Pei-Juan Shen

Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat

Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3(85-101)). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [125I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.

Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain.

Relaxin‐3 (RLN3) and its native receptor, relaxin family peptide 3 receptor (RXFP3), constitute a newly identified neuropeptide system enriched in mammalian brain. The distribution of RLN3/RXFP3 networks in rat brain and recent experimental studies suggest a role for this system in modulation of arousal, stress, metabolism, and cognition. In order to facilitate exploration of the biology of RLN3/RXFP3 in complementary murine models, this study mapped the neuroanatomical distribution of the RLN3/RXFP3 system in mouse brain. Adult, male wildtype and RLN3 knock‐out (KO)/LacZ knock‐in (KI) mice were used to map the central distribution of RLN3 gene expression and RLN3‐like immunoreactivity (‐LI). The distribution of RXFP3 mRNA and protein was determined using [35S]‐oligonucleotide probes and a radiolabeled RXFP3‐selective agonist ([125I]‐R3/I5), respectively. High densities of neurons expressing RLN3 mRNA, RLN3‐associated β‐galactosidase activity and RLN3‐LI were detected in the nucleus incertus (or nucleus O), while smaller populations of positive neurons were observed in the pontine raphé, the periaqueductal gray and a region adjacent to the lateral substantia nigra. RLN3‐LI was observed in nerve fibers/terminals in nucleus incertus and broadly throughout the pons, midbrain, hypothalamus, thalamus, septum, hippocampus, and neocortex, but was absent in RLN3 KO/LacZ KI mice. This RLN3 neural network overlapped the regional distribution of RXFP3 mRNA and [125I]‐R3/I5 binding sites in wildtype and RLN3 KO/LacZ KI mice. These findings provide further evidence for the conserved nature of RLN3/RXFP3 systems in mammalian brain and the ability of RLN3/RXFP3 signaling to modulate “behavioral state” and an array of circuits involved in arousal, stress responses, affective state, and cognition. J. Comp. Neurol. 518:4016–4045, 2010.

Swim stress excitation of nucleus incertus and rapid induction of relaxin-3 expression via CRF1 activation.

Relaxin-3 (RLX3), a newly identified member of the relaxin peptide family, is distinguished by its enriched expression in GABA projection neurons of the pontine nucleus incertus (NI), which are postulated to participate in forebrain neural circuits involved in behavioural activation and stress responses. In this regard, corticotrophin-releasing factor-1 receptor (CRF(1)) is abundantly expressed by NI neurons; central CRF administration activates c-fos expression in NI; and various stressors have been reported to increase NI neuron activity. In studies to determine whether a specific neurogenic stressor would activate RLX3 expression, we assessed the effect of a repeated forced swim (RFS) on levels of RLX3 mRNA and heteronuclear (hn) RNA in rat NI by in situ hybridization histochemistry of exon- and intron-directed oligonucleotide probes, respectively. Exposure of rats to an RFS (10 min at 23 degrees C, 24 h apart), markedly increased RLX3 mRNA levels in NI at 30-60 min after the second swim, before a gradual return to basal levels over 2-4 h, while RLX3 hnRNA levels were significantly up-regulated at 60-120 min post-RFS, following a transient decrease at 30 min. Systemic treatment of rats with a CRF(1) antagonist, antalarmin (20 mg/kg, i.p.) 30 min prior to the second swim, blunted the stress-induced effects on RLX3 transcripts. Relative levels of RLX3-immunostaining in NI neurons appeared elevated at 3 h post-swim, but not at earlier time points (30-60 min). These results suggest that acute stress-induced CRF secretion can rapidly alter RLX3 gene transcription by activation of CRF(1) present on NI neurons. More generally, these studies support a role for RLX3 neural networks in the normal neural and physiological response to neurogenic stressors in the rat.

Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmission

Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as γ‐aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5‐hydroxytryptamine, 5‐HT). The dorsal raphé nucleus (DRN) contains ≈ 50% of the 5‐HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5‐HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor‐1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1‐immunoreactivity (IR) was detected in a substantial population of 5‐HT‐immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1‐IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5‐HT‐IR to that in the rat, but GalR1‐IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1‐IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5‐HT neuron activity in the DRN is primarily regulated via 5‐HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward‐rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5‐HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5‐HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression.

Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression

Neocortex contains very few galanin neurons but receives a moderate galanin innervation from various subcortical loci. Recent data suggest that galanin helps regulate the tonic neuronal excitability of hippocampus and probably cerebral cortex but relatively little is known about the anatomy and functional regulation of cortical galanin systems. Therefore, we examined, in the rat, the effect of the intense but benign stimulus, cortical spreading depression (CSD), on the expression of galanin and galanin receptors (GalR1 and GalR2) in the neocortex and associated regions, revealing complex, multicellular responses. Thus, following acute, unilateral KCl‐induced CSD, a delayed and transient induction (onset after 48 h, lasting ∼24 h) of galanin mRNA and peptide production occurred across the ipsilateral cerebral cortex in activated oligodendrocyte progenitor cells (OPCs), identified by specific NG2 proteoglycan immunostaining. An increase in GalR1 mRNA, immunoreactivity and receptor binding occurred in neurons within layers II and V of neocortex and in piriform cortex at 7–28 days after CSD, associated with a long‐lasting depletion of galanin‐positive nerve fibres in these regions. In contrast, GalR2 mRNA expression was largely unaltered after CSD. Additional novel findings in normal, adult brain were the detection of galanin mRNA and immunoreactivity in OPCs within the medial corpus callosum and in immature progenitor cells in the subventricular zone and rostral migratory stream. GalR1 and GalR2 mRNA was also present in these latter regions. These findings and the complex modulation of galanin and galanin receptors in multiple cell types (neurons/OPCs) following acute cortical activation/depression further demonstrate the potential plasticity of neuronal and non‐neuronal galanin systems under physiological and pathological conditions and strongly suggest additional functions for this pleiotropic peptide in mammalian brain.

Characterization of the Rat INSL3 Receptor

Abstract: Human LGR8, initially discovered as a low‐affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full‐length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.

Comparative localization of leucine-rich repeat-containing G-protein-coupled receptor-7 (RXFP1) mRNA and [33P]-relaxin binding sites in rat brain: Restricted somatic co-expression a clue to relaxin action?

Relaxin is a polypeptide hormone with established actions associated with reproductive physiology, but until recently the precise nature of the relaxin receptor and its transmembrane signaling mechanisms had remained elusive. In 2002 however, the leucine-rich-repeat-containing G-protein-coupled receptor-7 (now classified as RXFP1) was identified as a cognate receptor for relaxin, with activation resulting in stimulation of intracellular cAMP production. These findings, along with the presence and putative actions of relaxin within the CNS and earlier descriptions of relaxin binding sites in brain, suggest the importance and feasibility of determining if these relaxin binding sites represent leucine-rich-repeat-containing G-protein-coupled receptor-7 and their precise comparative distribution. Thus, the current study reports the distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the rat brain using in situ hybridization histochemistry of [(35)S]-labeled oligonucleotides and the comparative distribution of [(33)P]-human relaxin binding sites. The extensive, topographical distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the adult rat brain correlated very closely to that of [(33)P]-relaxin binding sites. Leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA was expressed by neurons in several brain regions, including the olfactory bulb, cerebral cortex, thalamus, hippocampus, hypothalamus, midbrain, pons and medulla. Receptor transcripts were most abundant in areas such as the basolateral amygdala, subiculum, deep layers of the cingulate, somatosensory and motor cortices and intralaminar/midline thalamic nuclei. These areas also contained very high densities of [(33)P]-relaxin binding sites, suggesting a largely somatic localization of leucine-rich-repeat-containing G-protein-coupled receptor-7 protein and site of action for relaxin peptide. The central distribution of relaxin-producing neurons has been described, while data on the topography of nerve terminals that contain and secrete the peptide are currently lacking; but overall these findings strongly suggest that leucine-rich-repeat-containing G-protein-coupled receptor-7 is the cognate receptor for relaxin in the rat brain, and support a role for relaxin-leucine-rich-repeat-containing G-protein-coupled receptor-7 signaling in various somatosensory, autonomic and neurohumoral pathways, which warrants further investigation.

Delayed, but prolonged increases in astrocytic clusterin (ApoJ) mRNA expression following acute cortical spreading depression in the rat: evidence for a role of clusterin in ischemic tolerance

Clusterin is a sulfated glycoprotein produced by neurons and by resting and activated astrocytes that has several putative functions, including protective responses to brain injury. Cortical spreading depression (CSD) is a powerful yet largely benign stimulus that acutely is capable of providing long-lasting ischemic tolerance. The current study investigated possible alterations in expression of clusterin mRNA in the cerebral cortex of the rat at various times after unilateral CSD. Using semiquantitative in situ hybridization histochemistry, significant increases (30-100%; P< or =0.05) in clusterin mRNA were detected in layers I-III and IV-VI of the ipsilateral cortex at 1, 2, 7 and 14 (layers I-III only) days after CSD. Transcript levels in the ipsilateral cortex were again equivalent to contralateral (control) levels at 28 days after CSD. These molecular anatomical studies also revealed that both neurons and nonneuronal cells (presumed reactive astrocytes) increased their expression of clusterin mRNA following CSD. Notably the time-course of increases in clusterin mRNA after CSD (1-14 days) overlaps that during which CSD reportedly provides neuroprotection against subsequent cerebral ischemia. These findings along with other evidence suggest that increased clusterin production and secretion, particularly by astrocytes, could be neuroprotective-perhaps via one or more of its putative actions that include inhibition of complement activation and cytolysis, effects on chemotaxis and apoptosis, and actions as an anti-stress protein chaperone.

Neuronal‐NOS adaptor protein expression after spreading depression: implications for NO production and ischemic tolerance

Cortical spreading depression (CSD) is characterized by slowly propagating waves of neuronal/astrocytic depolarization and metabolic changes, followed by a period of quiescent neuronal and electroencephalographic activity. CSD acts as a preconditioning stimulus in brain, reducing cell death when elicited up to several days prior to an ischemic insult. Precise mechanisms associated with this neuroprotection are not known, although CSD increases the expression of a number of potentially neuroprotective genes/proteins. The nitric oxide (NO) system may be of particular importance, as it is acutely activated and chronically up‐regulated in cerebral cortex by CSD, and NO can ameliorate and exacerbate cell death under different conditions. Several molecules have recently been identified that modulate the production and/or cellular actions of NO, but it is not known whether their expression is altered by CSD. Therefore, the present study examined the effect of CSD on the spatiotemporal expression of PIN, CAPON, PSD‐95, Mn‐SOD and Cu/Zn‐SOD mRNA in the rat brain. In situ hybridization using specific [35S]‐labelled oligonucleotides revealed that levels of PIN mRNA were significantly increased in the cortex and claustrum (∼30–180%; p ≤ 0.01) after 6 h and 1 and 2 days, but were again equivalent to contralateral (control) cortical values at 7, 14 and 28 days. CAPON mRNA levels were increased (∼30–180%; p ≤ 0.05) in the ipsilateral cortical hemisphere at 6 h and 2 days post treatment, but not at the other times examined. In contrast, levels of PSD‐95, Mn‐ and Cu/Zn‐SOD mRNA were not altered at any time after CSD. These results suggest that following CSD, nNOS activity and NO levels may be tightly regulated by both transcriptional and translational alterations in a range of nNOS adaptor proteins, which may contribute to CSD‐induced neuroprotection against subsequent ischemia.

Differential increases in chromogranins, but not synapsin I, in cortical neurons following spreading depression: implications for functional roles and transmitter peptide release.

Experimental damage of cerebral cortex induces a slow‐moving depolarization and subsequent depression of activity called cortical spreading depression (CSD) which is associated with various ionic, metabolic and genomic changes. Chromogranins are a family of water‐soluble acidic proteins with a widespread distribution in secretory, large dense‐core vesicles of neurons. We have earlier reported that secretogranin II (SgII) mRNA is increased in cerebral cortex hours after a unilateral craniotomy which would have induced CSD. To investigate further the regulation of chromogranin systems and the nature of genomic and biochemical changes produced by CSD, this study examined the temporal changes in chromogranin A (CgA), chromogranin B (CgB) and SgII mRNAs and CgB and SgII immunoreactivity (IR) in cerebral cortex and hippocampus following unilateral