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 2ϩ , 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 M) and were completely absent after exposure to the competitive CRF receptor antagonists ␣-helical CRF Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide with diverse physiological and behavioral functions. It is the principal regulator of the hypothalamo-pituitary-adrenal axis and is an important modulator of autonomic and behavioral responses to stressful stimuli (Owens and Nemeroff, 1991;Contarino et al., 1999), including anxiety and aversive states associated with drug withdrawal (Heinrichs et al., 1995;Sarnyai et al., 1995) and stressinduced relapse to drug-seeking behavior (Shaham et al., 1997). One mechanism through which CRF may modulate a broad spectrum of physiological and behavioral responses is via actions on ascending neuromodulatory systems, such as serotonergic systems.Several lines of evidence support the hypothesis that CRF plays a role in regulating serotonergic neurotransmission. First, moderate to high densities of CRF-immunoreactive neuronal cell bodies and fibers are associated with serotonergic neurons in brainstem raphe structures (Cummings et al., 1983;Sakanaka et al., 1986Sakanaka et al., , 1987Austin et al., 1997;Ruggiero et al., 1999). Second, CRF 1 and CRF 2 receptor binding sites, receptor mRNA expression, and CRF 1 receptor-immunoreactive neurons have been identified in raphe nuclei (De Souza et al., 1985;Chalmers et al., 1995;Vaughan et al., 1995;Bonaz and Rivest, 1998;Bittencourt and Sawchenko, 2000;Chen et al., 2000), raising the possibility that CRF or CRFlike peptides may have direct receptor-mediated actions on serotonergic neurons. Third, stress-related stimuli, particularly behavioral paradigms associated with increased anxiety or conditioned fear (Pezzone et al., 1993;Silveira et al., 1993;Beck and Fibiger, 1995;Matsuda et al., 1996;Beckett et al., 1997;Campeau and Watson, 1997;Kollack-Walker et al., 1997;Martinez et al., 1998;Nikulina et al., 1998;Chung et al., 1999Chung et al., , 2000Grahn et al., 1999), including opiate withdrawal (Chieng et al., 1995;Chahl et al., 1996) and intracerebroventricular infusion of CRF or CRF-like peptides (Vaughan et al., 1995;Bittencourt and Sawchenko, 2000), activate immediate-early gene expression with...