Dual-Transmitter Systems Regulating Arousal, Attention, Learning and Memory

An array of neuromodulators, including monoamines and neuropeptides, regulate most behavioural and physiological traits. In the past decade, dramatic progress has been made in mapping neuromodulatory circuits, in analysing circuit dynamics, and interrogating circuit function using pharmacogenetic, optogenetic and imaging methods This review will focus on several distinct neural networks (acetylcholine/GABA/glutamate; histamine/GABA; orexin/glutamate; and relaxin-3/GABA) that originate from neural hubs that regulate wakefulness and related attentional and cognitive processes, and highlight approaches that have identified dual transmitter roles in these behavioural functions. Modulation of these different neural networks might be effective treatments of diseases related to arousal/sleep dysfunction and of cognitive dysfunction in psychiatric and neurodegenerative disorders

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

Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons

This is a pre-print of an article published in Brain Structure and Function. The final authenticated version is available online at: https://doi.org/10.1007/s00429-018-1763-5In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the Gi/o-protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling

Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala

The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or ‘hubs’) within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin, calretinin and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In contrast, sparse anterogradely-labeled and relaxin-3-immunoreactive fibers were observed in other amygdala nuclei, including the lateral, central and basal nuclei and the nucleus accumbens lacked any innervation. Using synaptophysin as a synaptic marker, we identified relaxin-3 positive synaptic terminals in the medial amygdala, BST and endopiriform nucleus of amygdala. Our findings demonstrate the existence of topographic NI and relaxin-3-containing projections to specific nuclei of the extended amygdala, consistent with a likely role for this putative integrative arousal system in the regulation of amygdala-dependent social and emotional behaviors

Validation of ‘Somnivore’, a Machine Learning Algorithm for Automated Scoring and Analysis of Polysomnography Data

Manual scoring of polysomnography data is labor-intensive and time-consuming, and most existing software does not account for subjective differences and user variability. Therefore, we evaluated a supervised machine learning algorithm, SomnivoreTM, for automated wake–sleep stage classification. We designed an algorithm that extracts features from various input channels, following a brief session of manual scoring, and provides automated wake-sleep stage classification for each recording. For algorithm validation, polysomnography data was obtained from independent laboratories, and include normal, cognitively-impaired, and alcohol-treated human subjects (total n = 52), narcoleptic mice and drug-treated rats (total n = 56), and pigeons (n = 5). Training and testing sets for validation were previously scored manually by 1–2 trained sleep technologists from each laboratory. F-measure was used to assess precision and sensitivity for statistical analysis of classifier output and human scorer agreement. The algorithm gave high concordance with manual visual scoring across all human data (wake 0.91 ± 0.01; N1 0.57 ± 0.01; N2 0.81 ± 0.01; N3 0.86 ± 0.01; REM 0.87 ± 0.01), which was comparable to manual inter-scorer agreement on all stages. Similarly, high concordance was observed across all rodent (wake 0.95 ± 0.01; NREM 0.94 ± 0.01; REM 0.91 ± 0.01) and pigeon (wake 0.96 ± 0.006; NREM 0.97 ± 0.01; REM 0.86 ± 0.02) data. Effects of classifier learning from single signal inputs, simple stage reclassification, automated removal of transition epochs, and training set size were also examined. In summary, we have developed a polysomnography analysis program for automated sleep-stage classification of data from diverse species. Somnivore enables flexible, accurate, and high-throughput analysis of experimental and clinical sleep studies

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm

Functional neuroanatomy of the rat nucleus incertus–medial septum tract : implications for the cell-specific control of the septohippocampal pathway

The medial septum (MS) is critically involved in theta rhythmogenesis and control of the hippocampal network, with which it is reciprocally connected. MS activity is influenced by brainstem structures, including the stress-sensitive, nucleus incertus (NI), the main source of the neuropeptide relaxin-3 (RLN3). In the current study, we conducted a comprehensive neurochemical and electrophysiological characterization of NI neurons innervating the MS in the rat, by employing classical and viral-based neural tract-tracing and electrophysiological approaches, and multiplex fluorescent in situ hybridization. We confirmed earlier reports that the MS is innervated by RLN3 NI neurons and documented putative glutamatergic (vGlut2 mRNA-expressing) neurons as a relevant NI neuronal population within the NI–MS tract. Moreover, we observed that NI neurons innervating MS can display a dual phenotype for GABAergic and glutamatergic neurotransmission, and that 40% of MS-projecting NI neurons express the corticotropin-releasing hormone-1 receptor. We demonstrated that an identified cholecystokinin (CCK)-positive NI neuronal population is part of the NI–MS tract, and that RLN3 and CCK NI neurons belong to a neuronal pool expressing the calcium-binding proteins, calbindin and calretinin. Finally, our electrophysiological studies revealed that MS is innervated by A-type potassium current-expressing, type I NI neurons, and that type I and II NI neurons differ markedly in their neurophysiological properties. Together these findings indicate that the MS is controlled by a discrete NI neuronal network with specific electrophysiological and neurochemical features; and these data are of particular importance for understanding neuronal mechanisms underlying the control of the septohippocampal system and related behaviors

Localization of relaxin-3 in brain of Macaca fascicularis: identification of a nucleus incertus in primate

Relaxin-3 (RLN3) is a highly conserved, ancestral member of the insulin/relaxin peptide family. RLN3 mRNA is highly expressed in rat, mouse, and human brain and molecular genetic and pharmacological studies suggest that RLN3 is the cognate ligand for the relaxin family peptide-3 receptor (RXFP3). The distribution of RLN3/RXFP3 networks has been determined in rat and mouse brain, but not in higher species. In this study we describe the distribution of RLN3 neurons in the brain of macaque (Macaca fascicularis) using in situ hybridization histochemistry and immunohistochemistry. RLN3 mRNA and high levels of RLN3-like immunoreactivity (-LI) were observed in neurons within a ventromedial region of the central gray of the pons and medulla that appears to represent the primate analog of the nucleus incertus (NI) described in lower species. Nerve fibers and terminals containing RLN3-LI were observed throughout brain regions identical to those known to receive afferents from the NI in the rat, including the septum, hippocampus, entorhinal cortex, lateral, dorsomedial and ventromedial hypothalamus, supramammillary and interpeduncular nuclei, anterodorsal, paraventricular and reuniens thalamic nuclei, lateral habenula, central gray, and dorsal raphe, solitary tract, and ambiguus nuclei. Experimental studies in the rat strongly implicate a role of this neuropeptide-receptor system in arousal, feeding, and metabolism, learning and memory, and central responses to psychological stressors. These new anatomical findings support the proposition that the RLN3 system is similarly involved in the integration and modulation of behavioral activation and arousal and responses to stress in nonhuman primates and humans