Key points• The nucleus incertus (NI) is a stress and arousal responsive, hindbrain region involved in ascending control of septohippocampal theta rhythm.• NI neurons express high levels of the neuropeptide relaxin-3 and corticotrophin-releasing factor (CRF) receptor-1 (CRF-R1).• We report the first in-depth characterization of NI neurons, using in vivo and in vitro electrophysiological techniques, which reveal a population of relaxin-3-containing NI neurons activated by CRF via postsynaptic CRF-R1 and a non-relaxin-3 neuron population inhibited or unaffected by CRF.• Relaxin-3 NI neurons exhibit strong phase-locked firing with the ascending phase of hippocampal theta oscillations.• These findings suggest the NI is a heterogeneous neuronal population and key site of CRF action with the capacity to modulate cognition in response to stress.Abstract The nucleus incertus (NI) of the rat hindbrain is a putative node in the ascending control of the septohippocampal system and hippocampal theta rhythm and is stress and arousal responsive. NI contains GABA neurons that express multiple neuropeptides, including relaxin-3 (RLN3) and neuropeptide receptors, including corticotrophin-releasing factor receptor-1 (CRF-R1), but the precise anatomical and physiological characteristics of NI neurons are unclear. Therefore, we examined the firing properties of NI neurons and their responses to CRF, the correlation of these responses with occurrence of relaxin-3, and NI neuron morphology in the rat. Most NI neurons excited by intracerebroventricular CRF infusion were RLN3-positive (9 of 10), whereas all inhibited cells were RLN3-negative (8 of 8). The spontaneous firing of RLN3 (n = 6) but not non-RLN3 neurons (n = 6) was strongly modulated and phase-locked with the initial ascending phase of hippocampal theta oscillations. In brain slices, the majority of recorded NI neurons (15 of 19) displayed excitatory responses to CRF, which uniformly increased action potential frequency and membrane potential depolarization in the presence of tetrodotoxin, indicating a direct, postsynaptic action of CRF on NI neurons. This excitation was associated with reduction in the slow component of afterhyperpolarization and a strong depolarization. Quantitative analysis in naïve rats of validated CRF-R1, RLN3 and neuronal nuclear antigen (NeuN) immunoreactivity revealed 52% of NI neurons as CRF-R1 positive, of which 53% were RLN3 positive, while 48% of NI neurons lacked CRF-R1 and RLN3. All RLN3 neurons expressed CRF-R1. CRF neurons that projected to the NI were identified in lateral preoptic hypothalamus, but not in paraventricular hypothalamus, bed nucleus of stria terminalis or central amygdala. Our findings suggest NI is an important site for CRF modulation of hippocampal theta rhythm via effects on GABA/RLN3 transmission.

This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra-PVN injections of the neuropeptide relaxin-3 or selective relaxin-3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin-3, acting through its cognate G-protein-coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin-3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3-A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3-A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri-PVN and sparse intra-PVN relaxin-3-immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin-3 neurons, identify a strong inhibitory influence of relaxin-3-RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

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Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin-3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.

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Index Coding has received considerable attention recently motivated in part by applications such as fast video-on-demand and efficient communication in wireless networks and in part by its connection to Network Coding. Optimal encoding schemes and efficient heuristics were studied in various settings, while also leading to new results for Network Coding such as improved gaps between linear and non-linear capacity as well as hardness of approximation. The basic setting of Index Coding encodes the side-information relation, the problem input, as an undirected graph and the fundamental parameter is the broadcast rate β, the average communication cost per bit for sufficiently long messages (i.e. the non-linear vector capacity). Recent nontrivial bounds on β were derived from the study of other Index Coding capacities (e.g. the scalar capacity β 1 ) by Bar-Yossef et al (2006), Lubetzky and Stav (2007) and Alon et al (2008). However, these indirect bounds shed little light on the behavior of β: there was no known polynomial-time algorithm for approximating β in a general network to within a nontrivial (i.e. o(n)) factor, and the exact value of β remained unknown for any graph where Index Coding is nontrivial.Our main contribution is a direct information-theoretic analysis of the broadcast rate β using linear programs, in contrast to previous approaches that compared β with graph-theoretic parameters. This allows us to resolve the aforementioned two open questions. We provide a polynomial-time algorithm with a nontrivial approximation ratio for computing β in a general network along with a polynomial-time decision procedure for recognizing instances with β = 2. In addition, we pinpoint β precisely for various classes of graphs (e.g. for various Cayley graphs of cyclic groups) thereby simultaneously improving the previously known upper and lower bounds for these graphs. Via this approach we construct graphs where the difference between β and its trivial lower bound is linear in the number of vertices and ones where β is uniformly bounded while its upper bound derived from the naive encoding scheme is polynomially worse.

Many physiological processes fluctuate throughout the day/night and daily fluctuations are observed in brain and peripheral levels of several hormones, neuropeptides and transmitters. In turn, mediators under the “control” of the “master biological clock” reciprocally influence its function. Dysregulation in the rhythmicity of hormone release as well as hormone receptor sensitivity and availability in different tissues, is a common risk-factor for multiple clinical conditions, including psychiatric and metabolic disorders. At the same time circadian rhythms remain in a strong, reciprocal interaction with the hypothalamic-pituitary-adrenal (HPA) axis. Recent findings point to a role of circadian disturbances and excessive stress in the development of obesity and related food consumption and metabolism abnormalities, which constitute a major health problem worldwide. Appetite, food intake and energy balance are under the influence of several brain neuropeptides, including the orexigenic agouti-related peptide, neuropeptide Y, orexin, melanin-concentrating hormone and relaxin-3. Importantly, orexigenic neuropeptide neurons remain under the control of the circadian timing system and are highly sensitive to various stressors, therefore the potential neuronal mechanisms through which disturbances in the daily rhythmicity and stress-related mediator levels contribute to food intake abnormalities rely on reciprocal interactions between these elements.