Distribution and targets of the relaxin-3 innervation of the septal area in the rat

Neural tracing studies have revealed that the rat medial and lateral septum are targeted by ascending projections from the nucleus incertus, a population of tegmental GABA neurons. These neurons express the relaxin-family peptide, relaxin-3, and pharmacological modulation of relaxin-3 receptors in medial septum alters hippocampal theta rhythm and spatial memory. In an effort to better understand the basis of these interactions, we have characterized the distribution of relaxin-3 fibers/terminals in relation to different septal neuron populations identified using established protein markers. Dense relaxin-3 fiber plexuses were observed in regions of medial septum containing hippocampal-projecting choline acetyltransferase (ChAT)-, neuronal nitric oxide synthase (nNOS)-, and parvalbumin (PV)-positive neurons. In lateral septum (LS), relaxin-3 fibers were concentrated in the ventrolateral nucleus of rostral LS and the ventral nucleus of caudal LS, with sparse labeling in the dorsolateral and medial nuclei of rostral LS, dorsal nucleus of caudal LS, and ventral portion nuclei. Relaxin-3 fibers were also observed in the septofimbrial and triangular septal nuclei. In the medial septum, we observed relaxin-3-immunoreactive contacts with ChAT-, PV-, and glutamate decarboxylase-67-positive neurons that projected to hippocampus, and contacts between relaxin-3 terminals and calbindin- and calretinin-positive neurons. Relaxin-3 colocalized with synaptophysin in nerve terminals in all septal areas, and ultrastructural analysis revealed these terminals were symmetrical and contacted spines, somata, dendritic shafts, and occasionally other axonal terminals. These data predict that this GABA/peptidergic projection modulates septohippocampal activity and hippocampal theta rhythm related to exploratory navigation, defensive and ingestive behaviors, and responses to neurogenic stressors. J. Comp. Neurol. 520:1903–1939, 2012. © 2011 Wiley Periodicals, Inc.

Arousal neural pathways of the brain are associated with modulation of behavior in accordance with environmental requirements and a key node in the regulation of arousal is the forebrain septal area. Ascending connections from the medial septum to the hippocampus are proposed to provide “pacemaker” control of hippocampal theta rhythm (Vertes and Kocsis,1997; Hangya et al.,2009), which may underpin goal-oriented behavior (Vinogradova,1995) and plastic changes occurring during the formation of cognitive maps (O'Keefe,1993), whereas descending projections from the lateral septum target a wide variety of subcortical circuits related to visceral and metabolic functions, ranging from aggression, social and sexual behavior, to circadian rhythms (Albert and Chew,1980; Risold and Swanson,1997a; Veenema and Neumann,2007).

The septal area plays a central role in controlling hippocampal function, and the importance of the medial septum for “pacemaking” of hippocampal theta rhythm was noted in early studies (Pestche and Stumpf,1962; Andersen et al.,1979; Vinogradova,1995). This view was strengthened by more recent EEG recordings in freely moving rats that demonstrated that the integrity of the entire medial and lateral septum-hippocampal network is critical for the generation of theta rhythm (Nerad and McNaughton,2006). There has also been a consensus over many years that the different types of neurons in the septal area play specific roles in generating theta synchrony, with slow-firing cholinergic neurons facilitating hippocampal firing, and parvalbumin GABAergic neurons that innervate GABAergic hippocampal interneurons driving disinhibition of pyramidal or granule cell inhibition, allowing hippocampal synchrony (Freund and Antal,1988; Freund and Gulyas,1997; Toth et al., 1997a; Wu et al.,2000), although more recent studies have questioned the relative importance of different neuron populations in awake animals (e.g., Simon et al.,2006).

Neural tract-tracing studies in the rat by our laboratory and others have demonstrated that the septal area is targeted by ascending projections arising from the nucleus incertus (Goto et al.,2001; Olucha-Bordonau et al.,2003). Neurons of the nucleus incertus contain GABA and a range of peptides, such as cholecystokinin, neurotensin, neuromedin B, and atrial natriuretic peptide (Kubota et al.,1983; Ryan et al., 1995; Olucha-Bordonau et al.,2003; see Ryan et al.,2011, for review). Recent studies have revealed that a large population of nucleus incertus neurons express high levels of the peptide relaxin-3 (RLN3), which is primarily expressed in this region, in addition to smaller adjacent tegmental and midbrain cell groups (Burazin et al.,2002; Bathgate et al.,2003; Tanaka et al.,2005; Ma et al.,2007). The nucleus incertus provides a distinct pattern of ascending projections to raphé nuclei, periaqueductal gray, supramammillary nucleus, several hypothalamic nuclei, midline intralaminar nuclei, habenula, amygdala, hippocampus, the septal area, and the prefrontal cortex (Goto et al.,2001; Olucha-Bordonau et al.,2003). This pattern of efferents overlaps extensively with the forebrain distribution of RLN3-containing nerve fibers (Tanaka et al.,2005; Ma et al.,2007). The native receptor for RLN3 is G-protein coupled receptor-135 (GPCR135) (Liu et al.,2003) or “RXFP3” (Bathgate et al.,2006) and the regional topography of RXFP3 in rat brain is largely consistent with the distribution of RLN3-positive fibers (Ma et al.,2007).

The strong connections of the nucleus incertus with a number of brain areas involved in brainstem-diencephalic modulation of hippocampal theta rhythm, such as the median raphé, supramammillary nucleus and the medial septum (Vertes et al., 1993a; Vertes and Kocsis,1997), led us to hypothesize a role for the nucleus incertus in theta rhythm activation. We subsequently demonstrated that stimulation of nucleus incertus in urethane-anesthetized rats increased theta and decreased delta activity of the hippocampus, whereas electrolytic lesion of the nucleus incertus abolished hippocampal theta induced by stimulation of the nucleus reticularis pontis oralis (RPO) (Nunez et al.,2006), a key brainstem generator of hippocampal theta rhythm (Vertes,1981, 1982; Nunez et al.,1991; Vertes et al., 1993b; Vertes and Kocsis,1997). The hippocampal area in which field potentials were recorded receives only sparse inputs from the nucleus incertus, and it was concluded that the influence of the nucleus incertus on hippocampal theta rhythm was most likely mediated by its effects within the medial septum and/or other lower brain structures. In fact, the nucleus incertus is presumed to be the major relay station of RPO inputs to the medial septum (and hippocampus), as there are no direct projections from the RPO to hippocampus (Teruel-Marti et al.,2008). Additionally, RPO stimulation results in theta synchronization in the hippocampus and nucleus incertus, at the same frequency and with a high degree of coherence (Cervera-Ferri et al.,2011). Furthermore, because the nucleus incertus is an RLN3 locus in the brain, we hypothesized that RLN3 might contribute to these effects. Consistent with the presence of RLN3 and RXFP3 in the medial septum, injections of a selective RXFP3 agonist peptide (R3/I5; Liu et al.,2005) into this area increased theta activity of the hippocampal field potential in urethane-anesthetized rats, which was significantly attenuated by prior injection of a selective RXFP3 antagonist peptide, R3(BΔ23-27)R/I5 (Kuei et al.,2007; Ma et al.,2009b). R3/I5 infusion into the medial septum also increased hippocampal theta in rats in a familiar home cage environment, whereas R3(BΔ23-27)R/I5 decreased hippocampal theta in rats exploring a novel enriched context (Ma et al.,2009b). These data support a significant contribution of nucleus incertus and RLN3 inputs to the septum in regulating a fundamental brain activity and associated complex behaviors, and therefore characterization of the anatomical and cellular interactions between these inputs and their targets is required.

The goal of the current study, therefore, was to map the distribution of RLN3 positive-fibers throughout the rat septum in relation to particular “landmark” neuron populations. This was achieved in a series of double-labeling experiments using a characterized RLN3 antiserum and antisera for established protein markers expressed by neurons in the septal area. We examined whether RLN3-positive fibers made close contacts with the major septal neuron types in triple- and quadruple-labeling studies combined with confocal microscopy analysis. We also examined the colocalization of RLN3 staining with that for the presynaptic marker, synaptophysin (Jahn et al.,1985), to assess the presence of RLN3 within synapses in the septum. Finally, we conducted ultrastructural analyses of RLN3-positive synapses in the septal area using electron microscopy. The data obtained provide strong anatomical evidence for a role of RLN3 in modulating the activity of specific neurons in the septum that have direct connections with the hippocampus, which may underlie the effects of RLN3/RXFP3 signaling on hippocampal theta rhythm and associated complex behaviors