PubMed Health⌕ Search

Biomedical subjects

A L Gundlach

Publications and source records attributed to A L Gundlach.

At least 19 recordsLinked to original sources
PubMed2006

Galanin treatment offsets the inhibition of bone formation and downregulates the increase in mouse calvarial expression of TNFalpha and GalR2 mRNA induced by chronic daily injections of an injurious vehicle.

We have previously shown that after bone fracture, galanin (GAL) and GAL receptor expression is increased in osteoblast-like cells of callus; however, the role of elevated GAL/GAL receptors in this instance of bone injury is not known. We hypothesize that in injury, GAL may facilitate bone formation by suppressing the production of cytokines such as TNFalpha and IL-1alpha, thereby affecting bone collagen formation and collagenolysis by key matrix metalloproteinases (MMPs). In studies to explore this hypothesis, we used a mouse calvarial injection model to (1) investigate whether mild injury caused by a daily subcutaneous injection of a glycerol-containing vehicle onto calvaria affected osteoblast/bone formation-associated histomorphometric parameters and gene expression (mRNA encoding GAL, GAL receptors, TNFalpha, IL-1beta, collagen type I, MMP-2 and -13) compared to non-injected, control mice and (2) determine the effect of GAL+vehicle treatment on these entities. Five groups of 4-week-old mice were used: a non-injected control group; a vehicle (50/50 solution of 10 mM PBS+0.025% BSA/5.4 M glycerol)-treated group; and 3 GAL-treated groups (0.2, 2 and 20 ng doses). Solutions were injected subcutaneously onto calvaria in a 10 mul volume, every day for 2 weeks. Vehicle injection reduced calvarial periosteal osteoblast cell height (P<0.001), osteoblast number (P<0.001) and osteoid thickness (P<0.01), relative to values in non-injected animals at 2 weeks. Vehicle injection also inhibited BFR in this periosteal bone relative to values in non-injected animals at both 1 and 2 weeks (P<0.05 and P<0.001, respectively). Increasing concentrations of GAL reversed the above-listed inhibitory effects caused by vehicle. This reversal was demonstrated by a dose-dependent effect of GAL on osteoblast cell height (Pearson's r=0.330; P<0.05), osteoblast number (Pearson's r=0.715; P=0.000), osteoid thickness (Pearson's r=0.516; P=0.000) and BFR (Pearson's r=0.525; P<0.05) after 2 weeks of GAL+vehicle treatment; with the 20 ng/day GAL+vehicle injection schedule returning these measured parameters toward non-injected control values. All GAL+vehicle treatments had no effect on calvarial expression of GAL, GALR1, GALR3, collagen type 1 and MMP-2 mRNAs compared to levels in vehicle-injected controls. GAL treatment did, however, produce dose-dependent effects on calvarial expression of GALR2 (Pearson's r=0.763; P=0.000), MMP-13 (Pearson's r=0.806; P=0.000), IL-1beta (Pearson's r=0.807; P=0.000) and TNFalpha (Pearson's r=0.542; P=0.000) mRNAs with 20 ng/day of GAL+vehicle producing the strongest reversal of vehicle-associated changes. Thus, the 20 ng/day GAL+vehicle regimen offset the inhibition of osteoblastic activity, and therefore bone formation caused by daily glycerol-containing vehicle injection. This effect on bone formation may be due in part to the peptide suppressing the formation and associated activity of TNFalpha, IL-1beta and MMP-13, as TNFalpha and IL-1beta are known inhibitors of bone formation and MMP-13 is involved in collagenolysis. Furthermore, these effects may be due to the action of GAL via GALR2, as it was the only GAL receptor affected by this GAL treatment regimen. These results indicate that GAL can facilitate bone formation associated with injury and reveal potential efficacy for GAL in treating osseous conditions where bone formation may be inhibited due to excess TNFalpha and IL-1beta production.

Animals↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Animals↗
PubMed2006

Leucine-rich repeat-containing G-protein-coupled receptor 8 in mature glomeruli of developing and adult rat kidney and inhibition by insulin-like peptide-3 of glomerular cell proliferation.

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3-LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3-LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3-LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.

Animals↗
PubMed2005

[125I]-Galanin binding in brain of wildtype, and galanin- and GalR1-knockout mice: strain and species differences in GalR1 density and distribution.

Widespread production of knockout and transgenic mice has led to an increased use of mice as animal models for studies of normal- and patho-physiology. Hence, the precise mapping of central transmitter/peptide systems in the mouse has become essential for the interpretation of functional studies and for the correct correlation with findings obtained in the rat, primates and/or human. In this regard, the current study reports the autoradiographic localization of [(125)I]-galanin (GAL) binding sites in brain of the common C57BL/6J and 129OlaHsd mouse strains, as well as in GAL and galanin receptor-1 (GalR1) knockout (KO) mice. In C57BL/6J and 129OlaHsd mice, [(125)I]-GAL binding sites were detected throughout the brain, including moderate-high relative densities in the basal ganglia (caudate putamen, nucleus [n.] accumbens, olfactory tubercle, substantia nigra), limbic regions (septum, bed n. stria terminalis, ventral hippocampus, amygdala), cingulate, retrosplenial, entorhinal cortex, centro-lateral/medial thalamic n., preoptic/lateral hypothalamus, midbrain (superior colliculus, periaqueductal gray), pons/medulla oblongata (parabrachial, pontine reticular and solitary tract n.) and cerebellar cortex. [(125)I]-GAL binding levels were low or absent in main olfactory bulb, neocortex, ventrolateral/geniculate thalamic n., dorsal hippocampus, inferior colliculus and cranial motor n. In simultaneous determinations, relative [(125)I]-GAL binding site densities in brain were generally lower in C57BL/6J than in 129OlaHsd mice, while the density and distribution of central binding in the GAL-KO mouse was essentially identical to that in its background-129OlaHsd strain. In contrast, no specific [(125)I]-GAL binding was detected in any region of GalR1-KO mouse brain, revealing that under the experimental conditions used, the peptide ligand binding is predominantly (exclusively) to the GalR1 subtype. This evaluation of GAL receptor site distribution in mouse brain has revealed similarities and some differences with the equivalent system in rat and provides a valuable reference for future comparative studies of central GAL transmission.

Animals↗
PubMed2005

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum.

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.

Aging↗
PubMed2003

Atrial natriuretic peptide expression is increased in rat cerebral cortex following spreading depression: possible contribution to sd-induced neuroprotection.

Cortical spreading depression (CSD) is characterised by slowly propagating waves of cellular depolarization and depression and involves transient changes in blood flow, ion balance and metabolism. In cerebral ischaemia, peri-infarct CSD-like depolarization potentiates infarct growth, whereas preconditioning with a CSD episode protects against subsequent ischaemic insult. Thus, many of the long-lasting molecular changes that occur in CSD-affected tissue are presumed to be part of a 'neuroprotective cascade.' 3',5'-Cyclic guanosine monophosphate (cGMP) has been shown to be a neuroprotective mediator and the nitric oxide system, which increases cGMP production by soluble guanylate cyclase, is up-regulated by CSD. Atrial and C-type natriuretic peptide (ANP/CNP) are present in cerebral cortex and their actions are mediated via particulate guanylate cyclase receptors and cGMP production. Therefore, in further efforts to characterise the role of cGMP-related systems in CSD and neuroprotection, this study investigated possible changes in cortical natriuretic peptide expression following acute, unilateral CSD in rats. Using in situ hybridisation, significant 20-80% increases in ANP mRNA were detected in layers II and VI of ipsilateral cortex at 6 h and 1-14 days after CSD. Ipsilateral cortical levels were again equivalent to control contralateral values after 28 days. Assessment of cortical concentrations of ANP immunoreactivity by radioimmunoassay revealed a significant 57% increase at 7 days after CSD. Despite using a sensitive signal-amplification protocol, authentic ANP-like immunostaining was readily detected in subcortical nerve fibres, but was not reliably detected in normal or CSD-affected neocortex, suggesting the presence of very low levels, and/or active or differential processing of the peptide. Cortical CNP mRNA levels are not altered by CSD, indicating the specificity of the observed effects.Overall, these novel findings demonstrate a prolonged increase in cortical ANP expression after an acute episode of CSD. The overlap between the described time course of CSD-induced protection against ischaemic insult and demonstrated increases in ANP levels, suggest that ANP (like nitric oxide) may contribute to CSD-induced neuroprotection, via effects on cGMP production and other signal-transduction pathways.

Animals↗
PubMed2001

Regulation by osmotic stimuli of galanin-R1 receptor expression in magnocellular neurones of the paraventricular and supraoptic nuclei of the rat.

Neurones of the supraoptic nucleus (SON) and the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) express galanin and [125I]galanin binding sites. Although the precise role(s) of galanin in these different cell populations is still unknown, it has been shown to regulate the electrophysiological, neurochemical and secretory activity of magnocellular neurones. In light of the well-described effects of hyperosmotic stimuli, such as salt-loading on magnocellular neurone activity and galanin synthesis and release, and the recent identification of multiple galanin receptors in brain, this study assessed the possible regulation of galanin receptor subtype expression in the PVN/SON of salt-loaded, dehydrated and food-deprived rats. Gal-R1 mRNA was abundant in the SON (and magnocellular PVN) of control rats and levels were increased in these same cells after 4 days of salt-loading (2% NaCl solution as drinking water) or water deprivation. The density of specific [125I]galanin(1-29) binding and the intensity of Gal-R1-like immunostaining were also increased in the characteristically enlarged, magnocellular neurones of the PVN and SON after these treatments. Gal-R2 mRNA was detected in the parvocellular PVN, but levels were not altered by the hyperosmotic stimuli. In contrast, food deprivation (4 days), which has been shown to reduce levels of several neurochemical markers in magnocellular neurones, produced a significant reduction in Gal-R1 (and galanin) mRNA levels in the SON, but no consistent change in neurone size, [125I]galanin binding levels, or Gal-R1 immunostaining. Along with previous findings from this and other laboratories, these data suggest that the expression of galanin and Gal-R1 receptors is regulated in parallel with functional and morphological changes in hypothalamic magnocellular neurones. Furthermore, Gal-R1 immunoreactivity was primarily detected in somatodendritic areas and thus galanin may influence the activity of these cells, particularly vasopressin synthesis/release, via autocrine or paracrine activation of Gal-R1 receptors, especially during long-lasting stimulation.

Animals↗
PubMed2001

Galanin-like peptide mRNA in neural lobe of rat pituitary. Increased expression after osmotic stimulation suggests a role for galanin-like peptide in neuron-glial interactions and/or neurosecretion.

Galanin-like peptide (GALP) was recently identified in the porcine hypothalamus, pituitary gland and gut, and has reported selectivity for the GalR2, c.f. the GalR1 receptor. GALP cDNAs have been cloned from pig, rat and human, and GALP mRNA expression is restricted to the arcuate nucleus in normal rat brain. This study examined the regional and cellular distribution of GALP mRNA in the rat pituitary gland, and subsequently determined the effect of osmotic stimulation on GALP transcript levels. GALP mRNA was not detected in the anterior or intermediate lobes, but moderate levels of GALP mRNA were present in the neural (posterior) lobe, in presumed pituicytes, of normal male and female rats. Osmotic stimulation by dehydration or salt loading produced a time-dependent increase in GALP mRNA levels in the neural lobe. Thus, dehydration for 4 days increased GALP mRNA 40-fold, while salt loading for 4, 7 or 10 days increased GALP levels 14-, 21- and 25-fold, respectively (p < or = 0.001). Levels of vasopressin (VP) mRNA in the neural lobe were also increased by these treatments, consistent with previous reports. Galanin (GAL) and GalR2 receptor mRNAs were not detected in the neural lobe, under normal or osmotic stimulation conditions. In addition, GALP mRNA levels in the arcuate nucleus were not altered in dehydrated or salt-loaded rats; and GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus, despite the characteristic up-regulation of VP and GAL mRNA in these cells. In view of the established anatomy and function of VP/oxytocin neurons in the hypothalamo-neurohypophysial system and the role played by pituicytes in their regulation, the likely synthesis/release of GALP by these specialized astrocytes strongly suggests a role for this novel peptide in regulation of pituicyte morphology/function and/or neurohormone release.

Animals↗
PubMed2000

Differential modulatory effects of alpha- and beta-adrenoceptor agonists and antagonists on cortical immediate-early gene expression following focal cerebrocortical lesion-induced spreading depression.

Unilateral, focal cerebrocortical lesion (FCL) and associated spreading depression (SD) increase immediate-early gene (IEG) expression throughout the ipsilateral hemisphere. Noradrenergic transmission is involved in the regulation of basal- and stimulation-induced expression of IEGs in cerebral cortex; and is modulated by both injury and SD. The present study further investigated the association between the noradrenergic system and cortical adaptive responses, by examining basal and FCL(SD)-induced cortical IEG expression following acute treatment with alpha(1)-, alpha(2)- and beta(1/2)-adrenoceptor (AR) agonists or antagonists. Activation of alpha(1)-ARs by NVI-085, or beta-ARs by salbutamol, increased cortical NGFI-A, c-jun and c-fos mRNA levels, whereas inhibition of alpha(1)-ARs by prazosin, or beta-ARs by propranolol, had no marked effect. The alpha(2)-AR agonists, clonidine and UK14304 also had no effect on basal IEG levels, while blockade of alpha(2)-ARs by methoxyidazoxan significantly increased NGFI-A and c-fos expression, but decreased c-jun mRNA levels. This latter effect confirms the complex and differential nature of IEG regulation in brain. In FCL(SD) rats, all AR agonists generally produced a supra-additive (synergistic) effect on expression of the examined IEGs, compared with drug-treatment or FCL alone. Prazosin reduced FCL(SD)-induced elevations of c-jun and c-fos, but not NGFI-A, mRNA. Methoxyidazoxan enhanced NGFI-A and c-fos mRNA expression after FCL(SD), but reduced c-jun. Propranolol enhanced all lesion-induced IEG levels. These results confirm that alpha(1)- and beta-ARs normally mediate a stimulatory, and alpha(2)-ARs a net inhibitory, influence on cortical cell activity (reflected by NGFI-A, c-fos expression); and demonstrate that alterations in noradrenergic tone modulate the level of cellular activation during and after SD, which is primarily elicited by K(+)/glutamate via NMDA receptors and Ca(2+)-associated mechanisms. In turn, noradrenergic transmission and interactions with excitatory systems are likely to be important in responses to brain injury, including regulation of IEGs and their downstream target genes.

Adrenergic alpha-Agonists↗
PubMed2000

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.

Age Factors↗
PubMed2000

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 [(35)S]-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.

Animals↗
PubMed1999

Localization of GDNF/neurturin receptor (c-ret, GFRalpha-1 and alpha-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 alpha-1 and alpha-2 (GFRalpha-1 and GFRalpha-2). In the present study the localization of c-ret and GFRalpha-1 and GFRalpha-2 mRNAs was assessed in the developing rat brain from postnatal day 4 to 70 by in situ hybridization histochemistry, using specific [35S]-labeled oligonucleotides. GFRalpha-1 and GFRalpha-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. GFRalpha-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. GFRalpha-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. GFRalpha-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). GFRalpha-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 GFRalpha-1 and GFRalpha-2 mRNAs in adult brain suggests possible non-trophic functions of GDNF/NTN, as described for other neurotrophins, such as brain-derived neurotrophic factor.

Animals↗
PubMed1999

Identification of imidazoline-receptor binding sites in cortex and medulla of the bovine adrenal gland. Colocalization with MAO-A and MAO-B.

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) in bovine adrenal gland were determined using [3H]clonidine, [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]2-BFI), and [3H]rilmenidine. In light of strong evidence that I-RBS and monoamine amine oxidase enzymes are linked, the selective radioligands [3H]RO41-1049 and [3H]RO19-6327 were used to label the distribution of MAO-A and -B enzymes, respectively. [3H]Clonidine (12 nM) labeled sites in two discrete regions of the bovine adrenal gland, the zona glomerulosa (39 +/- 7 fmol/mg tissue equivalent) and inner medulla (34 +/- 1 fmol/mg tissue). Binding was nonadrenergic (i.e., not inhibited by 100 nM methoxyidazoxan) and inhibited by 60-70% by 100 nM 2-BFI, the selective I2-RBS, suggesting binding predominantly to an I2-RBS. [3H]2-BFI (5 nM), the selective I2-RBS ligand, also labeled a high density of binding sites in the zona glomerulosa (57 +/- 9 fmol/mg) and chromaffin cells in the inner medulla (53 +/- 4 fmol/mg). These sites, however, were insensitive to clonidine (100 nM). By contrast, [3H]rilmenidine (40 nM) labeled I-RBS in all regions of the adrenal gland, that is, the zonae glomerulosa (59 +/- 10 fmol/mg), fasciculata (78 +/- 10 fmol/mg) and reticularis (63 +/- 7 fmol/mg), and outer and inner medullary chromaffin cells (42 +/- 1 and 55 +/- 2 fmol/mg, respectively). Binding to sites in the zona glomerulosa was partially inhibited (16%) by 100 nM 2-BFI. These results are consistent with previous studies indicating that [3H]rilmenidine labels an I2-RBS and additional I-RBS in rat brain and kidney. The distribution of [3H]RO19-6327 (5 nM) binding resembled that of [3H]2-BFI and [3H]clonidine binding with high densities of MAO-B enzyme located in the zona glomerulosa and chromaffin cells of the inner medulla (55 +/- 7 and 76 +/- 6 fmol/mg tissue, respectively), suggesting the colocalization of MAO-B enzyme with I2-RBS. [3H]RO41-1049 (20 nM) binding to MAO-A was highest in the zona reticularis (196 +/- 7 fmol/mg tissue) compared to the zonae glomerulosa and fasciculata (90 +/- 12 and 116 +/- 14 fmol/mg tissue) and inner medulla (149 +/- 38 fmol/mg tissue). Although the existence of I-RBS in bovine adrenal chromaffin cells is well established, this is the first description of I-RBS in the adrenal cortex. Further investigations are now required to determine whether imidazolines can affect adrenal function via actions at these sites.

Adrenal Cortex↗
PubMed1999

Prolonged induction of neuronal NOS expression and activity following cortical spreading depression (SD): implications for SD- and NO-mediated neuroprotection.

Cortical spreading depression (CSD) is associated with various short- and long-term physiological and neurochemical changes and has been shown to confer an increased susceptibility to accompanying ischemic injury or provide protection against a subsequent experimental ischemia. Nitric oxide is involved in the processes of ischemic injury and under certain conditions mediates cellular protection. To investigate the possibility that CSD-induced alterations in nitric oxide synthase (NOS) expression and activity occur and might be associated with the time-dependent enhancement or prevention by CSD of ischemic damage, this study examined the spatiotemporal changes in nNOS expression and activity in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl and were killed at various times thereafter. CSD increased both nNOS mRNA and protein levels throughout layers II-III of cortex. nNOS mRNA in the affected neocortex was significantly increased by 30-90% at 2, 7, and 14 days (P < or = 0.05) compared with contralateral levels, but was not significantly above control values at 1-6 h, 1 day, and 28 days after CSD induction. Levels of [3H]-L-N(G)-nitroarginine binding to NOS were increased by 40-170% 7, 14, and 28 days (P < or = 0.01) after CSD in a similar, but delayed, profile to nNOS mRNA. Levels of nNOS-immunoreactivity were also increased in both neurons and astrocytes of ipsilateral cortex 7 and 14 days after CSD--confirmed by double-immunofluorescence localization. Ex vivo NOS activity in layers I-III of ipsilateral cortex was also increased by 30-50% (P < or = 0.01) at 7 and 14 days after CSD, times coincident with reported maximal ischemic protection. These results demonstrate that nNOS is up-regulated by cellular depolarization/depression occurring during CSD, or by resultant stimuli and suggest that "CSD-conditioned" cortex may be capable of producing appropriate levels of NO to mediate or contribute to protective/adaptive responses to subsequent physical ischemic injury.

Animals↗
PubMed1998

Galanin-galanin receptor systems in the hypothalamic paraventricular and supraoptic nuclei. Some recent findings and future challenges.

Galanin and galanin receptors are widely distributed within the central nervous system, but historically much research has been focused on hypothalamic galanin systems including those in the preoptic area, paraventricular nucleus (PVN), supraoptic nucleus (SON), and median eminence. In early studies, galanin mRNA, immunoreactivity, and binding sites were detected in neurons of the SON and both the magnocellular and parvocellular regions of the PVN, all of which also contain vasopressin, oxytocin, and several other peptides. This article briefly reviews some important recent studies of the electrophysiologic effects of galanin on magno-cellular neurons in vitro; regulation of galanin expression by the physiologic stimulus of lactation; the role of parvocellular galanin systems in energy balance, body weight, and obesity; and the regional and cellular localization of galanin and galanin receptor mRNAs in the PVN/SON. In relation to the latter issue, two distinct galanin receptor subtypes, GalR1 and GalR2, have now been cloned and characterized. In situ hybridization histochemical studies of rat brain by several groups have consistently demonstrated GalR1 mRNA in the SON and PVN, in the magnocellular and parvocellular regions. By contrast, our recent experiments using [35S]-labeled oligonucleotide probes detected GalR2 mRNA enriched in the parvocellular, not the magnocellular regions of the PVN, and the transcripts were not detected in the SON, whereas studies by other using a digoxigenin-labeled RNA probe have detected GalR2 mRNA in the SON (and PVN). Nonetheless, given the known effects of hyperosmotic stimuli, changes in metabolic status, and various hormones on galanin synthesis and release and the ability of galanin to regulate the electrical and secretory activity of magnocellular neurons, it will be of interest to determine any possible (differential) regulation of galanin receptor subtype expression and the pre- and postsynaptic roles of GalR1 and GalR2 receptors in magnocellular and parvocellular neurons.

Animals↗
PubMed1998

Increased striatal proenkephalin mRNA subsequent to production of spreading depression in rat cerebral cortex: activation of corticostriatal pathways?

Cortical Spreading Depression (CSD) is a slowly propagating wave of depolarization and negative interstitial DC potential, that when induced in the rat brain extends across the entire homolateral hemisphere. Despite evidence that CSD does not penetrate into subcortical regions, neurochemical changes in areas anatomically connected to cortex have been reported. In this study in situ hybridization histochemistry was used to examine the levels of cholecystokinin (CCK), proenkephalin (ENK) and prodynorphin (DYN) mRNA in cortex and forebrain basal ganglia following KCl-induced CSD. Unilateral CSD was induced by topical application of 3 M KCl ( approximately 10 microliter) onto the right parietal cortex for 10 min and rats were then killed 1-6 h and 1-28 days later. CCK mRNA levels were increased (P<0.01) in the ipsilateral neocortex 3 h after CSD (13% above levels in contralateral side), reached a peak at 2 days ( approximately 70%) and were still elevated at 7 (30%) but not, 14 or 28 days later. Unilateral CSD also produced a rapid and sustained increase (P<0.05) in ENK mRNA in ipsilateral piriform cortex (from 3 h to 2 days; 70-250% above contralateral), and a delayed increase in caudate putamen and olfactory tubercle at 1 and 2 days ( approximately 25% in both regions), but levels were again equivalent to control at 7 days and beyond. In contrast, no marked changes in neocortical ENK mRNA, or DYN mRNA in both cortex and basal ganglia, were observed under these conditions. These findings demonstrate that CSD has specific, rapid and long-lasting effects on neuropeptide expression in neocortex and subcortical areas. CSD-induced changes in mesostriatal ENK mRNA are proposed to reflect synaptic activation of local neurons via cortical afferent projections.

Animals↗