Do centrally administered neuropeptides access cognate receptors?: an analysis in the central corticotropin-releasing factor system

Abstract

To determine the extent to which centrally administered corticotropin-releasing factor (CRF) activates neurons that express CRF receptors (CRF-Rs), we followed the kinetics and distribution (relative to those of CRF-Rs) of Fos induction seen in response to intracerebroventricular (icv) injection of the peptide (1-10 microg). CRF provoked widespread Fos expression: its strength was dose-related, it peaked at 2 hr after injection, and it was antagonized in a dose-dependent manner by coinjection of CRF-R antagonists. The activation pattern closely mimicked the distribution of CRF-R1 mRNA, in including widespread Fos induction throughout the cortical mantle, in cell groups involved in sensory information processing, and in the cerebellum and several of its major afferents and targets. Dual labeling revealed extensive correspondence of CRF-stimulated Fos-immunoreactivity (Fos-ir) and CRF-R1 mRNA at these and other loci. Unique sites of CRF-R2 expression were relatively unresponsive to CRF but were more so after icv administration of urocortin (UCN), a new mammalian CRF-related peptide. Both CRF and UCN elicited activational responses in cell groups that are involved in central autonomic control but that express neither CRF-R, including the central amygdaloid and paraventricular hypothalamic nuclei, and brainstem catecholaminergic cell groups. The results support an ability of CRF-related peptides in the ventricular system to access receptor-expressing cells directly but leave open questions as to the basis for the recruitment of central autonomic structures, many of which have been identified as stress-related sites of CRF action.

Fig. 1.

Dose-related Fos-ir induction in response to central CRF. Bright-field photomicrographs are of immunoperoxidase preparations to show Fos-ir expression in the piriform cortex (PIR) and adjoining regions of saline-treated controls, and rats given icv injections of 1 or 10 μg CRF 2 hr before they were killed. Relative to low levels of expression seen in vehicle-treated animals, 1 μg CRF provokes Fos-ir in piriform cortex, the endopiriform nucleus (EPd), and the nucleus of the lateral olfactory tract (NLOT); substantially more robust responses of similar topography are seen in rats receiving 10 μg doses of peptide. Amyg, Amygdala. All photomicrographs 30× magnification.

Fig. 2.

Coinjection of a CRF receptor antagonist interferes with central CRF-induced Fos expression in rat brain. Bright-field photomicrographs are of immunoperoxidase preparations to show Fos-ir expression in the ventrolateral medulla of rats that received icv injections of 1 μg CRF alone (top) or with 10 μg (middle) or 100 μg [DPhe¹², Nle^21,38] r/hCRF_12–41. Major sites of peptide-stimulated Fos induction in the lateral reticular (LRN) and spinal trigeminal (SpV) nuclei are markedly diminished in animals coinjected with 10 μg, and essentially abolished in rats treated with 100 μg, of the antagonist. All sections are from animals killed at 2 hr after icv injection, the time of maximal Fos induction in most brain regions. IO, Inferior olivary complex; py, pyramidal tract;stv, spinal tract of the trigeminal nerve. All photomicrographs 30× magnification.

Fig. 3.

Effects of icv injections at the brain-fluid interfaces. Bright-field photomicrographs show Fos-ir expression in the septal region, near the site of icv injection (top four panels) and caudal brainstem (bottom) in rats killed 2 hr after treatment. Injection of 1 μg oCRF_9–33, a peptide fragment that is bound with low affinity by each of the known CRF binding moieties, provokes little evidence of Fos induction even near the site of infusion (top). This contrasts with the effects of 1 μg injections of CRF or UCN (Fig. 7). Injection of 10 μg of the CRF receptor antagonist [D-Phe¹², Nle^21,38] rat/human CRF_12–41(D-Phe) evokes activational responses primarily in the ependymal lining of the ventricular system (ep) near the site of infusion and in immediately adjoining cells but only sporadically in deeper aspects of the brain parenchyma. High doses of the antagonist alone (100 μg D-Phe;middle) produce more robust labeling of the ependyma and periventricular regions, although deep parenchymal labeling is prominent near the site of infusion on the ipsilateral (Ipsi) but not the contralateral (Contra) side of the brain. High doses of the antagonist also result in extensive labeling of the ependyma throughout the ventricular system, as evidenced by labeling seen near the medullary spinal transition area (bottom) and additionally in the meninges (men) and in cells at and just deep to the pial surface of the brain (bottom). Note that labeling at the ependymal and pial surfaces spreads substantially to include cells in deeper regions of the parenchyma only near the site of icv injection.XII, Hypoglossal nucleus; AP, area postrema; cc, central canal; DMX, dorsal motor nucleus; IO, inferior olive;LSv, lateral septal nucleus ventral; Lsi, lateral septal nucleus intermediate; LRN, lateral reticular nucleus; MS, medial septal nucleus;NTS, nucleus of the solitary tract; sctv, spinocerebellar tract; vl, lateral ventricle. All photomicrographs 75× magnification, except bottom right(100×).

Fig. 4.

Relationship of forebrain sites of CRF-induced Fos-ir to loci of CRF-R1 mRNA expression. Patterns of Fos induction in various brain regions seen at 2 hr after icv injection of 1 μg CRF (bright-field photomicrographs, left) in relation to the distribution of CRF-R1 mRNA expression in the same regions (dark-field photomicrographs, right). At each level, the correspondence between the distribution of the two markers is striking and extends to the laminar and/or subnuclear levels. All major sites of Fos induction shown here express CRF-R1, except for the lateral part of the central nucleus of the amygdala (CeA), which expresses neither CRF receptor. I–VI, Isocortical layers; AON, anterior olfactory nucleus;BLA, basolateral nucleus of the amygdala;BMA, basomedial nucleus of the amygdala;CP, caudoputamen; ec, external capsule;epl, external plexiform layer (olfactory bulb);EPd, dorsal endopiriform nucleus; gl, glomerular layer (olfactory bulb); gr, granule cell layer (olfactory bulb); ic, internal capsule;m, mitral cell layer (olfactory bulb). All photomicrographs 30× magnification.

Fig. 5.

Some brainstem sites of CRF-induced Fos-ir in relation to loci of CRF-R1 mRNA expression. Shown are patterns of Fos induction in brainstem regions seen at 2 hr after icv injection of 1 μg CRF (bright-field, left) and patterns of CRF-R1 mRNA expression in the same regions (dark-field, right). Again, the distributions of the two markers are highly congruent, and most major areas in which Fos induction was detected also express CRF-R1, except for the locus coeruleus (LC), which expresses neither CRF-R, and the dorsal raphé nucleus (DR), aspects of which express CRF-R1 at low levels but CRF-R2 more robustly. aq, Cerebral aqueduct;DTN, dorsal tegmental nucleus; LDT, laterodorsal tegmental nucleus; mcp, middle cerebellar peduncle; mlf, medial longitudinal fasciculus;NI, nucleus incertus; PAG, periaqueductal gray; PG, pontine gray; TRN, tegmental reticular nucleus; V4, fourth ventricle;VLL, ventral nucleus of the lateral lemniscus. All photomicrographs 30× magnification.

Fig. 6.

Many neurons that are sensitive to icv CRF injection express CRF-R1 mRNA. Bright-field photomicrographs of combined immunohistochemical and hybridization histochemical preparations show localization of CRF-stimulated Fos-ir (brown nuclei) and CRF-R1 mRNA (blacksilver grains). Overlapping distributions are seen in field CA3 of the hippocampal formation, basolateral amygdaloid (BLA), medial septal (MS), and lateral reticular (LRN) nuclei, among many other regions. Examples of doubly labeled cells are indicated (arrows). All photomicrographs 300× magnification.

Fig. 7.

In the septal region, icv CRF- and UCN-induced Fos induction patterns preferentially conform to sites of CRF-R1 and CRF-R2 mRNA expression, respectively. Top, Sections through the septal region to show the differential distributions of CRF-R1 mRNA in the medial septal nucleus (MS) and CRF-R2 transcripts in the intermediate lateral septal nucleus (LSi). Bottom, Patterns of Fos induction seen 2 hr after icv injections of 1 μg CRF or UCN preferentially localize to regions enriched in CRF-R1 and CRF-R2 expression, respectively, but show overlap with the “nonpreferred” receptor distribution. vl, Lateral ventricle. All photomicrographs 75× magnification.

Fig. 8.

UCN provokes Fos induction comparable to that elicited by CRF in some, but not all, sites of CRF-R1 expression. Bright-field photomicrographs show immunoperoxidase material from animals killed 2 hr after icv injection of 1 μg CRF or UCN to compare Fos-ir induction patterns. In the ventral midbrain (top), CRF provokes activational responses in the substantia nigra (SNc, SNr), and the red (RN) and interpeduncular (IPN) nuclei, all of which are sites of CRF-R1 expression. UCN-stimulated Fos is seen principally in the interpeduncular nucleus, which alone among the structures shown is a site of substantial CRF-R2 expression. Both peptides provoke comparably robust activational responses in the lateral parabrachial nucleus (middle; concentrated in its external lateral subnucleus, el), a pivotal structure in the central autonomic system that expresses CRF-R1, but not CRF-R2, mRNA. In the cerebellar cortex (bottom), another unique site of CRF-R1 expression, icv UCN elicits a Fos-ir response the strength and distribution of which are similar to that provoked by CRF. Magnifications: top, 30×; middle andbottom, 50×.