Differential Expression of 5HT-1A, α1b Adrenergic, CRF-R1, and CRF-R2 Receptor mRNA in Serotonergic, γ-Aminobutyric Acidergic, and Catecholaminergic Cells of the Rat Dorsal Raphe Nucleus

The dorsal raphe nucleus (DR) contains the highest concentration of serotonin neurons in the brain and has extensive ascending projections that innervate most forebrain structures (Steinbusch, 1981). These projection neurons have been shown to regulate a wide variety of physiological responses and behaviors, including sleep–wake states, feeding, nociception, neuroendocrine responses, and motor activities (Jacobs and Azmitia, 1992). In addition, increasing evidence has implicated serotonin in affective conditions, such as depression and anxiety (Kahn et al., 1988; Graeff et al., 1996). However, the DR is not a homogeneous structure, and serotonin neurons have been classified into subpopulations according to their electrophysiological properties in behaving animals. For example, the activity of type I neurons that make up the majority of serotonergic neurons is correlated with the degree of behavioral arousal and motor activity (Jacobs and Fornal, 1999). In contrast, the firing rate of the small group of type II serotonergic neurons, found at the caudal interface of the DR and median raphe nucleus, does not correlate with changes in arousal or activity (Rasmussen et al., 1984). Although substantial numbers of DR neurons contain serotonin, many other neurotransmitters are also present, including γ-aminobutyric acid (GABA), dopamine, glutamate, and corticotropin-releasing factor (CRF), and the distribution of cellular neurochemical phenotypes varies across the rostral-caudal extent of the nucleus (Gamrani et al., 1979; Stratford and Wirtshafter, 1990; Jacobs and Azmitia, 1992; Commons et al., 2003). In addition, both afferent and efferent projections exhibit a distinct topographic arrangement (Kohler and Steinbusch, 1982; Van Bockstaele et al., 1993; Peyron et al., 1996, 1998). Together these observations are consistent with the idea that distinct regions of the DR may subserve different functions. Experimental support for this idea has recently been obtained with the animal model of learned helplessness. In this model, animals that experience uncontrollable shock exhibit a behavioral profile different from that of animals that have control over and can escape an equivalent shock, including an interference with subsequent escape behavior (Overmier and Seligman, 1967; Irwin et al., 1980), increased conditioned fear responses (Osborne et al., 1975), and increased anxiety (Short and Maier, 1993). Converging evidence suggests that the expression of learned helplessness following an uncontrollable stressor requires activation of the serotonergic neurons of the DR, specifically, in the caudal half of the nucleus, and interventions that reduce that activation prevent the development and/or expression of learned helplessness behaviors (Maier et al., 1993, 1994, 1995; Maswood et al., 1998; Grahn et al., 1999; Greenwood et al., 2003). Administration of CRF into the caudal, but not rostral, DR impeded escape behavior and increased conditioned fear in a manner similar to uncontrollable stress (Hammack et al., 2002). Furthermore, the behavioral consequences of exposure to uncontrollable shock were prevented by administration of a CRF receptor antagonist into the DR before exposure to the shock (Hammack et al., 2002). Use of more selective agonists and antagonists revealed that these effects were due to activation of the CRF-R2 receptor (Hammack et al., 2003). Consistent with these effects is the observation that CRF increases serotonergic activity in a subpopulation of cells in the caudal DR (Lowry et al., 2000). In contrast, the activity of serotonergic cells in the rostral to mid-DR is reportedly inhibited by CRF (Kirby et al., 2000). Despite the growing evidence for rostral-caudal and dorsal-ventral variations in DR responses, relatively little is known about the relative distribution and colocalization patterns of neurotransmitters and receptors in the DR. The present dual in situ hybridization study was therefore carried out to assess whether there was a rostral-caudal difference in the distribution of mRNA encoding the CRF receptors (types 1 and 2) in the DR and to determine whether they are expressed in serotonergic, GABAergic, or catecholaminergic neurons. In addition, the distribution and colocalization pattern of the α1b adrenergic receptor (α1b ADR) mRNA was determined, because noradrenergic afferents to the DR exert a tonic excitatory effect on serotonergic neurons of the DR through an α1 ADR mechanism (Baraban and Aghajanian, 1980a,b, 1981; Yoshimura et al., 1985), intra-DR administration of a selective α1 ADR antagonist prevents the behavioral consequences of uncontrollable stress exposure (Grahn et al., 2002), and mRNA for the α1b subtype is highly expressed in the rat DR (Day et al., 1997). Finally, the colocalization pattern of serotonin 1A (5HT-1A) receptor mRNA was determined, because this autoreceptor plays a vital role in the direct inhibition of serotonergic activity (Sprouse and Aghajanian, 1987), because its activation can prevent and reverse the enhancement of fear conditioning and interference with escape produced by exposure to inescapable shock (Maier et al., 1995), and because its expression on nonserotonergic neurons has been reported recently (Kirby et al., 2003). The distribution of mRNA encoding these receptors in serotonergic, GABAergic, and catecholaminergic neurons at four rostral-caudal levels, and dorsal, ventral, and lateral divisions of the DR is described. The differential distribution patterns observed provide additional evidence that the DR has a functional topographic organization.