Anna Blasiak

Heterogeneous responses of nucleus incertus neurons to corticotrophin‐releasing factor and coherent activity with hippocampal theta rhythm in the rat

•  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.

Relaxin-3 innervation of the intergeniculate leaflet of the rat thalamus - neuronal tract-tracing and in vitro electrophysiological studies

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.

Distribution, physiology and pharmacology of relaxin-3/RXFP3 systems in brain

Relaxin‐3 is a member of a superfamily of structurally‐related peptides that includes relaxin and insulin‐like peptide hormones. Soon after the discovery of the relaxin‐3 gene, relaxin‐3 was identified as an abundant neuropeptide in brain with a distinctive topographical distribution within a small number of GABAergic neuron populations that is well conserved across species. Relaxin‐3 is thought to exert its biological actions through a single class‐A GPCR – relaxin‐family peptide receptor 3 (RXFP3). Class‐A comprises GPCRs for relaxin‐3 and insulin‐like peptide‐5 and other peptides such as orexin and the monoamine transmitters. The RXFP3 receptor is selectively activated by relaxin‐3, whereas insulin‐like peptide‐5 is the cognate ligand for the related RXFP4 receptor. Anatomical and pharmacological evidence obtained over the last decade supports a function of relaxin‐3/RXFP3 systems in modulating responses to stress, anxiety‐related and motivated behaviours, circadian rhythms, and learning and memory. Electrophysiological studies have identified the ability of RXFP3 agonists to directly hyperpolarise thalamic neurons in vitro, but there are no reports of direct cell signalling effects in vivo. This article provides an overview of earlier studies and highlights more recent research that implicates relaxin‐3/RXFP3 neural network signalling in the integration of arousal, motivation, emotion and related cognition, and that has begun to identify the associated neural substrates and mechanisms. Future research directions to better elucidate the connectivity and function of different relaxin‐3 neuron populations and their RXFP3‐positive target neurons in major experimental species and humans are also identified.

Are ultra-slow isoperiodic oscillations in rat intergeniculate leaflet neurons dependent on reciprocal connection with its contralaterally located counterpart?

We investigated effects of the electrical lesion and/or chemical inactivation of intergeniculate leaflet (IGL) neurons on the ultra-slow isoperiodic neuronal oscillation of the contralaterally located IGL. The spontaneous extracellular activity of neurons, recorded simultaneously in both leaflets of the lateral geniculate nucleus, showed an ultradian oscillatory pattern. In all our experiments, both the electrical lesion and the inactivation of neurons via the blockade of action potential generation did not cause any changes in the neuronal activity pattern in the contralaterally located geniculate leaflet. The obtained results show that a bilateral IGL connection is not necessary for the pattern of neuronal oscillation in the IGL. Hence the functional significance of a reciprocal connection between both lateral geniculate nucleus leaflets is still an open question.

In vitro extracellular recording of spontaneous activity of the intergeniculate leaflet neurons.

In the view of importance of intergeniculate leaflet in circadian rhythms processes and lack of information about electrophysiological properties of isolated intergeniculate leaflet (IGL) neurons, we carried out extracellular recordings of the spontaneous activity of rat IGL cells in vitro. Unlike other structures of visual thalamus, IGL neurons have the ability to generate a robust spontaneous neuronal activity when maintained in vitro. We have observed that in a standard incubation fluid IGL neurons display at least three distinct firing patterns: continuously irregular-with a wide variety of firing rates, tonic-with a very stable level of activity, and phasic (slow bursts)-with intermittent silent periods. Our study is the first electrophysiological demonstration of IGL neuronal activity in vitro.

Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin‐3–RXFP3 signalling

Relaxin‐3 is a stress‐responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3‐dependent, inhibitory action of relaxin‐3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin‐3. We observed a Gαi/o‐protein‐dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium‐sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin‐3‐immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin‐3‐containing fibres/terminals within the PVN.

The influence of carbachol on glutamate-induced activity of the intergeniculate leaflet neurons—In vitro studies

The intergeniculate leaflet (IGL) is a very important component of the mammalian circadian timing system. One of the best known, but still barely understood functions of the IGL, is the integration of photic (retina-derived) and non-photic information, conveyed to the suprachiasmatic nucleus (SCN)--the site of the circadian pacemaker. Glutamate, the main neurotransmitter released from the axonal endings of the retinal ganglion cells to the SCN and most probably to the IGL, is thought to be responsible for mediating the effects of light on the circadian clock. The influence of carbachol, a non-specific cholinergic agonist, on locomotor activity, c-fos expression in the SCN, and the activity of this structure has been previously studied. However, no information is available concerning the influence of acetylcholine on the activity of the IGL neurons. Therefore, the purpose of the present study was to analyze the influence of carbachol (equivalent of non-photic stimulus) on the glutamate-induced activity of the IGL neurons. Experiments were performed on thalamic rat brain slices, using extracellular, single unit recordings. After reaching a stable response to focally applied glutamate, carbachol was added to the recording medium. In the presence of the cholinergic agonist, glutamate-induced activity was decreased in 32% and increased in 13% of investigated cases. Carbachol failed to evoke any change in glutamate-induced activity in 55% of the recording cells. Our results are in agreement with previous, mainly behavioral studies, where the influence of non-photic stimulus on photic-induced changes in circadian locomotor activity was determined.

Interactions of Circadian Rhythmicity, Stress and Orexigenic Neuropeptide Systems: Implications for Food Intake Control

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.

Viral-mediated delivery of an RXFP3 agonist into brain promotes arousal in mice

Anatomical and functional studies of central relaxin-3/RXFP3 systems suggest they constitute an ascending arousal network. For example, relaxin-3 knockout mice display circadian hypoactivity compared to wild type littermate controls. In studies to explore the effect of chronic RXFP3 activation on behaviour, we engineered a lentiviral construct to constitutively secrete the RXFP3 agonist, R3/I5, and express a green fluorescent protein (GFP) marker in transduced cells. Intracerebroventricular injection of the lenti-R3/I5-GFP virus (-10(power)8 genomic copies in 2 microl) in adult C57BL/6J mice resulted in GFP expression within cells of the ventricle walls and choroid plexus over a period of 1-4 weeks, suggesting likely chronic R3/I5 secretion and RXFP3 activation in brain regions proximal to the ventricular system. Subsequent testing in automated locomotor cells on day 8 and 9 post-injection revealed that lenti-R3/I5-GFP treated mice displayed prolonged, elevated locomotor activity (-18% higher over the last 15 min on day 8, and over the entire 30 min test on day 9) compared to mice injected with a control lenti-GFP virus, which habituated normally to the novel environment (n = 18/12 respectively, p < 0.05). These findings are consistent with an earlier report of increased activity scores in rats acutely injected centrally with R3/I5, and further suggest a role for relaxin-3/RXFP3 signalling in promoting behavioural arousal.

Melanin-concentrating hormone and orexin systems in rat nucleus incertus: Dual innervation, bidirectional effects on neuron activity, and differential influences on arousal and feeding

ABSTRACT The rat nucleus incertus (NI) contains GABA/peptide‐projection neurons responsive to orexin (hypocretin)/orexin receptor‐2 (OX2) signalling. Melanin‐concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor‐1 (MCH1) expression, and neurophysiological and behavioural effects of MCH c.f. orexin‐A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub‐zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH‐containing fibres in NI. Single‐cell RT‐PCR detected MCH1 and OX2 mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co‐expression patterns of MCH1 and OX2 mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch‐clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1&mgr;M), representing a distinct population from OXA‐sensitive NI neurons (35%). Intra‐NI OXA infusion (600pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra‐NI MCH infusion (600pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre‐fasted rats tested during the dark/active phase, intra‐NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide‐immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours. HIGHLIGHTSNucleus incertus (NI) receives an innervation from MCH and orexin neurons.GABAergic and non‐GABAergic NI neurons express MCH1 and/or OX2 receptor mRNA.NI neurons display differential responses to MCH (inhibition) and OXA (excitation).OXA injection into NI enhanced light/inactive phase locomotor activity and feeding.MCH injection into NI did not alter light or dark phase activity or food intake.