Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP) : a novel mechanism for elevating ‘free’ CRF levels in human brain

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in ‘free’ hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of ‘free’ hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.     

Ultrastructural evidence for GABAergic brain stem projections to spinal motoneurons in the rat.

In the present ultrastructural study in the rat, it was determined whether GABA was present in projections descending from the ventromedial reticular formation of the lower brain stem to motoneuronal cell groups in the lumbar spinal cord. For this purpose, the anterograde transport of WGA-HRP was combined with the postembedding immunogold technique for GABA, with the advantage that both markers could be visualized simultaneously in a single terminal. In 4 rats, WGA-HRP was injected in the ventromedial part of the brain stem reticular formation at levels between the rostral inferior olive and the caudal part of the facial nucleus. Vibratome sections were cut from the lumbar spinal cord, reacted for WGA-HRP, and processed for electron microscopy. Ultrathin sections containing the lateral motoneuronal cell groups were cut and treated following the immunogold technique using a polyclonal antibody directed against GABA. It was found that nearly 40% of the terminal profiles that were labeled with WGA-HRP reaction products from the ventromedial brain stem were also labeled for GABA (double labeled). Most of the double-labeled terminals (81%) were F-type (containing many flattened vesicles), 12% were G-type (containing many granular vesicles), and 7% were S-type (containing many spherical vesicles). The majority of the double-labeled terminals contacted proximal dendrites. It is argued that the descending GABAergic projection produces a general inhibitory effect on spinal motoneurons, counteracting the general facilitation produced by the serotonergic projection derived from the same brain stem area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland.

Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [125I]ovine CRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovine CRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. Bmax) without an alteration in the affinity (i.e. Kd) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a loss of corticotropes in the anterior pituitary.     

Differential expression of orexin receptors 1 and 2 in the rat brain

Orexins (hypocretins) are neuropeptides synthesized in the central nervous system exclusively by neurons of the lateral hypothalamus. Orexin-containing neurons have widespread projections and have been implicated in complex physiological functions including feeding behavior, sleep states, neuroendocrine function, and autonomic control. Two orexin receptors (OX(1)R and OX(2)R) have been identified, with distinct expression patterns throughout the brain, but a systematic examination of orexin receptor expression in the brain has not appeared. We used in situ hybridization histochemistry to examine the patterns of expression of mRNA for both orexin receptors throughout the brain. OX(1)R mRNA was observed in many brain regions including the prefrontal and infralimbic cortex, hippocampus, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. OX(2)R mRNA was prominent in a complementary distribution including the cerebral cortex, septal nuclei, hippocampus, medial thalamic groups, raphe nuclei, and many hypothalamic nuclei including the tuberomammillary nucleus, dorsomedial nucleus, paraventricular nucleus, and ventral premammillary nucleus. The differential distribution of orexin receptors is consistent with the proposed multifaceted roles of orexin in regulating homeostasis and may explain the unique role of the OX(2)R receptor in regulating sleep state stability.     

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.     

The effect of CRF2 receptor antagonists on rat conditioned fear responses and c-Fos and CRF expression in the brain limbic structures

The influence of intracerebroventricular-administered selective corticotropin-releasing factor receptor 2 (CRF₂) antagonists (antisauvagine-30, astressin-2B), on rat anxiety-like behavior, expression levels of c-Fos and CRF, and plasma corticosterone levels were examined in the present study. In fear-conditioned animals, both CRF receptor antagonists enhanced a conditioned freezing fear response and increased the conditioned fear-elevated concentration of serum corticosterone. Exogenously administered antisauvagine-30 increased the aversive context-induced expression of c-Fos in the 1 and 2 areas of the cingulate cortex (Cg1, Cg2), the central amygdala (CeA) and parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), and it enhanced the effect of conditioned fear in the secondary motor cortex (M2) and medial amygdala (MeA). Immunocytochemistry demonstrated an increase in CRF expression in the Cg1, M2 areas of the cortex, and pPVN, and it revealed the effect of conditioned fear in the CeA 35 min after antisauvagine-30 administration and 10 min after the conditioned fear test. Furthermore, astressin-2B, another CRF₂ receptor antagonist, enhanced expression of c-Fos and CRF in the CeA and pPVN, and revealed the effect of conditioned fear in the Cg1. These data support a model in which an excess in CRF₁ receptor activation, combined with reduced CRF₂ receptor signaling, may contribute to stronger expression of anxiety-like responses.     

CRF and urocortin decreased brain stimulation reward in the rat: reversal by a CRF receptor antagonist

The present studies were designed to investigate the effects of corticotropin-releasing factor (CRF) receptor activation and antagonism on intracranial self-stimulation (ICSS) reward using a discrete-trial current-intensity threshold procedure. Bipolar electrodes were implanted in the lateral hypothalamus, and cannula guides were implanted above the lateral ventricle of male Wistar rats. Dose-effect functions were established for the effects on ICSS of the competitive CRF receptor agonist h/rCRF (0-5.0 microg, i.c.v. ), the CRF receptor agonist urocortin (0-5.0 microg, i.c.v.), and the CRF receptor antagonist [D-Phe(12), Nle(21,38), C(alpha) MeLeu(37)] h/rCRF(12-41) (0-5.0 microg, i.c.v.). Administration of h/rCRF or urocortin dose-dependently elevated ICSS thresholds without altering performance measures (latencies to respond to stimulation, extra and time-out responses). CRF was more potent than urocortin in terms of threshold dose-effects on ICSS thresholds compared to vehicle. Despite these apparent potency differences, percent effect sizes on ICSS thresholds were comparable at the highest doses of both peptides. In contrast to the significant threshold elevation effects of CRF and urocortin, the competitive CRF antagonist D-Phe CRF(12-41) had no effect on ICSS thresholds or performance measures. To determine the neuropharmacological specificity of the effect of CRF on brain stimulation reward, D-Phe CRF(12-41) was used to antagonize CRF-induced threshold elevations. Pretreatment with either the 5.0- or 10.0-microg doses of D-Phe CRF(12-41) effectively blocked CRF-induced reward threshold elevations (3.0 microg) without affecting other ICSS performance measures. These results indicate that CRF neurotransmission can modulate ICSS reward processes.     

Distribution and expression of CRF receptor 1 and 2 mRNAs in the CRF over-expressing mouse brain

Corticotropin-releasing factor (CRF) acts through CRF 1 and CRF 2 receptors (CRF1, CRF2). To test the hypothesis that CRF controls the expression of these receptors in a brain site- and receptor-type specific manner, we studied CRF1 mRNA and CRF2 mRNA expressions in mice with central CRF over-expression (CRF-OE) and using in situ hybridization. CRF1 and CRF2 mRNAs appear to be differentially distributed across the brain. The brain structures expressing the receptors are the same in wild-type (WT) and in CRF-OE mice. We therefore conclude that chronically elevated CRF does not induce or inhibit expression of these receptors in structures that normally do not or do, respectively, show these receptors. However, from counting cell body profiles positive for CRF1 and CRF2 mRNAs, clear differences appear in receptor expression between CRF-OE and WT mice, in a brain-structure-specific fashion. Whereas some structures do not differ, CRF-OE mice exhibit remarkably lower numbers of CRF1 mRNA-positive profiles in the subthalamic nucleus (-38.6%), globus pallidus (-31.5%), dorsal part of the lateral septum (-23.5%), substantia nigra (-22,8%), primary somatosensory cortex (-18.9%) and principal sensory nucleus V (-18.4%). Furthermore, a higher number of CRF2 mRNA-positive profiles are observed in the dorsal raphe nucleus (+32.2%). These data strongly indicate that central CRF over-expression in the mouse brain is associated with down-regulation of CRF1 mRNA and up-regulation of CRF2 mRNA in a brain-structure-specific way. On the basis of these results and the fact that CRF-OE mice reveal a number of physiological and autonomic symptoms that may be related to chronic stress, we suggest that CRF1 in the basal nuclei may be involved in disturbed information processing and that CRF2 in the dorsal raphe nucleus may play a role in mediating stress-induced release of serotonin by CRF.     

Differential expression of PACAP receptors in postnatal rat brain

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a multi-functional neuropeptide that acts through activation of three common G-protein coupled receptors (VPAC1, VPAC2 and PAC1). In this study, we have investigated the gene expression profile of PAC1 isoforms (Hop1, Hip, Hip-Hop) and VPAC1, VPAC2 receptors in distinct brain regions during different stages of rat postnatal development. Using quantitative real time PCR approach we found that PAC1 isoforms were highly expressed in the cortex of newborns with marked decrease in expression during later stages of development. In contrast, mRNA levels of VPAC1, VPAC2 receptors were markedly lower in newborns in comparison to later developmental stages. Expression of PAC1 isoforms predominated in the hippocampus, while expression of VPAC1 was more prominent in the cortex and VPAC2 in the striatum and hippocampus. In addition we found that during early stages of postnatal development the expression of PAC1 receptor in the hippocampus was significantly higher in females than in males. No sex dependent differences in expression were observed for the VPAC1 and VPAC2 receptors. In summary, differential expression of PAC1, VPAC1 and VPAC2 receptors during postnatal development as well as gender dependent differences of PAC1 receptor expression in the hippocampus, will contribute to our understanding of the role of PACAP/VIP signaling system in normal brain development and function.     

Further chemical differentiation of type I and type II adrenocorticosteroid receptors in mouse brain cytosol: evidence for a new class of glucocorticoid receptors

There are at least two classes of intracellular receptors for adrenocorticosteroid hormones in brain. Type I receptors have a high affinity for the naturally occurring gluco- and mineralocorticoids, corticosterone (CORT) and aldosterone (ALDO), respectively, and a very low affinity for synthetic glucocorticoids such as dexamethasone (DEX). type II receptors have a high affinity for the synthetic glucocorticoids, a lower affinity for CORT and a very low affinity for ALDO. In recent studies with mouse brain cytosol we have found a number of other biochemical differences between these two receptor types. In the present study, brain cytosol from adrenalectomized mice was prepared in HEPES buffer and subjected to various potentially inactivating treatments prior to assessment of Type I and Type II receptor specific binding capacity by incubation for 24 h at 0 degrees C with [3H]ALDO +/- [1H]RU 26988 (to prevent or permit the cross-binding of [3H]ALDO to Type II receptors) or [3H]DEX +/- [1H]Prorenone (to prevent or permit the cross-binding of [3H]DEX to Type I receptors), respectively. These studies revealed that 10-20% of the high-affinity (Kd = 3 nM) [3H]DEX specific binding capacity remained even after extensive, high concentration and repeated pretreatments with dextran-coated charcoal (DDC. to remove endogenous sulfhydryl-reducing reagents and other biochemicals). These procedures had little effect on Type I receptors. Further analyses revealed that DCC-resistant [3H]DEX binders were not Type I receptors since they were not saturated by [1H]Prorenone. These binders were also not inactivated by aging steroid-free cytosol at 0 degree C or by treating it with buffers containing 0.3 M KCl. Since these     

Efferent projections of the nucleus of the solitary tract to peri-locus coeruleus dendrites in rat brain: evidence for a monosynaptic pathway.

Locus coeruleus (LC) neurons respond to autonomic influences, are activated by physiological stressors, and discharge in parallel with peripheral sympathetic nerves. The circuitry underlying modulation of LC activity by physiological manipulations (i.e., hemodynamic stress, hypovolumia) remains unclear. Specifically, monosynaptic projections from primary baroreceptor centers to the LC have been suggested by electrophysiological studies but have not been unequivocally established. Light microscopic anterograde tract-tracing studies have previously shown that neurons originating in the nucleus of the solitary tract (NTS) project to a region of the rostrodorsal pontine tegmentum, which contains noradrenergic dendrites of the LC; however, it is not known whether these NTS efferents specifically target LC dendrites. Therefore, we combined peroxidase labeling of biotinylated dextran amine (BDA) or Phaseolus vulgaris-leucoagglutinin (PHA-L) from the NTS with gold-silver labeling for tyrosine hydroxylase (TH) in the rostrolateral peri-LC region. Injections placed into neighboring nuclei (nucleus gracilis, hypoglossal nucleus) served as controls. Only injections centered in the NTS produced anterograde labeling in peri-LC regions containing TH processes. By electron microscopy, BDA- or PHA-L-labeled axon terminals originating from the NTS contained small, clear, and some large dense-core vesicles and formed heterogeneous synaptic contacts characteristic of both excitatory- and inhibitory-type transmitters. Approximately 19% of the BDA and PHA-L axon terminals examined originating from the commissural portion of the NTS formed synaptic specializations with dendrites exhibiting TH immunoreactivity in the peri-LC. These results demonstrate that neurons projecting from the cardiovascular-related portion of the NTS target noradrenergic dendrites, indicating that barosensitive NTS neurons may directly modulate the activity of LC neurons and may serve to integrate autonomic responses in brain by influencing the widespread noradrenergic projections of the LC. In addition, these findings demonstrate that extranuclear dendrites are an important termination site for afferents to the LC.     

Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors

The distribution of serotonin-2 (5-HT2) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with four ligands: [3H]ketanserin, [3H]mesulergine, [3H]LSD and [3H]spiperone, which are reported to show high affinity for 5-HT2 receptors. Co-incubation with increasing concentrations of several well-known 5-HT2-selective drugs, such as pirenperone, cinanserin and ketanserin, resulted in an inhibition of the binding of the four 3H-labeled ligands to the same areas. However, all of them recognized, in addition to 5-HT2 sites, other populations of binding sites. Receptor densities were quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Our results reveal a heterogeneous distribution of 5-HT2 receptor densities in the rat brain. Very high concentrations were localized in the claustrum, olfactory tubercle and layer IV of the neocortex. The anterior olfactory nucleus, piriform cortex and layer I of neocortex were also rich in 5-HT2 receptors. Intermediate concentrations of receptors were found in caudate putamen, nucleus accumbens, layer V of neocortex, ventral dentate gyrus and mammillary bodies. Areas containing only low concentrations of receptors included the thalamus, hippocampus, brainstem, medulla, cerebellum and spinal cord. The specificity of the different ligands used is discussed in terms of the other populations of sites recognized by them. The distribution of 5-HT2 receptors here reported is discussed in correlation with (a) the known distribution of serotoninergic terminals, (b) the specific anatomical systems and (c) the central effects reported to be mediated by 5-HT2-selective drugs.     

Role of CRF(1) and CRF(2) receptors in fear and anxiety.

Fear and anxiety are common emotions that can be triggered by stress. This paper reviews the work examining the role played by specific corticotropin-releasing factor (CRF) receptors in mediating the expression of these emotions. Several lines of evidence taken from CRF₁ transgenic knockout mice, CRF₁ antisense oligonucleotide studies, and CRF₁ receptor antagonist work suggest that the anxiety inducing effects of CRF are mediated by the CRF₁ receptor. Of these three methodological approaches, the work using transgenic CRF₁ knockout mice appears to be the most consistent. In contrast, the work using specific CRF₁ antagonists has produced somewhat varied results that may be explained, in part, by the testing method. When animals are stressed prior to behavioral testing, CRF₁ receptor antagonists appear to have anxiolytic-like effects. In addition, chronic dosing with CRF₁ antagonists may have more potent anxiolytic-like effects, especially in animal models of spontaneous anxiety, than acute dosing procedures. Spontaneous anxiety is defined as behavior that is elicited entirely by the testing situation without current or prior aversive or explicitly induced stress. CRF₁ antisense oligonucleotide work is difficult to interpret because of potential toxicological side effects produced by the antisense oligonucleotide and, in some cases, the absence of verifiable reductions in CRF₁ receptor densities after treatment. Similar methods—CRF₂ knockouts, CRF₂ antisense oligonucleotides, and CRF₂ antagonists—were used to evaluate the function of CRF₂ receptors in emotionality. In comparison to the large number of CRF₁ receptor studies, fewer CRF₂ receptor investigations have been conducted and these studies have yielded mixed results. However, recent work demonstrating a robust reduction in CRF₂ receptors using a CRF₂ antisense oligonucleotide with minimal toxicity, and dose response studies using a peptide CRF₂ antagonist suggest that CRF₂ receptors play a role in stress-induced and spontaneous anxiety. Furthermore, inhibiting the actions of both CRF₁ and CRF₂ receptors produces a greater reduction in stress-induced behavior than inhibition of either receptor alone. Thus, current data suggest that CRF₁ and CRF₂ receptors are involved in the mediation of fear and anxiety behavior.     

Distribution of cannabinoid receptors in rat brain determined with aminoalkylindoles.

Extensive mapping of the cannabinoid receptor in rat brain has been reported recently using synthetic cannabinoids. Another class of compounds, the aminoalkylindoles (AAIs), does not resemble the cannabinoids structurally. Ligand binding data on isolated membranes, however, indicate that AAIs bind to the cannabinoid receptor. The present experiments compared the binding of AAIs and synthetic cannabinoids in vitro and by receptor autoradiography. The AAIs bound to a receptor in rat cerebellum with high affinity (Kd = 15 nM), and synthetic cannabinoids were potent competitors for AAI binding sites. In the autoradiographic studies in rat brain, an AAI and a synthetic cannabinoid were used to compete for the binding of a radiolabeled AAI to compare regionally and quantitatively the inhibition of AAI binding by the two classes of compounds. The distribution of the AAI binding was very similar to that reported for synthetic cannabinoid binding. These data add further evidence that the aminoalkylindoles bind to the cannabinoid receptor. Furthermore, the autoradiographic data for AAI binding, in addition to the autoradiographic data for the synthetic cannabinoid, provide a high degree of confidence in the localization of the cannabinoid receptor in the rat brain.