Carlos Arias

Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse

Two G protein‐coupled receptors have been identified that bind corticotropin‐releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF‐R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF‐R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF‐R1. Major neuronal sites of CRF‐R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF‐R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF‐R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF‐R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF‐R2 mRNA in the nucleus of the solitary tract, and stress‐inducible expression of CRF‐R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF‐related peptides at a loci shown previously to lack a capacity for CRF‐R expression or CRF binding. Other such “mismatches” remain to be reconciled. J. Comp. Neurol. 428:191–212, 2000.

Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 CRF receptors.

Histochemical and axonal transport methods were used to clarify the central organization of cells and fibers that express urocortin (UCN), a recently discovered corticotropin‐releasing factor (CRF)‐related neuropeptide, which has been proposed as an endogenous ligand for type 2 CRF receptors (CRF‐R2). Neurons that display both UCN mRNA and peptide expression were found to be centered in the Edinger‐Westphal (EW), lateral superior olivary (LSO), and supraoptic nuclei; lower levels of expression are seen in certain cranial nerve and spinal motoneurons and in small populations of neurons in the forebrain. Additional sites of UCN mRNA and peptide expression detected only in colchicine‐treated rats are considered to be minor ones. UCN‐immunoreactive projections in brain are predominantly descending and largely consistent with central projections attributed to the EW and LSO, targeting principally accessory optic, precerebellar, and auditory structures, as well as the spinal intermediate gray. Although neither the EW nor LSO are known to project to the forebrain, UCN‐ir neurons in the EW were identified that project to the lateral septal nucleus, which houses a prominent UCN‐ir terminal field. Although substantial UCN‐ir projections were observed to several brainstem cell groups that express CRF‐R2, including the dorsal raphe and interpeduncular nuclei and the nucleus of the solitary tract (NTS), most prominent seats of CRF‐R2 expression were found to contain inputs immunopositive for piscine urotensin I, but not rat UCN. The results define a central UCN system whose organization suggests a principal involvement in motor control and sensorimotor integration; its participation in stress‐related mechanisms would appear to derive principally by virtue of projections to the spinal intermediolateral column, the NTS, and the paraventricular nucleus. Several observations, including the lack of a pervasive relationship of UCN‐ir projections with CRF‐R2‐expressing targets, support the existence of still additional CRF‐related peptides in mammalian brain. J. Comp. Neurol. 415:285–312, 1999.

Evidence for an Intramedullary Prostaglandin-Dependent Mechanism in the Activation of Stress-Related Neuroendocrine Circuitry by Intravenous Interleukin-1

We have provided evidence that the stimulatory effects of intravenous interleukin-1 (IL-1) on neurosecretory neurons in the paraventricular nucleus (PVH) that express corticotropin-releasing factor (CRF) depend specifically on the integrity of catecholaminergic projections originating in caudal medulla. Here we report on experiments designed to test alternative means by which circulating IL-1 might access medullary aminergic neurons, including mechanisms involving sensory components of the vagus, the area postrema, or perivascular cells bearing IL-1 receptors. Neither abdominal vagotomy nor area postrema lesions reliably altered Fos expression induced in the medulla or PVH in response to a moderately suprathreshold dose of IL-1β. Cytokine-stimulated increases in CRF mRNA in the PVH were also unaffected by either ablation. By contrast, systemic administration of the cyclooxygenase inhibitor indomethacin resulted in parallel dose-related attenuations of IL-1 effects in hypothalamus and medulla. Microinjections of prostaglandin E2 (PGE2; ≥10 ng) in rostral ventrolateral medulla, the principal seat of IL-1-sensitive neurons that project to the PVH, provoked discrete patterns of cellular activation in hypothalamus and medulla that mimicked those seen in response to intravenous IL-1. We interpret these findings as supporting the hypothesis that paracrine effects of PGE2 released from perivascular cells in the medulla as a consequence of IL-1 stimulation and, acting through prostanoid receptors on or near local aminergic neurons that project to the PVH, contribute to the stimulatory effects of increased circulating IL-1 on neurons constituting the central limb of the hypothalamo–pituitary–adrenal axis.

Regional Differentiation of the Medial Prefrontal Cortex in Regulating Adaptive Responses to Acute Emotional Stress

The medial prefrontal cortex (mPFC) is an important neural substrate for integrating cognitive-affective information and regulating the hypothalamo–pituitary–adrenal (HPA) axis response to emotional stress. mPFC modulation of stress responses is effected in part via the paraventricular hypothalamic nucleus (PVH), which houses both autonomic (sympathoadrenal) and neuroendocrine (HPA) effector mechanisms. Although the weight of evidence suggests that mPFC influences on stress-related PVH outputs are inhibitory, discordant findings have been reported, and such work has tended to treat this cortical region as a unitary structure. Here we compared the effects of lesions of the dorsal versus ventral aspects of mPFC, centered in the prelimbic and infralimbic fields, respectively, on acute restraint stress-induced activation of PVH cell groups mediating autonomic and neuroendocrine responses. Lesions to the dorsal mPFC enhanced restraint-induced Fos and corticotropin-releasing factor (CRF) mRNA expression in the neurosecretory region of PVH. Ablation of the ventral mPFC decreased stress-induced Fos protein and CRF mRNA expression in this compartment but increased Fos induction in PVH regions involved in central autonomic control. Repetition of the experiments in rats bearing retrograde tracer deposits to label PVH-autonomic projections confirmed that ventral mPFC lesions selectively increased stress-induced Fos expression in identified preautonomic neurons. Finally, hormonal indices of HPA activation in response to acute stress were augmented after dorsal mPFC lesions and attenuated after ventral mPFC lesions. These results suggest that dorsal and ventral aspects of the mPFC differentially regulate neuroendocrine and autonomic PVH outputs in response to emotional stress.

Distribution of the EP3 prostaglandin E2 receptor subtype in the rat brain: Relationship to sites of interleukin-1–induced cellular responsiveness

The activation of neurosecretory neurons that express corticotropin‐releasing hormone (CRH) in response to increased circulating levels of interleukin‐1β (IL‐1β) depends on prostaglandin E2 (PGE2) acting locally within the brain parenchyma. To identify potential central targets for PGE2 relevant to pituitary‐adrenal control, the distribution of mRNA encoding the PGE2 receptor subtype EP3 (EP3R) was analyzed in rat brain. Hybridization histochemistry revealed prominent labeling of cells in discrete portions of the olfactory system, iso‐ and hippocampal cortices, and subcortical telencephalic structures in the septal region and amygdala. Labeling over the midline, intralaminar, and anterior thalamic groups was particularly prominent. EP3R expression was enriched in the median preoptic nucleus and adjoining aspects of the medial preoptic area (MPO) implicated in thermoregulatory/febrile responses and sleep induction. EP3R‐expressing cells were also prominent in brainstem cell groups involved in nociceptive information processing/modulation (periaqueductal gray, locus coeruleus (LC), parabrachial nucleus (PB), caudal raphé nuclei), arousal and wakefulness (LC, midbrain raphé and tuberomammillary nuclei); and in conveying interoceptive input, including systemic IL‐1 signals, to the endocrine hypothalamus (nucleus of the solitary tract (NTS) and rostral ventrolateral medulla [VLM]). Combined hybridization histochemical detection of EP3R mRNA with immunolocalization of IL‐1β–induced Fos protein expression identified cytokine‐sensitive, EP3R‐positive cells in the medial NTS, rostral VLM, and, to a lesser extent, aspects of the MPO. These findings are consistent with the view that increased circulating IL‐1 may stimulate central neural mechanisms, including hypothalamic CRH neurons, through an EP3R‐dependent mechanism involving PGE2‐mediated activation of cells in the caudal medulla and/or preoptic region. J. Comp. Neurol. 428:5–20, 2000.

The Creb1 coactivator Crtc1 is required for energy balance and fertility.

The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that act on the signal transducer and activator of transcription 3 (Stat3). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element–binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction—Crtc1−/− mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient ob/ob mice, while leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptins effects on satiety and fertility. Crtc1 overexpression in hypothalamic cells increased Cartpt and Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr, and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility.

A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction.

G protein–gated potassium (Kir3) channels are important for controlling neuronal excitability in the brain. Using a proteomics approach, we have identified a unique rodent intracellular protein, sorting nexin 27 (SNX27), which regulates the trafficking of Kir3 channels. Like most sorting nexins, SNX27 possesses a functional PX domain that selectively binds the membrane phospholipid phosphatidylinositol-3-phosphate (PI3P) and is important for trafficking to the early endosome. SNX27, however, is the only sorting nexin to contain a PDZ domain. This PDZ domain discriminates between channels with similar class I PDZ-binding motifs, associating with the C-terminal end of Kir3.3 and Kir3.2c (−ESKV), but not with that of Kir2.1 (−ESEI) or Kv1.4 (−ETDV). SNX27 promotes the endosomal movement of Kir3 channels, leading to reduced surface expression, increased degradation and smaller Kir3 potassium currents. The regulation of endosomal trafficking via sorting nexins reveals a previously unknown mechanism for controlling potassium channel surface expression.

Ovine genomic urocortin: cloning, pharmacologic characterization, and distribution of central mRNA

Urocortin (Ucn), the newest member of the corticotropin-releasing factor (CRF) family of peptides, has been demonstrated to have significant physiologic and behavioral effects following its peripheral and central administration, respectively. In order to assess the differences in Ucn across species, an 18-kb sheep genomic DNA fragment encoding urocortin was isolated by the hybridization screening of a lambda phage library with a probe generated from rat urocortin (rUcn) cDNA. The sheep clone contains a region that is 84% and 88% homologous to the coding region of rUcn and human Ucn (hUcn), respectively and encodes an ovine Ucn (oUcn) that is predicted to be identical to the rat peptide. Competitive binding assays demonstrated oUcn to have a high affinity (Ki=0.1 nM) for the sheep CRF-binding protein (CRF-BP) and localization studies by in situ hybridization have shown that the distribution of oUcn messenger RNA in sheep brain shares with that of rUcn in rat brain a predominant locus of expression in the Edinger-Westphal nucleus of the midbrain, though some secondary sites of expression reported in rat are not conserved. These findings demonstrate that, even across diverse species, Ucn is highly conserved with respect to its structure and pharmacology unlike CRF where significant amino acid substitutions between the rat/human and sheep peptides may underlie differences in neuroendocrine regulation.

Ultrastructural localization of the corticotropin-releasing factor-binding protein in rat brain and pituitary.

Preembedding immunoperoxidase staining methods were used to permit ultrastructural analyses of the distribution in rat brain and pituitary of the corticotropin‐releasing factor–binding protein (CRF‐BP), a moiety distinct from CRF receptors, but which is nonetheless capable of binding the peptide and reversibly neutralizing its biological actions. In anterior pituitary, CRF‐BP immunoreactivity (ir) was detected in corticotropelike cells, with reaction product associated principally with secondary lysosomes and multivesicular bodies and not at all with secretory granules. In brain, marked regional differences in the subcellular pattern of CRF‐BP staining were evident. In isocortex, where BP/peptide colocalization is rare, BP‐ir was distributed in cells and processes in a manner similar to that of a prototypic neuropeptide, including in terminals commonly engaging in synaptic contacts with unlabeled dendritic profiles. In the bed nucleus of the stria terminalis, a site that contains overlapping accumulations of CRF‐BP‐ir projections and CRF‐ir perikarya, BP staining was restricted to vesicle‐laden varicosities that rarely engaged in synaptic contacts with somatic or dendritic elements but were frequently apposed to unlabeled axon varicosities and terminals. In the ventromedial medulla, a site of partial CRF/BP overlap, most cells displayed a subcellular localization CRF‐BP‐ir like that seen in cortex, whereas in others the distribution shared similarities with that observed in pituitary. The results suggest that the function of the CRF‐BP may differ in different cellular contexts. In cellular targets of CRF or in neurons in which peptide and BP coexist, the CRF‐BP may play a role in processing and degradation of CRF and/or ligand–receptor complexes. In other areas of the central nervous system, the BP seems positioned to serve as a transmitter/modulator at conventional synapses or as an autocrine or paracrine modulator of local CRF effects. J. Comp. Neurol. 413:241–254, 1999.

Protein synthesis blockade differentially affects the stress- induced transcriptional activation of neuropeptide genes in parvocellular neurosecretory neurons

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are synergistically interacting ACTH secretagogues that are co-expressed by parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVH). To shed light on the mechanisms that mediate the stress-induced transcriptional activation of these neuropeptide genes, quantitative hybridization histochemical methods were used to assess the effects of systemic treatment with the protein synthesis inhibitor, cycloheximide, on the ether stress-induced upregulation of primary CRF and AVP transcripts, in vivo. Pretreatment with cycloheximide prevented the induction of FOS, but not CREB phosphorylation, normally seen in response to acute ether exposure, and significantly attenuated the stress-induced rise in AVP, but not CRF, heteronuclear RNA expression in the parvocellular division of the PVH. These results support the view that distinct molecular mechanisms govern the expression of the two principal corticotropin-releasing factors, in vivo.