Andrew L. Gundlach

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain.

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene‐3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene‐3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT‐PCR and 3′ and 5′ RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.

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.

Relaxin: new peptides, receptors and novel actions

Relaxin has long been known as a hormone of pregnancy. Until recently, little was known of potential roles for relaxin in non-pregnant females and males. The identification of a new gene encoding relaxin-3 (RLN3), the discovery of the elusive relaxin receptor and a novel role for relaxin-1 in regulating the normal turnover of collagen has provided us with unique insights into potential new roles for this peptide family. The Rln3 gene appears to be predominantly expressed in the brain, and mapping studies indicate a highly developed network of Rln3, Rln1 and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides might have important roles in the central nervous system. Rln1-knockout mice show progressive tissue fibrosis as they age, and this fibrosis leads to functional changes in both the heart and lungs. Hence, the biological significance of this enigmatic peptide family is expanding, as are its potential clinical uses.

Galanin-Like Peptide (GALP) mRNA Expression Is Restricted to Arcuate Nucleus of Hypothalamus in Adult Male Rat Brain

Galanin-like peptide (GALP) is a novel 60-amino acid neuropeptide, isolated from porcine hypothalamus and subsequently identified in rats and humans, which has reported selectivity for the Gal-R2 galanin receptor [Ohtaki T et al: J Biol Chem 1999; 274: 37041–37045]. In the current study, the regional and cellular distribution of GALP mRNA in rat brain has been investigated by in situ hybridization of [35S]-labelled oligonucleotide probes. In a thorough screening of adult male rat brain, GALP mRNA expression was detected only throughout the rostrocaudal extent of the arcuate nucleus (ARC) with the most abundant hybridization signal in the posterior, periventricular zones. GALP mRNA-positive neurons were mostly localized in the ventromedial division of the ARC, with many closely adjacent to the wall of the third ventricle. Smaller numbers of labelled neurons were also found in ventrolateral areas. The distribution of GALP mRNA was somewhat complementary to that for galanin (GAL) mRNA in the ARC, but contrasted with the broad distribution of this transcript throughout the hypothalamus. GAL mRNA was also distributed along the rostrocaudal extent of the ARC, but was most abundant in the anterior to middle levels and in ventrolateral regions. Interestingly, somatostatin mRNA expression appeared to overlap the distribution of GALP mRNA in posterior, ventromedial regions of the ARC. Thus, in adult rat brain GALP mRNA expression was restricted to a discrete subpopulation of neurons in the ARC, with a unique localization pattern unlike GAL or many other known peptide- and transmitter-containing cells in this region. GALP could, however, be co-expressed in sub-populations of other neuronal phenotypes (e.g. somatostatin neurons) or within cells that express Gal-R2 receptors. In view of the established anatomy and function of the ARC and the restricted localization of GALP mRNA, this novel peptide is likely to play a role in regulation of anterior pituitary hormone secretion, or in regulation of other hypothalamic peptide and transmitter systems.

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.

Distribution of GABAA receptor subunit mRNAs in rat lumbar spinal cord

The expression of various GABAA receptor subunit mRNAs (alpha 1, alpha 2, alpha 3, alpha 5, beta 1, beta 2, beta 3, gamma 2, delta) was studied in the adult rat lumbar spinal cord by in situ hybridization. Of these, only alpha 2, alpha 3, beta 3 and gamma 2 mRNAs are expressed at significant levels. The alpha 3, beta 3 and gamma 2 transcripts are present in many neurons throughout the Rexed laminae, whereas the alpha 2 mRNA is restricted to motor neurons and adjacent cells.

Galanin‐R1 and ‐R2 receptor mRNA expression during the development of rat brain suggests differential subtype involvement in synaptic transmission and plasticity

The present study employed 35S‐labelled oligonucleotides and in situ hybridization to examine the distribution in the developing rat brain of mRNA encoding two galanin receptor subtypes, i.e. Gal‐R1 and Gal‐R2. Gal‐R1 and/or Gal‐R2 mRNA was detected at embryonic day (E) 20 and from postnatal day (P) 0–70. Gal‐R1 mRNA was highly expressed in olfactory regions, ventral hippocampal CA fields, dorsomedial thalamic areas and many hypothalamic nuclei at all ages studied. In adult brain, Gal‐R2 mRNA was most abundant in the dentate gyrus, anterior and posterior hypothalamus, raphe and spinal trigeminal nuclei, and in the dorsal motor nucleus of the vagus. At P0–P7, Gal‐R2 mRNA was more widely distributed and abundant than at other ages, with highest levels of expression detected throughout the neocortex and thalamus. Thus, Gal‐R2 transcripts had a more restricted distribution than Gal‐R1 and were differentially abundant at different ages, while the distribution and relative abundance of Gal‐R1 mRNA did not alter substantially during postnatal development. In general, Gal‐R1 and ‐R2 mRNAs were localized in regions previously shown to contain [125I]‐galanin binding sites and galanin‐positive terminals in adult brain. Galanin‐immunostaining was assessed in postnatal brain to determine whether peptide innervation correlated with observed transient receptor expression, but was not particularly enriched in Gal‐R2 mRNA‐positive areas of P4 or P7 brain. These results, together with earlier findings [e.g. Burazin, T. C. D. & Gundlach, A. L. (1998) J. Neurochem., 71, 879–882], suggest that Gal‐R1 receptors have a broad role in normal synaptic transmission, while Gal‐R2 receptors, in addition to a similar role in particular pathways, may be involved in processes prominent during the establishment and maturation of synaptic connections in developing brain and during neural damage and repair in the mature nervous system.

Preproneuropeptide Y messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat is increased by food deprivation or dehydration

The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro‐NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro‐NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro‐NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro‐GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.

Localization of GDNF/neurturin receptor (c-ret, GFRα-1 and α-2) mRNAs in postnatal rat brain: differential regional and temporal expression in hippocampus, cortex and cerebellum

Recent studies have identified a multi-component receptor system for the neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and its homolog, neurturin (NTN), comprising the signaling tyrosine kinase, Ret and multiple GPI-linked binding proteins, GDNF family receptor α-1 and α-2 (GFRα-1 and GFRα-2). In the present study the localization of c-ret and GFRα-1 and GFRα-2 mRNAs was assessed in the developing rat brain from postnatal day 4 to 70 by in situ hybridization histochemistry, using specific []-labeled oligonucleotides. GFRα-1 and GFRα-2 mRNAs were differentially distributed throughout the brain at all ages studied, particularly in cerebral cortex, hippocampus, substantia nigra and regions of the thalamus and hypothalamus — both distributions overlapping but different to that of c-ret mRNA. C-ret mRNA was abundant in areas such as the lateral habenula, reticular thalamic nucleus, substantia nigra pars compacta, cranial motor nuclei, and the Purkinje cell layer of the cerebellum. GFRα-1 mRNA was abundant in dorsal endopiriform nucleus, medial habenula, reticular thalamic nucleus, pyramidal and granule cell layers of the hippocampus, substantia nigra pars compacta and in cranial motor nuclei. GFRα-2 mRNA was highly expressed in many regions including olfactory bulb, lateral olfactory tract nucleus, neocortical layers IV and VI, septum, zona incerta, and arcuate and interpeduncular nuclei. GFRα-2 mRNA was detected in the pyramidal cell layers (CA3) of hippocampus at P4 and P7, but was no longer detectable at P14 and beyond, including P70 (adult). GFRα-2 mRNA was also detected in Purkinje cells throughout the cerebellum in young postnatal rats, but was enriched in the posterior lobes at P28 and P70. These localization studies support evidence of GDNF/NTN as target-derived and autocrine/paracrine trophic factors in developing brain pathways and earlier suggestions of unique and complex signaling mechanisms for these factors via a family of receptors. Strong expression of GFRα-1 and GFRα-2 mRNAs in adult brain suggests possible non-trophic functions of GDNF/NTN, as described for other neurotrophins, such as brain-derived neurotrophic factor.