Behavioral and anatomical interactions between dopamine and corticotropin-releasing factor in the rat

Abstract

The neuropeptide corticotropin-releasing factor (CRF) is believed to play a role in a number of psychiatric conditions, including anxiety disorders and depression. In the present study, male Sprague Dawley rats were used to examine the behavioral effects of altering dopamine transmission on CRF-enhanced startle, a behavioral assay believed to reflect stress- or anxiety-like states. Systemic administration of the selective dopamine D1 receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] (0, 0.01, 0.05, 0.1, 0.5 mg/kg) dose dependently blocked the effect of CRF (1 microg, i.c.v.) on startle at doses that had no effect on baseline startle response. Immunohistochemical studies showed that most CRF-containing cells in the dorsolateral division of the bed nucleus of the stria terminalis (BSTld), part of the critical brain area mediating CRF-enhanced startle, are surrounded by a dense plexus of tyrosine hydroxylase (TH)-positive fibers. Intra-BSTld injections of the retrograde tracer Fluorogold (FG) into the TH field identified neurons in the major dopaminergic areas (A8-A10), but not the major noradrenergic areas [A5, A6 (locus ceruleus), A7], as a significant source of TH-positive innervation. The majority of FG-filled cells double-labeled for TH were found in the dorsocaudal A10 cell group (A10dc) located in the periaqueductal gray area. Together, these data suggest that neuronal regulation of the BSTld by specific dopaminergic pathways and receptors may be an important mechanism for controlling CRF-dependent moods and affective states. These data also suggest that compounds with D1 receptor antagonist properties might have anxiolytic-like effects that could be useful for treating conditions associated with hyperactive CRF systems.

Figure 1.

Behavioral studies. A, Effect of pretreatment (subcutaneous) with the dopamine D₁ receptor antagonist SCH 23390 (SCH; doses in mg/kg) on the enhancement of startle by CRF (1 μg, i.c.v.). There was a significant dose-dependent reduction in CRF-enhanced startle by SCH 23390 (0.05 and 0.1 mg/kg) during peak CRF effects on startle; data are collapsed across blocks of 50 min for statistical comparisons. B, Effect of SCH 23390 on baseline startle responding. Baseline startle was not significantly different from vehicle controls (VEH-VEH group) at any dose of SCH 23390 (SCH) except for the high dose (0.5 mg/kg) during the first 50 min block of time. C, D, Full time course data are expressed as the percentage difference from the VEH-VEH group for the effect of SCH 23390 (SCH) on CRF-enhanced startle (C) and baseline startle (D). *p < 0.05, **p < 0.005, ***p < 0.0005 compared with VEH-VEH group; ^†p < 0.05, ^††p < 0.005 compared with VEH-CRF group.

Figure 2.

Anatomical studies. A, Coronal section (200×) of FITC-labeled CRF neurons in the BSTld. B, Same section showing TRITC-labeled TH-positive fibers in the BSTld. C, Merged image of A and B shows strong colocalization of CRF neurons with TH-positive fibers in the BSTld. To determine the source of the TH-positive innervation to the BSTld, discrete deposits of the retrograde tracer FG were made. D, Coronal section (100×) of an FG deposit in the BSTld; the deposit center is marked with an arrow. E, Light-microscope image of the adjacent brain section to that shown in D; the section was processed to determine the location of the FG deposit in relation to CRF neurons (brown-labeled cells) in the BSTld. F, Merged image of D and E shows the FG deposit to be restricted to the CRF-dense part of the BSTld with little spread into the adjacent caudate–putamen (CPu). The lateral ventricle (LV), AC, and prominent blood vessels (marked with asterisks) were used to align the two images for merging. G, Coronal section (200×) of retrogradely labeled FG-filled cells in the A10dc PAG area. H, Same section showing that most FG-filled cells in the PAG were also TH positive (indicated by arrows in G and H). I, Fewer FG-filled cells were seen in sections through the VTA, of which most were also TH positive (J). K, Coronal section (200×) showing retrogradely labeled cells at the level of the LC. FG-filled cells were not found in the LC but were seen in the adjacent medial parabrachial nucleus (MPB). L, Same section from K showing the location of the FG-filled cells (marked by arrows) in relation to TH-positive neurons (known to be noradrenergic) in the LC. Aq, Aqueduct of Sylvius; IPN, interpeduncular nucleus; 4V, fourth ventricle; Me5, mesencephalic 5 nucleus. Scale bars: A–C, G–L, 50 μm; D–F, 100 μm.

Figure 3.

Representative illustration of the location of an FG deposit (black area marked by an arrow) in the BSTld. FG-filled cells (open circles) and FG-filled cells double-labeled for TH (filled circles) were found throughout the major dopaminergic areas (A8-A10) but not the major noradrenergic areas (A5-A7). Neurons in the A10dc area were the greatest source of TH-positive innervation to the BSTld. A significant number of FG-filled and double-labeled neurons was also seen in the PV (Fig. 2G,H) surrounding the aqueduct of Sylvius (Aq). For clarity of the illustration, mapping of the PV efferents to the BSTld has been omitted. Coronal sections were taken from the atlas of Paxinos and Watson (1997) and are in millimeters caudal to bregma. BSTlv, Ventrolateral bed nucleus of the stria terminalis; CPu, caudate–putamen; MFB, medial forebrain bundle; LPB, lateral parabrachial nucleus; MPB, medial parabrachial nucleus; Me5, mesencephalic 5 nucleus; 3V, third ventricle; LV, lateral ventricle.

Figure 4.

A model of DA-CRF interactions underlying CRF effects on startle. Based on the results of the present study, together with other anatomical, electrophysiological, and behavioral studies (see Discussion for details), we proposed a BST-PAG feedforward circuit (adapted from Koob, 1999) that could mediate CRF effects on startle and account for the blockade of CRF-enhanced startle by SCH 23390. Activation of this circuit may be initiated by intracerebroventricular (icv) CRF (iC) effects on CRF neurons in the BSTld or A10dc DA neurons in the PAG. D₁ receptor-dependent, CRF-mediated effects could exit this feedforward loop and be transmitted to the primary acoustic startle pathway in the brainstem via a direct projection from the lateral BST to the PnC. Accordingly, blockade of D₁ receptors in the BST by SCH 23390 may sufficiently interrupt this feedforward activation leading to a reduction in CRF-enhanced startle. As reflected in our behavioral data, the small D₁-independent effect of intracerebroventricular CRF on startle may be mediated by a direct effect of CRF on neurons in the PnC. CRN, Cochlear root neurons.