain. Brain Behavior and Evolution 83 8292.

    • Search Google Scholar
    • Export Citation
  • Gillies GE, Linton EA & Lowry PJ 1982 Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299 355357. (doi:10.1038/299355a0)

    • Search Google Scholar
    • Export Citation
  • Girotti M, Weinberg MS & Spencer RL 2009 Diurnal expression of functional and clock-related genes throughout the rat HPA axis: system-wide shifts in response to a restricted feeding schedule. American Journal of Physiology: Endocrinology and Metabolism 296 E888E897. (doi:10.1152/ajpendo.90946.2008)

    • Search Google Scholar
    • Export Citation
  • Gold PW, Licinio J, Wong ML & Chrousos GP 1995 Corticotropin releasing hormone in the pathophysiology of melancholic and atypical depression and in the mechanism of action of antidepressant drugs. Annals of the New York Academy of Sciences 771 716729. (doi:10.1111/j.1749-6632.1995.tb44723.x)

    • Search Google Scholar
    • Export Citation
  • Green JD & Harris GW 1947 The neurovascular link between the neurohypophysis and adenohypophysis. Journal of Endocrinology 5 136146. (doi:10.1677/joe.0.0050136)

    • Search Google Scholar
    • Export Citation
  • Guillemin R 1978 Peptides in the brain: the new endocrinology of the neuron. Science 202 390402. (doi:10.1126/science.212832)

  • Gustafsson JA, Carlstedt-Duke J, Poellinger L, Okret S, Wikstrom AC, Bronnegard M, Gillner M, Dong Y, Fuxe K & Cintra A 1987 Biochemistry, molecular biology, and physiology of the glucocorticoid receptor. Endocrine Reviews 8 185234. (doi:10.1210/edrv-8-2-185)

    • Search Google Scholar
    • Export Citation
  • Gutknecht E, Vauquelin G & Dautzenberg FM 2010 Corticotropin-releasing factor receptors induce calcium mobilization through cross-talk with Gq-coupled receptors. European Journal of Pharmacology 642 19. (doi:10.1016/j.ejphar.2010.05.027)

    • Search Google Scholar
    • Export Citation
  • Hasegawa K, Wakino S, Kimoto M, Minakuchi H, Fujimura K, Hosoya K, Komatsu M, Kaneko Y, Kanda T & Tokuyama H et al. 2013 The hydrolase DDAH2 enhances pancreatic insulin secretion by transcriptional regulation of secretagogin through a Sirt1-dependent mechanism in mice. FASEB Journal 27 23012315. (doi:10.1096/fj.12-226092)

    • Search Google Scholar
    • Export Citation
  • Hata Y, Slaughter CA & Sudhof TC 1993 Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366 347351. (doi:10.1038/366347a0)

    • Search Google Scholar
    • Export Citation
  • Henry FE, Sugino K, Tozer A, Branco T & Sternson SM 2015 Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss. eLife 4 e09800. (doi:10.7554/elife.09800)

    • Search Google Scholar
    • Export Citation
  • Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC & Cullinan WE 2003 Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Frontiers in Neuroendocrinology 24 151180. (doi:10.1016/j.yfrne.2003.07.001)

    • Search Google Scholar
    • Export Citation
  • Hooper A & Maguire J 2016 Characterization of a novel subtype of hippocampal interneurons that express corticotropin-releasing hormone. Hippocampus 26 4153. (doi:10.1002/hipo.22487)

    • Search Google Scholar
    • Export Citation
  • Hrabovszky E, Wittmann G, Turi GF, Liposits Z & Fekete C 2005 Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone neurons of the rat contain vesicular glutamate transporter-2. Endocrinology 146 341347. (doi:10.1210/en.2004-0856)

    • Search Google Scholar
    • Export Citation
  • Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, Lonnerberg P & Linnarsson S 2014 Quantitative single-cell RNA-seq with unique molecular identifiers. Nature Methods 11 163166. (doi:10.1038/nmeth.2772)

    • Search Google Scholar
    • Export Citation
  • Kahaly GJ & Hansen MP 2016 Type 1 diabetes associated autoimmunity. Autoimmunity Reviews 15 644648. (doi:10.1016/j.autrev.2016.02.017)

  • Keegan CE, Herman JP, Karolyi IJ, O’Shea KS, Camper SA & Seasholtz AF 1994 Differential expression of corticotropin-releasing hormone in developing mouse embryos and adult brain. Endocrinology 134 25472555. (doi:10.1210/en.134.6.2547)

    • Search Google Scholar
    • Export Citation
  • Kiss JZ, Mezey E & Skirboll L 1984 Corticotropin-releasing factor-immunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. PNAS 81 18541858. (doi:10.1073/pnas.81.6.1854)

    • Search Google Scholar
    • Export Citation
  • Krashes MJ, Shah BP, Madara JC, Olson DP, Strochlic DE, Garfield AS, Vong L, Pei H, Watabe-Uchida M & Uchida N et al. 2014 An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507 238242. (doi:10.1038/nature12956)

    • Search Google Scholar
    • Export Citation
  • Lantos TA, Gorcs TJ & Palkovits M 1995 Immunohistochemical mapping of neuropeptides in the premamillary region of the hypothalamus in rats. Brain Research: Brain Research Reviews 20 209249. (doi:10.1016/0165-0173(94)00013-F)

    • Search Google Scholar
    • Export Citation
  • Lee Y & Davis M 1997 Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropin-releasing hormone on the acoustic startle reflex. Journal of Neuroscience 17 64346446.

    • Search Google Scholar
    • Export Citation
  • Lloyd RB & Nemeroff CB 2011 The role of corticotropin-releasing hormone in the pathophysiology of depression: therapeutic implications. Current Topics in Medicinal Chemistry 11 609617. (doi:10.2174/1568026611109060609)

    • Search Google Scholar
    • Export Citation
  • Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N & Martersteck EM et al. 2015 Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161 12021214. (doi:10.1016/j.cell.2015.05.002)

    • Search Google Scholar
    • Export Citation
  • Mains RE, Eipper BA & Ling N 1977 Common precursor to corticotropins and endorphins. PNAS 74 30143018. (doi:10.1073/pnas.74.7.3014)

  • Maj M, Gartner W, Ilhan A, Neziri D, Attems J & Wagner L 2010 Expression of TAU in insulin-secreting cells and its interaction with the calcium-binding protein secretagogin. Journal of Endocrinology 205 2536. (doi:10.1677/JOE-09-0341)

    • Search Google Scholar
    • Export Citation
  • Makara GB, Stark E, Karteszi M, Palkovits M & Rappay G 1981 Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat. American Journal of Physiology 240 E441E446.

    • Search Google Scholar
    • Export Citation
  • Makino S, Schulkin J, Smith MA, Pacak K, Palkovits M & Gold PW 1995 Regulation of corticotropin-releasing hormone receptor messenger ribonucleic acid in the rat brain and pituitary by glucocorticoids and stress. Endocrinology 136 45174525. (doi:10.1210/en.136.10.4517)

    • Search Google Scholar
    • Export Citation
  • McEwen BS 2007 Physiology and neurobiology of stress and adaptation: central role of the brain. Physiological Reviews 87 873904. (doi:10.1152/physrev.00041.2006)

    • Search Google Scholar
    • Export Citation
  • McEwen BS 2012 The ever-changing brain: cellular and molecular mechanisms for the effects of stressful experiences. Developmental Neurobiology 72 878890. (doi:10.1002/dneu.20968)

    • Search Google Scholar
    • Export Citation
  • McEwen BS & Stellar E 1993 Stress and the individual. Mechanisms leading to disease. Archives of Internal Medicine 153 20932101. (doi:10.1001/archinte.1993.00410180039004)

    • Search Google Scholar
    • Export Citation
  • McNally GP & Akil H 2002 Role of corticotropin-releasing hormone in the amygdala and bed nucleus of the stria terminalis in the behavioral, pain modulatory, and endocrine consequences of opiate withdrawal. Neuroscience 112 605617. (doi:10.1016/S0306-4522(02)00105-7)

    • Search Google Scholar
    • Export Citation
  • Meister B, Cortes R, Villar MJ, Schalling M & Hokfelt T 1990 Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels. Cell and Tissue Research 260 279297. (doi:10.1007/BF00318631)

    • Search Google Scholar
    • Export Citation
  • Merchenthaler I, Vigh S, Petrusz P & Schally AV 1982 Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain. American Journal of Anatomy 165 385396. (doi:10.1002/aja.1001650404)

    • Search Google Scholar
    • Export Citation
  • Merchenthaler I, Vigh S, Petrusz P & Schally AV 1983 The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats. Regulatory Peptides 5 295305. (doi:10.1016/0167-0115(83)90287-2)

    • Search Google Scholar
    • Export Citation
  • Miller DB & O’Callaghan JP 2002 Neuroendocrine aspects of the response to stress. Metabolism 51 510. (doi:10.1053/meta.2002.33184)

  • Miyazaki T, Yamasaki M, Uchigashima M, Matsushima A & Watanabe M 2011 Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum. European Journal of Neuroscience 33 8294. (doi:10.1111/j.1460-9568.2010.07472.x)

    • Search Google Scholar
    • Export Citation
  • Mizuno TM, Makimura H & Mobbs CV 2003 The physiological function of the agouti-related peptide gene: the control of weight and metabolic rate. Annals of Medicine 35 425433. (doi:10.1080/07853890310012076)

    • Search Google Scholar
    • Export Citation
  • Mulder J, Zilberter M, Spence L, Tortoriello G, Uhlen M, Yanagawa Y, Aujard F, Hokfelt T & Harkany T 2009 Secretagogin is a Ca2+-binding protein specifying subpopulations of telencephalic neurons. PNAS 106 2249222497. (doi:10.1073/pnas.0912484106)

    • Search Google Scholar
    • Export Citation
  • Mulder J, Spence L, Tortoriello G, Dinieri JA, Uhlen M, Shui B, Kotlikoff MI, Yanagawa Y, Aujard F & Hokfelt T et al. 2010 Secretagogin is a Ca2+-binding protein identifying prospective extended amygdala neurons in the developing mammalian telencephalon. European Journal of Neuroscience 31 21662177. (doi:10.1111/j.1460-9568.2010.07275.x)

    • Search Google Scholar
    • Export Citation
  • Munck A, Guyre PM & Holbrook NJ 1984 Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrine Reviews 5 2544. (doi:10.1210/edrv-5-1-25)

    • Search Google Scholar
    • Export Citation
  • Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN & Numa S 1979 Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature 278 423427. (doi:10.1038/278423a0)

    • Search Google Scholar
    • Export Citation
  • Napier C & Pearce SH 2014 Current and emerging therapies for Addison’s disease. Current Opinion in Endocrinology, Diabetes and Obesity 21 147153. (doi:10.1097/MED.0000000000000067)

    • Search Google Scholar
    • Export Citation
  • Nijsen MJ, Croiset G, Diamant M, De Wied D & Wiegant VM 2001 CRH signalling in the bed nucleus of the stria terminalis is involved in stress-induced cardiac vagal activation in conscious rats. Neuropsychopharmacology 24 110. (doi:10.1016/S0893-133X(00)00167-6)

    • Search Google Scholar
    • Export Citation
  • Owens MJ, Bartolome J, Schanberg SM & Nemeroff CB 1990 Corticotropin-releasing factor concentrations exhibit an apparent diurnal rhythm in hypothalamic and extrahypothalamic brain regions: differential sensitivity to corticosterone. Neuroendocrinology 52 626631. (doi:10.1159/000125655)

    • Search Google Scholar
    • Export Citation
  • Palkovits M 2000 Stress-induced expression of co-localized neuropeptides in hypothalamic and amygdaloid neurons. European Journal of Pharmacology 405 161166. (doi:10.1016/S0014-2999(00)00549-5)

    • Search Google Scholar
    • Export Citation
  • Pang ZP & Sudhof TC 2010 Cell biology of Ca2+-triggered exocytosis. Current Opinion in Cell Biology 22 496505. (doi:10.1016/j.ceb.2010.05.001)

    • Search Google Scholar
    • Export Citation
  • Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE & Vale W 1994 Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. PNAS 91 87778781. (doi:10.1073/pnas.91.19.8777)

    • Search Google Scholar
    • Export Citation
  • Reul JM & Holsboer F 2002 Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Current Opinion in Pharmacology 2 2333. (doi:10.1016/S1471-4892(01)00117-5)

    • Search Google Scholar
    • Export Citation
  • Rhodes CH, Morrell JI & Pfaff DW 1981 Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin. Journal of Comparative Neurology 198 4564. (doi:10.1002/cne.901980106)

    • Search Google Scholar
    • Export Citation
  • Rivier C & Vale W 1983 Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretion in vivo. Endocrinology 113 939942. (doi:10.1210/endo-113-3-939)

    • Search Google Scholar
    • Export Citation
  • Roberts JL & Herbert E 1977 Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. PNAS 74 48264830. (doi:10.1073/pnas.74.11.4826)

    • Search Google Scholar
    • Export Citation
  • Rogstam A, Linse S, Lindqvist A, James P, Wagner L & Berggard T 2007 Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin. Biochemical Journal 401 353363. (doi:10.1042/BJ20060918)

    • Search Google Scholar
    • Export Citation
  • Romanov RA, Alpar A, Zhang MD, Zeisel A, Calas A, Landry M, Fuszard M, Shirran SL, Schnell R & Dobolyi A et al. 2015 A secretagogin locus of the mammalian hypothalamus controls stress hormone release. EMBO Journal 34 3654. (doi:10.15252/embj.201488977)

    • Search Google Scholar
    • Export Citation
  • Romanov RA, Zeisel A, Bakker J, Girach F, Hellysaz A, Tomer R, Alpar A, Mulder J, Clotman F & Keimpema E et al. 2016 Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nature Neuroscience [in press]. (doi:10.1038/nn.4462)

    • Search Google Scholar
    • Export Citation
  • Salata RA, Jarrett DB, Verbalis JG & Robinson AG 1988 Vasopressin stimulation of adrenocorticotropin hormone (ACTH) in humans. In vivo bioassay of corticotropin-releasing factor (CRF) which provides evidence for CRF mediation of the diurnal rhythm of ACTH. Journal of Clinical Investigation 81 766774. (doi:10.1172/JCI113382)

    • Search Google Scholar
    • Export Citation
  • Sapolsky RM, Romero LM & Munck AU 2000 How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21 5589. (doi:10.1210/er.21.1.55)

    • Search Google Scholar
    • Export Citation
  • Saraiva LR, Ibarra-Soria X, Khan M, Omura M, Scialdone A, Mombaerts P, Marioni JC & Logan DW 2015 Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq. Scientific Reports 5 18178. (doi:10.1038/srep18178)

    • Search Google Scholar
    • Export Citation
  • Sawchenko PE, Swanson LW & Vale WW 1984 Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat. PNAS 81 18831887. (doi:10.1073/pnas.81.6.1883)

    • Search Google Scholar
    • Export Citation
  • Schally AV 1978 Aspects of hypothalamic regulation of the pituitary gland. Science 202 1828. (doi:10.1126/science.99816)

  • Schwaller B 2010 Cytosolic Ca2+ buffers. Cold Spring Harbor Perspectives in Biology 2 a004051. (doi:10.1101/cshperspect.a004051)

  • Seasholtz AF, Valverde RA & Denver RJ 2002 Corticotropin-releasing hormone-binding protein: biochemistry and function from fishes to mammals. Journal of Endocrinology 175 8997. (doi:10.1677/joe.0.1750089)

    • Search Google Scholar
    • Export Citation
  • Shapiro E, Biezuner T & Linnarsson S 2013 Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews Genetics 14 618630. (doi:10.1038/nrg3542)

    • Search Google Scholar
    • Export Citation
  • Simmons DM & Swanson LW 2009 Comparison of the spatial distribution of seven types of neuroendocrine neurons in the rat paraventricular nucleus: toward a global 3D model. Journal of Comparative Neurology 516 423441. (doi:10.1002/cne.22126)

    • Search Google Scholar
    • Export Citation
  • Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P & Rothman JE 1993 SNAP receptors implicated in vesicle targeting and fusion. Nature 362 318324. (doi:10.1038/362318a0)

    • Search Google Scholar
    • Export Citation
  • Spiess J, Rivier J, Rivier C & Vale W 1981 Primary structure of corticotropin-releasing factor from ovine hypothalamus. PNAS 78 65176521. (doi:10.1073/pnas.78.10.6517)

    • Search Google Scholar
    • Export Citation
  • Stahl PL, Salmen F, Vickovic S, Lundmark A, Navarro JF, Magnusson J, Giacomello S, Asp M, Westholm JO & Huss M et al. 2016 Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353 7882. (doi:10.1126/science.aaf2403)

    • Search Google Scholar
    • Export Citation
  • Sutton RB, Fasshauer D, Jahn R & Brunger AT 1998 Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395 347353. (doi:10.1038/26412)

    • Search Google Scholar
    • Export Citation
  • Swaab DF, Pool CW & Nijveldt F 1975 Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophypopseal system. Journal of Neural Transmission 36 195215. (doi:10.1007/BF01253126)

    • Search Google Scholar
    • Export Citation
  • Swanson LW 1991 Biochemical switching in hypothalamic circuits mediating responses to stress. Progress in Brain Research 87 181200.

  • Swanson LW & Kuypers HG 1980 The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. Journal of Comparative Neurology 194 555570. (doi:10.1002/cne.901940306)

    • Search Google Scholar
    • Export Citation
  • Swanson LW, Sawchenko PE, Rivier J & Vale WW 1983 Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36 165186. (doi:10.1159/000123454)

    • Search Google Scholar
    • Export Citation
  • Swanson LW, Sawchenko PE & Lind RW 1986 Regulation of multiple peptides in CRF parvocellular neurosecretory neurons: implications for the stress response. Progress in Brain Research 68 169190. (doi:10.1016/s0079-6123(08)60238-1)

    • Search Google Scholar
    • Export Citation
  • Tobin V, Schwab Y, Lelos N, Onaka T, Pittman QJ & Ludwig M 2012 Expression of exocytosis proteins in rat supraoptic nucleus neurones. Journal of Neuroendocrinology 24 629641. (doi:10.1111/j.1365-2826.2011.02237.x)

    • Search Google Scholar
    • Export Citation
  • Tramu G, Croix C & Pillez A 1983 Ability of the CRF immunoreactive neurons of the paraventricular nucleus to produce a vasopressin-like material. Immunohistochemical demonstration in adrenalectomized guinea pigs and rats. Neuroendocrinology 37 467469. (doi:10.1159/000123595)

    • Search Google Scholar
    • Export Citation
  • Turkelson CM, Thomas CR, Arimura A, Chang D, Chang JK & Shimizu M 1982 In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin. Peptides 3 111113. (doi:10.1016/0196-9781(82)90037-7)

    • Search Google Scholar
    • Export Citation
  • Uehara Y, Shimizu H, Ohtani K, Sato N & Mori M 1998 Hypothalamic corticotropin-releasing hormone is a mediator of the anorexigenic effect of leptin. Diabetes 47 890893. (doi:10.2337/diabetes.47.6.890)

    • Search Google Scholar
    • Export Citation
  • Vale W, Spiess J, Rivier C & Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213 13941397. (doi:10.1126/science.6267699)

    • Search Google Scholar
    • Export Citation
  • van der Maaten L & Hinton G 2008 Visualizing Data using t-SNE. Journal of Machine Learning Research 9 25792605.

  • Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, Prins GS, Perrin M, Vale W & Sawchenko PE 2000 Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. Journal of Comparative Neurology 428 191212. (doi:10.1002/1096-9861(20001211)428:2<191::AID-CNE1>3.0.CO;2-U)

    • Search Google Scholar
    • Export Citation
  • van Rossum EF, Voorhoeve PG, te Velde SJ, Koper JW, Delemarre-van de Waal HA, Kemper HC & Lamberts SW 2004 The ER22/23EK polymorphism in the glucocorticoid receptor gene is associated with a beneficial body composition and muscle strength in young adults. Journal of Clinical Endocrinology and Metabolism 89 40044009. (doi:10.1210/jc.2003-031422)

    • Search Google Scholar
    • Export Citation
  • Wagner L, Oliyarnyk O, Gartner W, Nowotny P, Groeger M, Kaserer K, Waldhausl W & Pasternack MS 2000 Cloning and expression of secretagogin, a novel neuroendocrine- and pancreatic islet of Langerhans-specific Ca2+-binding protein. Journal of Biological Chemistry 275 2474024751. (doi:10.1074/jbc.M001974200)

    • Search Google Scholar
    • Export Citation
  • Waters RP, Rivalan M, Bangasser DA, Deussing JM, Ising M, Wood SK, Holsboer F & Summers CH 2015 Evidence for the role of corticotropin-releasing factor in major depressive disorder. Neuroscience and Biobehavioral Reviews 58 6378. (doi:10.1016/j.neubiorev.2015.07.011)

    • Search Google Scholar
    • Export Citation
  • Watts AG, Sanchez-Watts G & Kelly AB 1999 Distinct patterns of neuropeptide gene expression in the lateral hypothalamic area and arcuate nucleus are associated with dehydration-induced anorexia. Journal of Neuroscience 19 61116121.

    • Search Google Scholar
    • Export Citation
  • Watts AG, Tanimura S & Sanchez-Watts G 2004 Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology 145 529540. (doi:10.1210/en.2003-0394)

    • Search Google Scholar
    • Export Citation
  • Whitnall MH, Mezey E & Gainer H 1985 Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles. Nature 317 248250. (doi:10.1038/317248a0)

    • Search Google Scholar
    • Export Citation
  • Wynn PC, Hauger RL, Holmes MC, Millan MA, Catt KJ & Aguilera G 1984 Brain and pituitary receptors for corticotropin releasing factor: localization and differential regulation after adrenalectomy. Peptides 5 10771084. (doi:10.1016/0196-9781(84)90174-8)

    • Search Google Scholar
    • Export Citation
  • Yang SY, Lee JJ, Lee JH, Lee K, Oh SH, Lim YM, Lee MS & Lee KJ 2016 Secretagogin impacts insulin secretion in pancreatic beta cells by regulating actin dynamics and focal adhesion. Biochemical Journal 473 17911803. (doi:10.1042/BCJ20160137)

    • Search Google Scholar
    • Export Citation
  • Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La Manno G, Jureus A, Marques S, Munguba H, He L & Betsholtz C et al. 2015 Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347 11381142. (doi:10.1126/science.aaa1934)

    • Search Google Scholar
    • Export Citation

 

Society for Endocrinology logo

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 87 87 41
PDF Downloads 95 95 59
  • View in gallery

    Molecular heterogeneity of Crh+ neurons in the hypothalamus. (A) Distribution of GFP+ neurons outside the PVN in the hypothalamus of Crh-IRES-Cre::GFP mice. (B) Single-cell RNA-seq distinguishes 62 neuronal subclasses along the PVN–arcuate axis of the hypothalamus (Romanov et al. 2015). Expression levels (horizontal axis) were plotted as means of log2 transformed mRNA copy numbers ± s.e.m. Red and green colors identify GABAergic (#14, #15 and #24) and glutamatergic clusters (#44 and #45) of hypothalamic neurons, which express Crh mRNA at levels exceeding 2× the s.e.m. *q < 0.05 (Wilcoxon rank-sum test corrected for multiple testing). (C) Among both GABA and glutamate neurons, subsets are endowed with Crh mRNAs. (D) CRH co-exists in Lhx6-GFP neurons (asterisks) particularly in the bed nucleus of stria terminalis and preoptic area. (E) Neurotransmitter heterogeneity of Crh+ neurons. Venn diagrams demonstrate the proportion of dual and triple neuronal phenotypes in GABA/glutamate (top), GABA/dopamine (middle) and GABA/glutamate/dopamine (bottom) neurons. This analysis was performed using a threshold for mRNA expression at a level of ≥2 mRNA transcripts for each gene. Percentage values indicate the proportion of Crh+ neurons falling into groups and intersections (‘dual phenotype’ categories). Scale bars = 250 µm (A), 10 µm (D). Adapted, with permission, from Romanov et al. (2016).

  • View in gallery

    Molecular determinants of CRH signaling in the mouse hypothalamus. (A) Neuronal subclusters expressing CRH receptors (Crhr1 and Crhr2) and CRH-binding protein (Crhbp). Note that some cells in clusters #14 (GABA) and #44 (glutamate) can express at least one receptor and binding protein for CRH signaling. Red and green colors identify respective GABAergic and glutamatergic clusters, which express Crh mRNA at levels exceeding 2× the s.e.m. Note that significant levels of gene expression were found only for Crhr2 and Crhbp but not for Crhr1 within the hypothalamus (*q < 0.05, Wilcoxon rank-sum test corrected for multiple testing). (B) Secretagogin expression among hypothalamic neurons. Cluster #44 (glutamate) coexpresses both Scgn and Crh genes, and this cluster localizes to the PVN (Romanov et al. 2015). (C) Secretagogin expression in the hypothalamus of laboratory rodents and humans. In mouse, secretagogin co-exists with neither oxytocin nor vasopressin. In contrast, a subset of vasopressin+ and oxytocin+ neurons can co-express secretagogin in rat and human, respectively. Color code: secretagogin (red), vasopressin (green) and oxytocin (blue). Scale bars = 20 µm. (A and B) adapted, with permission, from Romanov et al. (2016).

  • View in gallery

    Secretagogin affects hormone secretion. (A) Secretagogin knockdown in INS-1E insulinoma cells limits insulin release. (B and C) Secretagogin co-localizes with CRH at the median eminence (B) and accumulates as a membrane-associated synaptic component at the ultrastructural level (C). (D) Plasma ACTH levels 12 min after formalin (4%) injection into a paw of conscious adult mice. Red color: secretagogin knockdown in vivo. Note that secretagogin deletion occludes ACTH release. *, P < 0.05 vs. formalin-stressed controls. (E) The scheme showing involvement of secretagogin in hypothalamic CRH release, including potential protein-protein interactions. Scale bars = 10 µm (B) and 500 nm (C). Reproduced, with permission, from Romanov et al. (2015).

  • Adolf K, Wagner L, Bergh A, Stattin P, Ottosen P, Borre M, Birkenkamp-Demtroder K, Orntoft TF & Torring N 2007 Secretagogin is a new neuroendocrine marker in the human prostate. Prostate 67 472484. (doi:10.1002/pros.20523)

    • Search Google Scholar
    • Export Citation
  • Aguilera G, Nikodemova M, Wynn PC & Catt KJ 2004 Corticotropin releasing hormone receptors: two decades later. Peptides 25 319329. (doi:10.1016/j.peptides.2004.02.002)

    • Search Google Scholar
    • Export Citation
  • Alon T, Zhou L, Perez CA, Garfield AS, Friedman JM & Heisler LK 2009 Transgenic mice expressing green fluorescent protein under the control of the corticotropin-releasing hormone promoter. Endocrinology 150 56265632. (doi:10.1210/en.2009-0881)

    • Search Google Scholar
    • Export Citation
  • Alonso G, Szafarczyk A & Assenmacher I 1986 Immunoreactivity of hypothalamo-neurohypophysial neurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp): immunocytochemical evidence for a correlation with their functional state in colchicine-treated rats. Experimental Brain Research 61 497505. (doi:10.1007/bf00237574)

    • Search Google Scholar
    • Export Citation
  • Alpar A, Attems J, Mulder J, Hokfelt T & Harkany T 2012 The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage. Cellular Signalling 24 378387. (doi:10.1016/j.cellsig.2011.09.028)

    • Search Google Scholar
    • Export Citation
  • Aponte Y, Atasoy D & Sternson SM 2011 AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nature Neuroscience 14 351355. (doi:10.1038/nn.2739)

    • Search Google Scholar
    • Export Citation
  • Attems J, Preusser M, Grosinger-Quass M, Wagner L, Lintner F & Jellinger K 2008 Calcium-binding protein secretagogin-expressing neurones in the human hippocampus are largely resistant to neurodegeneration in Alzheimer’s disease. Neuropathology and Applied Neurobiology 34 2332. (doi:10.1111/j.1365-2990.2007.00854.x)

    • Search Google Scholar
    • Export Citation
  • Bauer M, Maj M, Wagner L, Cahill DJ, Linse S & O’Connell DJ 2011a Protein networks involved in vesicle fusion, transport, and storage revealed by array-based proteomics. Methods in Molecular Biology 781 4758. (doi:10.1007/978-1-61779-276-2_3)

    • Search Google Scholar
    • Export Citation
  • Bauer MC, O’Connell DJ, Maj M, Wagner L, Cahill DJ & Linse S 2011b Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion. Molecular BioSystems 7 21962204. (doi:10.1039/c0mb00349b)

    • Search Google Scholar
    • Export Citation
  • Ben-Jonathan N & Hnasko R 2001 Dopamine as a prolactin (PRL) inhibitor. Endocrine Reviews 22 724763. (doi:10.1210/edrv.22.6.0451)

  • Cam GR & Bassett JR 1983 The plasma levels of ACTH following exposure to stress or nicotine. Archives Internationales de Pharmacodynamie Et de Therapie 264 154167.

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Eriksson M & Hokfelt T 1989a Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus. Neuroendocrinology 49 309323. (doi:10.1159/000125133)

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Villar MJ, Goldstein M & Hokfelt T 1989b Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress. PNAS 86 95699573. (doi:10.1073/pnas.86.23.9569)

    • Search Google Scholar
    • Export Citation
  • Ceccatelli S, Cortes R & Hokfelt T 1991 Effect of reserpine and colchicine on neuropeptide mRNA levels in the rat hypothalamic paraventricular nucleus. Brain Research: Molecular Brain Research 9 5769. (doi:10.1016/0169-328X(91)90130-P)

    • Search Google Scholar
    • Export Citation
  • Choi DC, Furay AR, Evanson NK, Ostrander MM, Ulrich-Lai YM & Herman JP 2007 Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic-pituitary-adrenal axis activity: implications for the integration of limbic inputs. Journal of Neuroscience 27 20252034. (doi:10.1523/JNEUROSCI.4301-06.2007)

    • Search Google Scholar
    • Export Citation
  • Daftary SS, Boudaba C, Szabo K & Tasker JG 1998 Noradrenergic excitation of magnocellular neurons in the rat hypothalamic paraventricular nucleus via intranuclear glutamatergic circuits. Journal of Neuroscience 18 1061910628.

    • Search Google Scholar
    • Export Citation
  • Dautzenberg FM & Hauger RL 2002 The CRF peptide family and their receptors: yet more partners discovered. Trends in Pharmacological Sciences 23 7177. (doi:10.1016/S0165-6147(02)01946-6)

    • Search Google Scholar
    • Export Citation
  • Dautzenberg FM, Py-Lang G, Higelin J, Fischer C, Wright MB & Huber G 2001 Different binding modes of amphibian and human corticotropin-releasing factor type 1 and type 2 receptors: evidence for evolutionary differences. Journal of Pharmacology and Experimental Therapeutics 296 113120.

    • Search Google Scholar
    • Export Citation
  • Dietrich MO, Zimmer MR, Bober J & Horvath TL 2015 Hypothalamic Agrp neurons drive stereotypic behaviors beyond feeding. Cell 160 12221232. (doi:10.1016/j.cell.2015.02.024)

    • Search Google Scholar
    • Export Citation
  • Drescher VS, Chen HL & Romsos DR 1994 Corticotropin-releasing hormone decreases feeding, oxygen consumption and activity of genetically obese (ob/ob) and lean mice. Journal of Nutrition 124 524530.

    • Search Google Scholar
    • Export Citation
  • Du Vigneaud V 1954 Hormones of the posterior pituitary gland: oxytocin and vasopressin. Harvey Lectures 50 126.

  • Fuzesi T, Daviu N, Wamsteeker Cusulin JI, Bonin RP & Bains JS 2016 Hypothalamic CRH neurons orchestrate complex behaviours after stress. Nature Communications 7 11937. (doi:10.1038/ncomms11937)

    • Search Google Scholar
    • Export Citation
  • Gati G, Lendvai D, Hokfelt T, Harkany T & Alpar A 2014 Revival of calcium-binding proteins for neuromorphology: secretagogin typifies distinct cell populations in the avian brain. Brain Behavior and Evolution 83 8292.

    • Search Google Scholar
    • Export Citation
  • Gillies GE, Linton EA & Lowry PJ 1982 Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299 355357. (doi:10.1038/299355a0)

    • Search Google Scholar
    • Export Citation
  • Girotti M, Weinberg MS & Spencer RL 2009 Diurnal expression of functional and clock-related genes throughout the rat HPA axis: system-wide shifts in response to a restricted feeding schedule. American Journal of Physiology: Endocrinology and Metabolism 296 E888E897. (doi:10.1152/ajpendo.90946.2008)

    • Search Google Scholar
    • Export Citation
  • Gold PW, Licinio J, Wong ML & Chrousos GP 1995 Corticotropin releasing hormone in the pathophysiology of melancholic and atypical depression and in the mechanism of action of antidepressant drugs. Annals of the New York Academy of Sciences 771 716729. (doi:10.1111/j.1749-6632.1995.tb44723.x)

    • Search Google Scholar
    • Export Citation
  • Green JD & Harris GW 1947 The neurovascular link between the neurohypophysis and adenohypophysis. Journal of Endocrinology 5 136146. (doi:10.1677/joe.0.0050136)

    • Search Google Scholar
    • Export Citation
  • Guillemin R 1978 Peptides in the brain: the new endocrinology of the neuron. Science 202 390402. (doi:10.1126/science.212832)

  • Gustafsson JA, Carlstedt-Duke J, Poellinger L, Okret S, Wikstrom AC, Bronnegard M, Gillner M, Dong Y, Fuxe K & Cintra A 1987 Biochemistry, molecular biology, and physiology of the glucocorticoid receptor. Endocrine Reviews 8 185234. (doi:10.1210/edrv-8-2-185)

    • Search Google Scholar
    • Export Citation
  • Gutknecht E, Vauquelin G & Dautzenberg FM 2010 Corticotropin-releasing factor receptors induce calcium mobilization through cross-talk with Gq-coupled receptors. European Journal of Pharmacology 642 19. (doi:10.1016/j.ejphar.2010.05.027)

    • Search Google Scholar
    • Export Citation
  • Hasegawa K, Wakino S, Kimoto M, Minakuchi H, Fujimura K, Hosoya K, Komatsu M, Kaneko Y, Kanda T & Tokuyama H et al. 2013 The hydrolase DDAH2 enhances pancreatic insulin secretion by transcriptional regulation of secretagogin through a Sirt1-dependent mechanism in mice. FASEB Journal 27 23012315. (doi:10.1096/fj.12-226092)

    • Search Google Scholar
    • Export Citation
  • Hata Y, Slaughter CA & Sudhof TC 1993 Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366 347351. (doi:10.1038/366347a0)

    • Search Google Scholar
    • Export Citation
  • Henry FE, Sugino K, Tozer A, Branco T & Sternson SM 2015 Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss. eLife 4 e09800. (doi:10.7554/elife.09800)

    • Search Google Scholar
    • Export Citation
  • Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC & Cullinan WE 2003 Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Frontiers in Neuroendocrinology 24 151180. (doi:10.1016/j.yfrne.2003.07.001)

    • Search Google Scholar
    • Export Citation
  • Hooper A & Maguire J 2016 Characterization of a novel subtype of hippocampal interneurons that express corticotropin-releasing hormone. Hippocampus 26 4153. (doi:10.1002/hipo.22487)

    • Search Google Scholar
    • Export Citation
  • Hrabovszky E, Wittmann G, Turi GF, Liposits Z & Fekete C 2005 Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone neurons of the rat contain vesicular glutamate transporter-2. Endocrinology 146 341347. (doi:10.1210/en.2004-0856)

    • Search Google Scholar
    • Export Citation
  • Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, Lonnerberg P & Linnarsson S 2014 Quantitative single-cell RNA-seq with unique molecular identifiers. Nature Methods 11 163166. (doi:10.1038/nmeth.2772)

    • Search Google Scholar
    • Export Citation
  • Kahaly GJ & Hansen MP 2016 Type 1 diabetes associated autoimmunity. Autoimmunity Reviews 15 644648. (doi:10.1016/j.autrev.2016.02.017)

  • Keegan CE, Herman JP, Karolyi IJ, O’Shea KS, Camper SA & Seasholtz AF 1994 Differential expression of corticotropin-releasing hormone in developing mouse embryos and adult brain. Endocrinology 134 25472555. (doi:10.1210/en.134.6.2547)

    • Search Google Scholar
    • Export Citation
  • Kiss JZ, Mezey E & Skirboll L 1984 Corticotropin-releasing factor-immunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. PNAS 81 18541858. (doi:10.1073/pnas.81.6.1854)

    • Search Google Scholar
    • Export Citation
  • Krashes MJ, Shah BP, Madara JC, Olson DP, Strochlic DE, Garfield AS, Vong L, Pei H, Watabe-Uchida M & Uchida N et al. 2014 An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507 238242. (doi:10.1038/nature12956)

    • Search Google Scholar
    • Export Citation
  • Lantos TA, Gorcs TJ & Palkovits M 1995 Immunohistochemical mapping of neuropeptides in the premamillary region of the hypothalamus in rats. Brain Research: Brain Research Reviews 20 209249. (doi:10.1016/0165-0173(94)00013-F)

    • Search Google Scholar
    • Export Citation
  • Lee Y & Davis M 1997 Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropin-releasing hormone on the acoustic startle reflex. Journal of Neuroscience 17 64346446.

    • Search Google Scholar
    • Export Citation
  • Lloyd RB & Nemeroff CB 2011 The role of corticotropin-releasing hormone in the pathophysiology of depression: therapeutic implications. Current Topics in Medicinal Chemistry 11 609617. (doi:10.2174/1568026611109060609)

    • Search Google Scholar
    • Export Citation
  • Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N & Martersteck EM et al. 2015 Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161 12021214. (doi:10.1016/j.cell.2015.05.002)

    • Search Google Scholar
    • Export Citation
  • Mains RE, Eipper BA & Ling N 1977 Common precursor to corticotropins and endorphins. PNAS 74 30143018. (doi:10.1073/pnas.74.7.3014)

  • Maj M, Gartner W, Ilhan A, Neziri D, Attems J & Wagner L 2010 Expression of TAU in insulin-secreting cells and its interaction with the calcium-binding protein secretagogin. Journal of Endocrinology 205 2536. (doi:10.1677/JOE-09-0341)

    • Search Google Scholar
    • Export Citation
  • Makara GB, Stark E, Karteszi M, Palkovits M & Rappay G 1981 Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat. American Journal of Physiology 240 E441E446.

    • Search Google Scholar
    • Export Citation
  • Makino S, Schulkin J, Smith MA, Pacak K, Palkovits M & Gold PW 1995 Regulation of corticotropin-releasing hormone receptor messenger ribonucleic acid in the rat brain and pituitary by glucocorticoids and stress. Endocrinology 136 45174525. (doi:10.1210/en.136.10.4517)

    • Search Google Scholar
    • Export Citation
  • McEwen BS 2007 Physiology and neurobiology of stress and adaptation: central role of the brain. Physiological Reviews 87 873904. (doi:10.1152/physrev.00041.2006)

    • Search Google Scholar
    • Export Citation
  • McEwen BS 2012 The ever-changing brain: cellular and molecular mechanisms for the effects of stressful experiences. Developmental Neurobiology 72 878890. (doi:10.1002/dneu.20968)

    • Search Google Scholar
    • Export Citation
  • McEwen BS & Stellar E 1993 Stress and the individual. Mechanisms leading to disease. Archives of Internal Medicine 153 20932101. (doi:10.1001/archinte.1993.00410180039004)

    • Search Google Scholar
    • Export Citation
  • McNally GP & Akil H 2002 Role of corticotropin-releasing hormone in the amygdala and bed nucleus of the stria terminalis in the behavioral, pain modulatory, and endocrine consequences of opiate withdrawal. Neuroscience 112 605617. (doi:10.1016/S0306-4522(02)00105-7)

    • Search Google Scholar
    • Export Citation
  • Meister B, Cortes R, Villar MJ, Schalling M & Hokfelt T 1990 Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels. Cell and Tissue Research 260 279297. (doi:10.1007/BF00318631)

    • Search Google Scholar
    • Export Citation
  • Merchenthaler I, Vigh S, Petrusz P & Schally AV 1982 Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain. American Journal of Anatomy 165 385396. (doi:10.1002/aja.1001650404)

    • Search Google Scholar
    • Export Citation
  • Merchenthaler I, Vigh S, Petrusz P & Schally AV 1983 The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats. Regulatory Peptides 5 295305. (doi:10.1016/0167-0115(83)90287-2)

    • Search Google Scholar
    • Export Citation
  • Miller DB & O’Callaghan JP 2002 Neuroendocrine aspects of the response to stress. Metabolism 51 510. (doi:10.1053/meta.2002.33184)

  • Miyazaki T, Yamasaki M, Uchigashima M, Matsushima A & Watanabe M 2011 Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum. European Journal of Neuroscience 33 8294. (doi:10.1111/j.1460-9568.2010.07472.x)

    • Search Google Scholar
    • Export Citation
  • Mizuno TM, Makimura H & Mobbs CV 2003 The physiological function of the agouti-related peptide gene: the control of weight and metabolic rate. Annals of Medicine 35 425433. (doi:10.1080/07853890310012076)

    • Search Google Scholar
    • Export Citation
  • Mulder J, Zilberter M, Spence L, Tortoriello G, Uhlen M, Yanagawa Y, Aujard F, Hokfelt T & Harkany T 2009 Secretagogin is a Ca2+-binding protein specifying subpopulations of telencephalic neurons. PNAS 106 2249222497. (doi:10.1073/pnas.0912484106)

    • Search Google Scholar
    • Export Citation
  • Mulder J, Spence L, Tortoriello G, Dinieri JA, Uhlen M, Shui B, Kotlikoff MI, Yanagawa Y, Aujard F & Hokfelt T et al. 2010 Secretagogin is a Ca2+-binding protein identifying prospective extended amygdala neurons in the developing mammalian telencephalon. European Journal of Neuroscience 31 21662177. (doi:10.1111/j.1460-9568.2010.07275.x)

    • Search Google Scholar
    • Export Citation
  • Munck A, Guyre PM & Holbrook NJ 1984 Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrine Reviews 5 2544. (doi:10.1210/edrv-5-1-25)

    • Search Google Scholar
    • Export Citation
  • Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN & Numa S 1979 Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature 278 423427. (doi:10.1038/278423a0)

    • Search Google Scholar
    • Export Citation
  • Napier C & Pearce SH 2014 Current and emerging therapies for Addison’s disease. Current Opinion in Endocrinology, Diabetes and Obesity 21 147153. (doi:10.1097/MED.0000000000000067)

    • Search Google Scholar
    • Export Citation
  • Nijsen MJ, Croiset G, Diamant M, De Wied D & Wiegant VM 2001 CRH signalling in the bed nucleus of the stria terminalis is involved in stress-induced cardiac vagal activation in conscious rats. Neuropsychopharmacology 24 110. (doi:10.1016/S0893-133X(00)00167-6)

    • Search Google Scholar
    • Export Citation
  • Owens MJ, Bartolome J, Schanberg SM & Nemeroff CB 1990 Corticotropin-releasing factor concentrations exhibit an apparent diurnal rhythm in hypothalamic and extrahypothalamic brain regions: differential sensitivity to corticosterone. Neuroendocrinology 52 626631. (doi:10.1159/000125655)

    • Search Google Scholar
    • Export Citation
  • Palkovits M 2000 Stress-induced expression of co-localized neuropeptides in hypothalamic and amygdaloid neurons. European Journal of Pharmacology 405 161166. (doi:10.1016/S0014-2999(00)00549-5)

    • Search Google Scholar
    • Export Citation
  • Pang ZP & Sudhof TC 2010 Cell biology of Ca2+-triggered exocytosis. Current Opinion in Cell Biology 22 496505. (doi:10.1016/j.ceb.2010.05.001)

    • Search Google Scholar
    • Export Citation
  • Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE & Vale W 1994 Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. PNAS 91 87778781. (doi:10.1073/pnas.91.19.8777)

    • Search Google Scholar
    • Export Citation
  • Reul JM & Holsboer F 2002 Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Current Opinion in Pharmacology 2 2333. (doi:10.1016/S1471-4892(01)00117-5)

    • Search Google Scholar
    • Export Citation
  • Rhodes CH, Morrell JI & Pfaff DW 1981 Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin. Journal of Comparative Neurology 198 4564. (doi:10.1002/cne.901980106)

    • Search Google Scholar
    • Export Citation
  • Rivier C & Vale W 1983 Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretion in vivo. Endocrinology 113 939942. (doi:10.1210/endo-113-3-939)

    • Search Google Scholar
    • Export Citation
  • Roberts JL & Herbert E 1977 Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. PNAS 74 48264830. (doi:10.1073/pnas.74.11.4826)

    • Search Google Scholar
    • Export Citation
  • Rogstam A, Linse S, Lindqvist A, James P, Wagner L & Berggard T 2007 Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin. Biochemical Journal 401 353363. (doi:10.1042/BJ20060918)

    • Search Google Scholar
    • Export Citation
  • Romanov RA, Alpar A, Zhang MD, Zeisel A, Calas A, Landry M, Fuszard M, Shirran SL, Schnell R & Dobolyi A et al. 2015 A secretagogin locus of the mammalian hypothalamus controls stress hormone release. EMBO Journal 34 3654. (doi:10.15252/embj.201488977)

    • Search Google Scholar
    • Export Citation
  • Romanov RA, Zeisel A, Bakker J, Girach F, Hellysaz A, Tomer R, Alpar A, Mulder J, Clotman F & Keimpema E et al. 2016 Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nature Neuroscience [in press]. (doi:10.1038/nn.4462)

    • Search Google Scholar
    • Export Citation
  • Salata RA, Jarrett DB, Verbalis JG & Robinson AG 1988 Vasopressin stimulation of adrenocorticotropin hormone (ACTH) in humans. In vivo bioassay of corticotropin-releasing factor (CRF) which provides evidence for CRF mediation of the diurnal rhythm of ACTH. Journal of Clinical Investigation 81 766774. (doi:10.1172/JCI113382)

    • Search Google Scholar
    • Export Citation
  • Sapolsky RM, Romero LM & Munck AU 2000 How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21 5589. (doi:10.1210/er.21.1.55)

    • Search Google Scholar
    • Export Citation
  • Saraiva LR, Ibarra-Soria X, Khan M, Omura M, Scialdone A, Mombaerts P, Marioni JC & Logan DW 2015 Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq. Scientific Reports 5 18178. (doi:10.1038/srep18178)

    • Search Google Scholar
    • Export Citation
  • Sawchenko PE, Swanson LW & Vale WW 1984 Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat. PNAS 81 18831887. (doi:10.1073/pnas.81.6.1883)

    • Search Google Scholar
    • Export Citation
  • Schally AV 1978 Aspects of hypothalamic regulation of the pituitary gland. Science 202 1828. (doi:10.1126/science.99816)

  • Schwaller B 2010 Cytosolic Ca2+ buffers. Cold Spring Harbor Perspectives in Biology 2 a004051. (doi:10.1101/cshperspect.a004051)

  • Seasholtz AF, Valverde RA & Denver RJ 2002 Corticotropin-releasing hormone-binding protein: biochemistry and function from fishes to mammals. Journal of Endocrinology 175 8997. (doi:10.1677/joe.0.1750089)

    • Search Google Scholar
    • Export Citation
  • Shapiro E, Biezuner T & Linnarsson S 2013 Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews Genetics 14 618630. (doi:10.1038/nrg3542)

    • Search Google Scholar
    • Export Citation
  • Simmons DM & Swanson LW 2009 Comparison of the spatial distribution of seven types of neuroendocrine neurons in the rat paraventricular nucleus: toward a global 3D model. Journal of Comparative Neurology 516 423441. (doi:10.1002/cne.22126)

    • Search Google Scholar
    • Export Citation
  • Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P & Rothman JE 1993 SNAP receptors implicated in vesicle targeting and fusion. Nature 362 318324. (doi:10.1038/362318a0)

    • Search Google Scholar
    • Export Citation
  • Spiess J, Rivier J, Rivier C & Vale W 1981 Primary structure of corticotropin-releasing factor from ovine hypothalamus. PNAS 78 65176521. (doi:10.1073/pnas.78.10.6517)

    • Search Google Scholar
    • Export Citation
  • Stahl PL, Salmen F, Vickovic S, Lundmark A, Navarro JF, Magnusson J, Giacomello S, Asp M, Westholm JO & Huss M et al. 2016 Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353 7882. (doi:10.1126/science.aaf2403)

    • Search Google Scholar
    • Export Citation
  • Sutton RB, Fasshauer D, Jahn R & Brunger AT 1998 Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395 347353. (doi:10.1038/26412)

    • Search Google Scholar
    • Export Citation
  • Swaab DF, Pool CW & Nijveldt F 1975 Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophypopseal system. Journal of Neural Transmission 36 195215. (doi:10.1007/BF01253126)

    • Search Google Scholar
    • Export Citation