Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system

Abstract

In vivo studies suggest that the stress-related neuropeptide corticotropin-releasing factor (CRF) modulates serotonergic neurotransmission. To investigate the underlying mechanisms for this interaction, the present study examined the effects of CRF in vitro on dorsal raphe neurons that displayed electrophysiological and pharmacological properties consistent with a serotonergic phenotype. In the presence of either 1 or 2 mm Ca(2+), perfusion of ovine CRF or rat/human CRF rapidly and reversibly increased firing rates of a subpopulation (19 of 70, 27%) of serotonergic neurons predominantly located in the ventral portion of the dorsal raphe nucleus. For a given responsive neuron, the excitatory effects of CRF were reproducible, and there was no tachyphylaxis. Excitatory effects were dose-dependent (over the range of 0.1-1.6 micrometer) and were completely absent after exposure to the competitive CRF receptor antagonists alpha-helical CRF(9-41) or rat/human [d-Phe(12), Nle(21, 38), alpha-Me-Leu(37)]-CRF(12-41). Both the proportion of responsive neurons and the magnitude of excitatory responses to CRF in the ventral portion of the caudal dorsal raphe nucleus were markedly potentiated in slices prepared from animals previously exposed to isolation and daily restraint stress for 5 d. Immunohistochemical staining of the recorded slices revealed close associations between CRF-immunoreactive varicose axons and tryptophan hydroxylase-immunoreactive neurons in the area of the recordings, providing anatomical evidence for potential direct actions of CRF on serotonergic neurons. The electrophysiological properties and the distribution of responsive neurons within the dorsal raphe nucleus are consistent with the hypothesis that endogenous CRF activates a topographically organized mesolimbocortical serotonergic system.

Fig. 1.

CRF increases the firing rate of a subpopulation of serotonergic neurons in the dorsal raphe nucleus. A,B, The majority of serotonergic neurons studied in the dorsal raphe nucleus were unaffected by application of rhCRF or oCRF.PE (−), Removal of phenylephrine from the aCSF.C, In contrast, a small subpopulation of serotonergic neurons responded with a rapid, reversible increase in firing rate. In this example, the excitatory effects of oCRF were reversed by previous application of the long-acting CRF receptor antagonistd-Phe-CRF_12–41. Coapplication of 1.2 μm oCRF and 2 μmd-Phe-CRF_12–41 resulted in a decreased duration of the excitatory effects of CRF, whereas a subsequent coapplication of 1.2 μm oCRF and 4 μmd-Phe-CRF_12–41 resulted in a complete inhibition of the excitatory effects of oCRF. Responses to subsequent applications of 1.2 μm oCRF every 15 min up to 1 hr were also inhibited. D, Diagrammatic illustration summarizing the recorded locations of 70 serotonergic neurons studied during the application of rhCRF or oCRF to slices from group-housed rats (open symbols, nonresponsive neurons; filled symbols, responsive neurons). A higher percentage of neurons in the ventral and interfascicular regions of the dorsal raphe nucleus (DRV) were stimulated by CRF compared with neurons in the dorsal region of the dorsal raphe nucleus (DRD). Aq, Aqueduct; mlf, medial longitudinal fasciculus.

Fig. 2.

Comparison of the effects of CRF on the firing rates of serotonergic neurons in control and stressed rats, based on electrophysiological recordings in the ventral portion of the caudal dorsal raphe nucleus (DRV). A, Application of 400 nm (top) or 1.2 μm (bottom) oCRF had no effect on the firing rate of the majority of serotonergic neurons recorded from control animals. B, Application of 400 nm(top) or 1.2 μm (bottom) oCRF reversibly increased the firing rate of a proportion of serotonergic neurons recorded from stressed animals. C, CRF dose-dependently increased the firing rate of a serotonergic neuron in the ventral portion of the caudal dorsal raphe nucleus.D, Dose–response curves (0.4–2 μm oCRF) for three serotonergic neurons in the ventral portion of the caudal dorsal raphe nucleus in slices from stressed rats. E, Mean change in firing rate of the first cell tested in each animal at 400 and 1200 nm oCRF. Serotonergic neurons responded to 400 nm or 1.2 μm oCRF with greater increases in firing rate in stressed rats (stippled bars) compared with control rats (solid bars). *p≤ 0.01, **p ≤ 0.001.

Fig. 3.

Immunohistochemical double-labeling of associations between CRF-immunoreactive fibers (SG,blue reaction product) and tryptophan hydroxylase-immunoreactive neurons (DAB, brown reaction product) within tissues used previously for electrophysiological recordings. A, C, and Dare from the same 30 μm section at approximately bregma −8.00 mm.A, Schematic illustration of the location of the recording electrode during recording of the unit illustrated in Figure1C. B, Camera lucida drawing of tryptophan hydroxylase-immunoreactive neurons from an alternate section to that illustrated in A. Superimposed on this drawing are indications of the locations of dense bands of CRF-immunoreactive fibers (dotted ovals; illustrated for 5 cases), which ascend through the DpMe and then the VLPAG at progressively more caudal anatomical levels. These fibers, the pattern of tryptophan hydroxylase immunoreactive staining, and other anatomical features permitted precise identification of the rostrocaudal levels of recordings in these animals. C, Higher magnification of the interfascicular region of the dorsal raphe nucleus illustrated inA. CRF-immunoreactive varicose fibers were visible throughout the interfascicular region of the dorsal raphe nucleus. Illustrated are close associations between CRF-immunoreactive varicose fibers and some tryptophan hydroxylase-immunoreactive neurons (arrowheads), raising the possibility that synaptic specializations may exist between CRF fibers and a subpopulation of serotonergic neurons. D, Higher magnification of the dorsomedial portion of the dorsal raphe nucleus illustrated inA. CRF-immunoreactive varicose fibers were visible throughout the dorsal part of the dorsal raphe nucleus, although electrophysiological recordings failed to identify a significant population of CRF-responsive serotonergic neurons in the dorsal part of the dorsal raphe nucleus. E, Tryptophan hydroxylase-immunoreactive neuronal cell bodies and fibers and CRF-immunoreactive varicose fibers in the dorsal raphe nucleus at bregma −7.80 mm. Note the dense CRF-immunoreactive fibers throughout the VLPAG and the bundle of CRF-immunoreactive fibers in the DpMe (dotted oval). Also, tryptophan hydroxylase-immunoreactive neurons were more dense within the DRV than at bregma −8.00 mm (A). Rectangleindicates region illustrated in F. F, Higher magnification of the VLPAG and the ventrolateral part of the dorsal raphe nucleus. CRF-immunoreactive varicose fibers were visible throughout this region and were particularly dense in the VLPAG; pericellular baskets of CRF-immunoreactive varicosities resulted in a dense patch-like pattern of immunolabeling. Aq, Aqueduct; DLPAG, dorsolateral periaqueductal gray;DpMe, deep mesencephalic nucleus; DRD, dorsal raphe nucleus, dorsal part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; dtg, dorsal tegmental bundle;LPAG, lateral periaqueductal gray; me5, mesencephalic trigeminal tract; mlf, medial longitudinal fasciculus; VLPAG, ventrolateral periaqueductal gray. Scale bars: A, B, E, 400 μm; C, D, F, 50 μm.