Neuropeptides
Volume 46, Issue 1 , Pages 19-27, February 2012

Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats

  • Lovisa Holm

      Affiliations

    • Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden
    • ,
  • Susanne Hilke

      Affiliations

    • Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden
    • ,
  • Csaba Adori

      Affiliations

    • Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
    • ,
  • Elvar Theodorsson

      Affiliations

    • Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden
    • ,
  • Tomas Hökfelt

      Affiliations

    • Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
    • ,
  • Annette Theodorsson

      Affiliations

    • Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden
    • Division of Neurosurgery, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden
    • Corresponding Author InformationCorresponding author at: Division of Neurosurgery, Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden. Tel.: +46 (0)101037585; fax: +46 (0)101033240.

Received 20 June 2011; accepted 27 November 2011. published online 26 December 2011.

Article Outline

Abstract 

Injury to neurons results in up-regulation of galanin in some central and peripheral systems, and it has been suggested that this neuropeptide may play a protective and trophic role, primarily mediated by galanin receptor 2 (GalR2). The objective of the present study was to investigate galanin, GalR1, GalR2 and GalR3 gene expression in the female rat brain 7days after a 60-min unilateral occlusion of the middle cerebral artery followed by reperfusion. Quantitative real-time PCR was employed in punch-biopsies from the locus coeruleus, somatosensory cortex and dorsal hippocampal formation, including sham-operated rats as controls. Galanin gene expression showed a ∼2.5-fold increase and GalR1 a ∼1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side. Furthermore, the GalR1 mRNA levels decreased by 35% in somatosensory cortex of the ischemic hemisphere. Immunohistochemical analysis indicated a depletion of galanin from cell bodies and dendrites in the locus coeruleus after middle cerebral artery occlusion. The present results suggest that a stroke-induced forebrain lesion up-regulates synthesis of galanin and GalR1 in the locus coeruleus, a noradrenergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation. These results support the notion that galanin may play a role in the response of the central nervous system to injury.

Keywords: Cerebral ischemia, Galanin receptor, Neuropeptide, RT-PCR, Stroke, Transmitter coexistence

 

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1. Introduction 

Galanin, a 29/30 amino acid neuropeptide isolated from pig intestine (Tatemoto et al., 1983), is widely distributed in the peripheral and central nervous systems (Rokaeus et al., 1984, Skofitsch and Jacobowitz, 1985, Skofitsch and Jacobowitz, 1986, Melander et al., 1986a, Melander et al., 1986b, Melander et al., 1986c). Galanin is involved in several important biological functions including modulation of classical neurotransmitters and is markedly up-regulated in many neuronal tissues after nerve injury, either at the site of the lesion or at a distance (Ch’ng et al., 1985, Hökfelt et al., 1987, Villar et al., 1989, Cortes et al., 1990, Agoston et al., 1994, Gabriel et al., 1995, Holmes and Crawley, 1996, Zigmond et al., 1996, Brecht et al., 1997, Theodorsson and Theodorsson, 2005) as well as in neurodegenerative disease (Chan-Palay, 1988, Beal et al., 1990, Crawley, 1993, Mufson et al., 1993). This has led to the proposal that galanin may play a protective and trophic role following injury to neurons (Holmes et al., 2000, Kerr et al., 2000, O’Meara et al., 2000, Zigmond, 2001, Mahoney et al., 2003, Elliott-Hunt et al., 2004, Elliott-Hunt et al., 2007, Pirondi et al., 2005, Pirondi et al., 2010, Ding et al., 2006, Hobson et al., 2008).

The effects of galanin are mediated by the activation of three G-protein-coupled galanin receptor subtypes, galanin receptor (GalR) 1, GalR2 or GalR3 (Habert-Ortoli et al., 1994, Wang and Gustafson, 1998, O’Donnell et al., 1999, O’Donnell et al., 2003, Branchek et al., 2000, Burazin et al., 2000, Waters and Krause, 2000, Mennicken et al., 2002). Several galanin receptor subtype-selective ligands have now been developed (Mitsukawa et al., 2010). For example, the galanin fragment Gal (2–11) (AR-M1896) (Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-NH2) acts as an agonist with 500-fold selectivity for GalR2 compared to GalR1 (Liu et al., 2001).

However, Gal (2–11) can also bind and activate GalR3, as shown in transfected cell lines, with a similar affinity as to GalR2 (Lu et al., 2005).

The neuroprotective role of galanin is probably mediated through GalR2. For example, galanin or its fragment Gal (2–11) administered to cultured rat basal forebrain neurons or cell lines can protect against β-amyloid-induced cell death (Ding et al., 2006, Elliott-Hunt et al., 2007, Pirondi et al., 2010). Moreover, galanin has been found to attenuate seizure activity in part mediated through modulation of GalR2, attenuating glutamate release (Mazarati et al., 1998, Mazarati et al., 2000, Kokaia et al., 2001, Mazarati et al., 2004, Lerner et al., 2010). This is of interest since the hippocampal formation (HiFo) is extremely vulnerable to injury, in particularly in situations of excessive glutamate release which opens receptor-regulated ionophores (Benveniste, 2009).

Galanin is strongly expressed in the noradrenaline (NA) neurons in the locus coeruleus (LC) (Melander et al., 1986b, Xu et al., 1998a, Xu et al., 1998b) and inhibits the firing of these neurons, likely through GalR1 (Seutin et al., 1989, Sevcik et al., 1993, Pieribone et al., 1995, Xu et al., 1998a, Ma et al., 2001). The LC NA cells project i.a. to most cortical regions and the HiFo, and galanin has been shown to inhibit the release of glutamate in the latter region through a pre-synaptic mechanism (Ari and Lazdunski, 1989). Thus, by attenuating glutamate excitotoxicity galanin may play a protective role reducing cell death (Elliott-Hunt et al., 2004, Pirondi et al., 2005).

Previously we have shown that occlusion of the middle cerebral artery (MCAo) by a microclip caused a decrease in galanin-like immunoreactivity in the HiFo of the ischemic hemisphere 3days after a transient MCAo (Theodorsson and Theodorsson, 2005). Using the same model, we were surprised to find that intracerebroventricular (icv) administration of a GalR2/3 agonist, Gal (2–11), doubled the size of the ischemic lesion (Holm et al., 2011). To further elucidate the effect of MCAo in the female rat brain on the endogenous galanin system, we studied galanin and GalR1, GalR2 and GalR3 gene expression 7days after MCAo. Quantitative real-time PCR (qRT-PCR) was employed on punch biopsies from the LC, somatosensory cortex and the dorsal HiFo.

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2. Materials and methods 

2.1. Animal model 

The protocol was approved by the Local Ethics Committee for Animal Care and Use at Linköping University, Sweden. A total of 22 female rats (Sprague–Dawley, Taconic, Germany) were used and housed at the Linköping University Animal Department for at least one week before the start of the experiments. Two rats were kept in each cage at a constant room temperature (21°C), with free access to water and standard rat chow (Lactamin, Vadstena, Sweden), and with a 12-h light/dark cycle (light on at 8.00 am) before the experiments. At the age of 12–14weeks, body weight of 216–283g, the animals were randomly allocated to MCA occlusion (n=16) or sham (n=6) treatments and observation periods of 7days.

2.2. Vaginal smear 

Vaginal smears were used to assess the stage in the estrous cycle. Vaginal secretion was collected with a plastic pipette filled with 10μL of normal saline (0.9% NaCl; B. Braun Medical AB, Bromma, Sweden) by inserting the tip into the rat’s vagina. Saline was quickly released and immediately drawn back into it. The sample containing cells were placed on untreated glass microscopic slide and viewed at 20× and 40× magnifications. The surgery was employed, when the animals were in the di-estrous and estrous phase.

2.3. Occlusion of the MCA 

Occlusion of the MCA was performed as described earlier (Theodorsson et al., 2005). In brief, anesthesia was induced by 4% isoflurane (Forene®, Abbott, Scandinavia AB, Kista, Sweden) in a mixture (30/70%) of oxygen/nitrous oxide in an induction chamber. A soft endotracheal tube was inserted for controlled ventilation (Zoovent, CWC600AP, ULV Ltd., Newport, UK) using 1–1.5% isoflurane in a mixture of oxygen/nitrous oxide (as above). The tidal volume and ventilation frequency were carefully regulated using on-site monitoring of blood gases and acid/base status (AVL, OPTI 1 Medical Nordic AB, Stockholm, Sweden).

The rats were placed with their left side up on a thermostatic heating pad (Harvard Homeothermic Blanket system, Edenbridge, UK) to maintain the core/rectum temperature at 37.0±0.5°C. The left femoral artery was cannulated using a soft catheter Micro-renathane® tubing (MRE-025 Braintree Scientific, Inc., MA, USA) primed with saline containing heparin (100IU/mL, Lovens, Ballerup, Denmark) for registration of blood pressure and pulse [(AcqKnowledge software (BioPac system, Goleta, CA, USA) and Blood Pressure Transducer (56360, Stoelting, IL, USA)].

Using an operating microscope (Zeiss Opmi 6-H, oberkochen, West Germany), the left MCA was exposed transcranially (Tamura et al., 1981), removing part of the zygomatic bone but maintaining the temporal muscle and the facial and mandibular nerves. The MCA was occluded for 60min with a microclip between the rhino-cortical branch and the lenticulostriate artery (Theodorsson et al., 2005).

2.4. Measuring the size of the ischemic brain lesions 

The ischemic lesions were measured 7days after the 60min occlusion of the MCA. The rats were anesthetized by pure carbon dioxide and sacrificed by rat guillotine. The brain was carefully dissected out and cooled in 0.9% NaCl at 4°C. Two-millimeter thick coronary slices of the brain were cut out with razor blades directed by a rat brain matrix (RBM-4000, ASI Instrument Inc., Warren, MI, USA) using the bregma (B) as position 0, with two slices, B +2mm and B +4mm anterior to B and six slices, posterior to B (B −2, −4, −6, −8, −10 and −12mm, respectively).

The slices were freed from the dura mater and soaked for 10min in a solution of 2% 2,3,5-triphenyltetrazolium hydrochloride (TTC) in 0.1mol/L phosphate-buffered saline (PBS) (pH 7.4) in a small Petri dish, maintained at 37°C in a heater. Gentle stirring of the slices was used to ensure even exposure of the surfaces to staining. Excess TTC was then drained, and the seven slices (B +2mm to B −8mm) were scanned (ScanJet 2c, Hewlett–Packard, Cupertino, CA, USA).

The size of the brain lesion was measured using SigmaScan Pro version 5 (Systat Software Inc., Richmond, CA, USA). The image was divided into red color spectra to measure the total area of the slice, green color spectra for measuring the ischemic lesion area and blue color spectra (not used in this context). To sharpen the boundary between the slice itself and its surroundings, the intensity of the red spectra was maximized before the measurement. In the green spectra the outer boundaries of the ischemic area were marked in the “overlay draw mode” to demark the area from normal structures in the brain. To mark the ischemic area an automatically threshold of 40% in the green spectra was used in the function “fill mode” (Bederson et al., 1986, Goldlust et al., 1996).

2.5. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in brain punch biopsies 

Gene expression was analysed by RT-PCR in punch biopsies (2mm in diameter and 2mm thick) from the brain slices from LC (B −12mm), somatosensory cortex (B −2mm) and the dorsal HiFo (B −8mm) (Fig. 1). The biopsies were rapidly collected using a micro-dissection protocol and snap-frozen on dry ice and stored at −70°C. Total RNA was extracted using the RNeasy Micro kit (Qiagen, Sollentuna, Sweden), according to the manufacturer’s instructions, including deoxyribonuclease treatment with ribonuclease-free deoxyribonuclease set (RNase-Free DNase set, Qiagen). The concentrations and purity of the RNA were measured with NanoDrop ND-1000 Spectrophotometer (Saveen & Werner AB, Limhamn, Sweden). RNA samples were kept at −70°C prior to analysis.

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  • Fig. 1. 

    Lesion areas of coronal slices of the brains of female rats visualized by TTC staining after transient (60min) occlusion of the middle cerebral artery. The TTC staining colors viable (well-functioning mitochondria) tissue in red and ischemic tissue lesions in white.

A sample of 80ng of RNA from LC, dorsal HiFo and cortex, were reversely transcribed to single-stranded cDNA by random hexamer priming using High Capacity cDNA RT kit (Applied Biosystem, Stockholm, Sweden), in a volume of 20μL, using the following program: 25°C for 10min, 37°C for 60min, 85°C for 5s, 4°C ∞. The samples were diluted 1:10 and kept at −70°C prior to analysis.

For RT-PCR, 7μL of cDNA were added to TaqMan fast Universal PCR Master Mix (2X), RNase-free water and TaqMan Gene Expression Assay primer (Applied Biosystems) to achieve a final reaction volume of 20μL. The PCR protocol consisted of: initiation at 1 cycle at 95°C for 20s, followed by amplification for 40 cycles at 95°C for 1s and 60°C for 20s.

The following TaqMan Gene Expression assays were used: Galanin: Rn 00583681_m1 (Applied Biosystem); Galanin receptor 1 – FP: TGGCGCGCAGCAAAC, RP: GGCCAGGGTGAAGATGCT, probe: CCGTGTCCATGCTCG (Invitrogen AB, Lidingö, Sweden); Galanin receptor 2 – Rn 01773918_m1 (Applied Biosystem); Galanin receptor 3 – FP: CTGGACGTGGCCACCTT, RP: TAGGCCAGGCTCACCAC, probe: CCGCGGGCTACCTG (Invitrogen).

Ywahz Rn 00755072_m1 and Sdha Rn 00590475_m1 were used as endogenous reference genes for study of the effects of lesions (Gubern et al., 2009) and of the estrous cycle (Hvid et al., 2010).

The reactions were carried out in 96-well plates covered with optical adhesive film (Applied Biosystem). Each sample was analyzed in duplicate and average values were calculated. No-template reactions with water instead of cDNA were included as negative controls on all plates.

Gene expression was calculated using the ΔΔCt method (Ct=threshold cycle). Each gene was normalized with the corresponding average of Sdha and Ywahz expression in the same animal and expressed as the fold-difference in relation to the control group.

2.6. Immunohistochemistry 

Three rats with MCA occlusion and three controls were deeply anaesthetized with sodium pentobarbital (Mebumal; 50mg/kg, i.p.), and transcardially perfused with 50ml warm saline (0.9%; 37°C), followed by 50ml of a warm mixture of paraformaldehyde (4%; 37°C) with 0.4% picric acid in 0.16M phosphate buffer (pH 7.2) (Pease, 1962, Zamboni and De Martino, 1967) and then by 200ml of the same, but ice cold fixative. The brain was dissected out and immersed in the same fixative for 90min at 4°C and subsequently stored in 10% sucrose in phosphate-buffered saline (PBS; pH 7.4) containing 0.01% sodium azide (Sigma, St. Louis, MO) and 0.02% Bacitracin (Sigma) at 4°C for 2days. Tissues were frozen and cut in a cryostat (Microm, Heidelberg, Germany) at 12μm thickness and mounted onto alum-gelatin-coated slides. After cutting the sections were dried at room temperature (RT) for 30min, rinsed in PBS for 15min and incubated for 24h at 4°C in a humid chamber with primary antiserum. The galanin antiserum (Theodorsson and Rugarn, 2000) (were diluted in PBS containing 0.2% (w/v) bovine serum albumin, 0.03% Triton X-100 (Sigma). Immunoreactivity was visualized using a commercial kit (TSA Plus, NEN Life Science Products, Inc., Boston, MA, USA). Briefly, the slides were rinsed in TNT buffer (0.1M Tris–HCl, pH 7.5; 0.15M NaCl; 0.05% Tween 20) for 15min at RT, blocked with TNB buffer (0.1M Tris–HCl;pH 7.5; 0.15M NaCl; 0.5% Du Pont Blocking Reagent) for 30min at RT followed by 30min incubation with horseradish peroxidase-labeled secondary antibody (1:200) (Dako, Copenhagen, Denmark) diluted in TNB buffer (1:200). After a simple wash (15min) in TNT buffer all sections were exposed to biotinyl tyramide-fluorescein (1:100) diluted in amplification diluent for approximately 15min, and finally washed in TNT buffer for 30min at RT. Finally, all slides were cover-slipped with glycerol/PBS (9:1) containing 0.1% para-phenylenediamine (Johnson and Nogueira Araujo, 1981, Platt and Michael, 1983).

The sections were analyzed in a Nikon Eclipse E 600 fluorescence microscope (Tokyo, Japan) equipped with 10× (0.5N.A.) and 20× (0.75N.A.) objectives.

2.7. Statistical analysis 

Data were analyzed using two-tailed t-test for independent samples, only of the groups deemed of interest when the study was designed. A p-value <0.05 was considered statistically significant. Data are reported as mean±SEM.

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3. Results 

3.1. Monitoring of vital parameters 

Repeated measurements of the arterial blood gases before, during and after MCAo as well as continuous arterial blood pressure were within physiological ranges for all animals. The rats were maintained at a normothermic core/rectum temperature at 37.0±0.5°C for the duration of the experiment. No rats died peri- and postoperatively. Three rats were excluded from the study, since no ischemic lesion could be detected.

We made sure in the design of the study that the rats were included at di-estrous and estrous phase for analyses of gene-expression. Moreover, the reference genes used (Sdha and Ywahz) were not affected by the estrous-phases. This is important, since HiFo and cortex are sensitive to estrogen under various conditions and in various experimental paradigms (McEwen, 2002), and since galanin expression is influenced by estrogen not only in the pituitary gland (Vrontakis et al., 1987, Kaplan et al., 1988) but also in some brain regions (Gabriel et al., 1990).

3.2. Size of the ischemic brain lesion 

The mean lesion area as visualized with TTC staining 7days after 60min MCAo. The lesions were largest at B (6.2±1.7%) and B −2mm (5.7±0.9%) (Fig. 1, Fig. 2).

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  • Fig. 2. 

    Areas of ischemic lesions in coronal slices of the brains of female rats determined with the TTC staining method 7days after transient (60min) occlusion of the middle cerebral artery. The lesions were largest at B (6.2±1.7% and B −2mm (5.7±0.9%).

3.3. Middle cerebral artery occlusion induced changes in galanin and GalR1 gene-expression in the locus coeruleus and cortex 

The effect of a 60-min-MCAo on the gene expression of galanin, GalR1, GalR2 and GalR3 was measured after 7days in the LC, somatosensory cortex and in the dorsal HiFo by means of quantitative RT-PCR. The galanin gene expression in the LC was up-regulated ipsi-laterally by 259% (p<0.01) and GalR1 by 45% (p<0.05) (Fig. 3) as compared to the contra-lateral control side. No evidence was found for changes in GalR2 or GalR3 expression in this nucleus.

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  • Fig. 3. 

    Gene expression of galanin, galanin receptor1 (GalR1), GalR2 and GalR3 measured by RT-PCR in punch biopsies from locus coeruleus in female rats (n=22) 7days after 60min transient occlusion of the middle cerebral artery. The galanin gene expression in the locus coeruleus was up-regulated by 259% (p<0.01) and GalR1 by 45% (p<0.05) in the ischemic hemisphere as compared to the contra-lateral control side.

In the somatosensory cortex, GalR1 transcript levels decreased ipsi-laterally by 35% (p<0.05) in the ischemic hemisphere as compared to the contra-lateral control side (Fig. 4). We found no effects in the somatosensory cortex on galanin, GalR2 and GalR3 gene expression. The punch biopsies in the somatosensory cortex include the peri-ischemic area/penumbra.

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  • Fig. 4. 

    Gene expression of galanin, galanin receptor1 (GalR1), GalR2 and GalR3 measured by RT-PCR in punch biopsies from parietal cortex in female rats (n=22) 7days after 60min transient occlusion of the middle cerebral artery. In cortex, gene expression of GalR1 decreased by 35% (p<0.05) in the ischemic hemisphere as compared to the contra-lateral control side.

In dorsal HiFo a trend towards a decrease of galanin and GalR2 mRNAs was found in the ischemic hemisphere, however not reaching significance (p<0.08 and 0.07, respectively) (Fig. 5).

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  • Fig. 5. 

    Gene expression of galanin, galanin receptor1 (GalR1), GalR2 and GalR3 measured by RT-PCR in punch biopsies from dorsal of hippocampus in female rats (n=22) 7days after 60min transient occlusion of the middle cerebral artery.

3.4. Immunohistochemistry 

In two of the three control rats numerous strongly fluorescent cell bodies and many processes were observed throughout the rostro-caudal extent of the LC (Fig. 6A, C, and E). In contrast, in the rats receiving MCAo only few, weakly fluorescent cell bodies and fluorescent dots were seen (Fig. 6B, D, and F). A similar result was found in one of the three control rats (not shown).

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  • Fig. 6. 

    Immunofluorescence micrographs of three different rostro-caudal levels from locus coeruleus of a control female rat (A, C, and E) and a female rat 7days after a 60min transient occlusion of the middle cerebral artery (B, D, F) after incubation with galanin antiserum. Note: strongly fluorescent cell bodies and processes in controls (A, C, and E) versus weak fluorescence in stroke rat (B, D, and F). Bar indicates 100μm.

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4. Discussion 

In the present study, the expression of galanin and galanin receptors was examined in rats after the stroke model based on MCAo, using qPCR and of galanin using immunohistochemistry. We selected three brain regions, the somatosensory cortex which is partly lesioned by the occlusion (extending from the main lesion in the striatum), the HiFo which is apparently unaffected, and the LC which provides noradrenergic/galaninergic innervation of both previous regions, probably partly representing collaterals (Room et al., 1981). Thus, one and the same neuron can send afferents to both somatosensory cortex and HiFo.

The gene expression for galanin increased by 259% and for GalR1 by 45% in the LC in the ischemic hemisphere compared to the intact control side 7days after a 60-min-microclip occlusion of the MCA in female rats. Furthermore, a 35% decrease in the GalR1 transcript levels was found in the somatosensory cortex including the peri-infarction area/penumbra.

The immunohistochemical analysis showed a depletion of galanin-like immunoreactivity (Li) in the LC neurons of MCAo rats.

4.1. Distribution of the galanin system 

Under normal circumstances transcripts for galanin and galanin receptors could not be detected with in situ hybridization in cortex or HiFo (Jacobowitz and Skofitsch, 1990). However, after colchicine treatment galanin-positive cells can be seen in cortex, hippocampus and striatum (Skofitsch and Jacobowitz, 1985, Melander et al., 1986a, Melander et al., 1986b, Melander et al., 1986c), also in glia (Xu et al., 1992, Calza et al., 1998) and in the latter cell type in addition after spreading depression (Shen et al., 2003). Radioimmunoassay showed low concentrations of galanin-Li in cortical areas compared to e.g. hypothalamus (Skofitsch and Jacobowitz, 1986). Immunohistochemical and biochemical studies suggest that galanin in dorsal cortical and HiFo areas is mainly localized in afferents from the brain stem, especially in NA fibers originating in the LC (Melander et al., 1986c, Gabriel et al., 1995, Xu et al., 1998b). Taken together, these data show that, in addition to galanin mainly in afferents, there are local cortical/hippocampal systems that have the potential to synthesize galanin de novo.

4.2. Ischemia and the galanin system: Earlier and present studies 

A number of earlier studies in male rodents involving different stroke models and time periods have reported on expression of the galanin system after MCAo (Bond et al., 2002, Raghavendra Rao et al., 2002, Hwang et al., 2004, Lee et al., 2005, De Michele et al., 2006). Our present work, monitoring transcript levels, may to some extent be comparable with the two array studies (Bond et al., 2002, Raghavendra Rao et al., 2002). However, both monitored effects 24h and/or shorter after MCAo versus our 7days. Bond et al. (2002) analyzed whole hemispheres (versus our punches) using normotensive Sprague Dawley rats, occluding the MCA for 90min followed by reperfusion for 24h. They found that galanin mRNA was increased in the ipsilateral versus the contra-lateral “hemisphere”, however, dependent on reference gene.

A better comparison is with Raghavendra Rao et al. (2002) who dissected “MCA territory” and found significant increases both in GalR1, and especially galanin mRNA in cortex after 24h, versus our decrease in GalR1 mRNA in the same area after 7days. It may be speculated that Raghavendra Rao et al. (2002) detected an initial transient increase, possibly at least in part in glia (Xu et al., 1992, Calza et al., 1998, Shen et al., 2003), which then subsided after 7days as shown in our study, possibly due to tissue necrosis. In fact, in the Raghavendra Rao et al. (2002) study the GalR1 mRNA levels were lower at 24 than at 6h after MCAo, indicating a downward trend.

With regard to galanin peptide, De Michele et al. (2006) performed MCAo without reperfusion and analyzed rats (gender not given) 1, 4, 24h and 3days after insult. They report presence of galanin-positive neurons in the peri-infarct area both in cortex and, especially in the caudate nucleus after 3days, but such neurons were “barely” detected at 24h or in the infarct zone. In addition, galanin-Li was accumulated in fibers in the peri-infarct zone.

The other two immunohistochemical studies observed increases in neurons in HiFo of gerbils, that is a different species (Hwang et al., 2004, Lee et al., 2005), but in the 4-day-group galanin levels were back to base line. This may be relevant in relation to the decrease in galanin-Li observed in our two studies (Theodorsson and Theodorsson, 2005, Theodorsson et al., 2008). Since the HiFo, at least in rat, is not directly affected by the lesion, this may indicate increased release of galanin from noradrenergic/galaninergic LC afferents in this area (Xu et al., 1998b) in response to the ischemic injury/stress (see below). If so, the present increase in galanin mRNA in the LC neurons may in part be the result of a feed-back compensatory activation/production of galanin to replace peptide released in the HiFo and from LC soma and dendrites (see below).

An intriguing finding in relation to stroke and the galanin system is the increased ischemic brain lesion caused by the continuous infusion of a galanin agonist, Gal (2–11), for 3 or 7days (Holm et al., 2011). This agonist mainly acts through GalR2, which in many other studies has been shown to exert trophic effects (see above and below). Interestingly, in the present study there was a trend towards lower GalR2 mRNA levels in the HiFo. Taken together, other studies showing changes in the galanin system were carried out at relatively short intervals after artery occlusion, whereby changes decreased with time. None of the studies included a 7-day post-occlusion period.

4.3. A possible scenario 

The selected two forebrain regions (somatosensory cortex and the HiFo) are differently affected by the MCAo: the former is lesioned, the latter is not. Both regions are innervated by ascending LC neurons, probably at least partly via collaterals (Room et al., 1981). Thus, one and the same LC neuron may project to both regions, with some collaterals damaged, others not. This damage may in turn lead to up-regulation of galanin synthesis (increased transcript levels), as seen after bulbectomy, that is after injury to nerve terminals originating from the LC neurons (Holmes and Crawley, 1996), as also seen in other systems (Hökfelt et al., 1987, Villar et al., 1989, Cortes et al., 1990).

Also another mechanism, mutually not exclusive, may underlie galanin up-regulation in LC. Thus, this nucleus is an important part of the central stress system activated by many forms of stress (Heilig, 2004, Valentino and Van Bockstaele, 2008), and stress-induced increase in galanin transcript levels in LC has in fact been reported (Holmes et al., 1995, Sweerts et al., 1999). It is likely that also MCAo induces a considerable stress, activating the LC.

The activation may lead to enhanced galanin release in the forebrain, possibly in agreement with the decrease in galanin levels in the HiFo after MCAo reported by (Theodorsson and Theodorsson, 2005). A likely activity-dependent decrease/disappearance of galanin in/from cortical/hippocampal nerve terminals has been observed in immunohistochemical studies after reserpine treatment (Xu et al., 1998b) and seizure activity (Mazarati et al., 1998). The increased activity could in turn result in feed-back induced increase in galanin synthesis to replace released peptide.

However, there is presumably also increased galanin release from soma and dendrites in the LC, resulting in inhibition of firing via GalR1 and opening of potassium channels (Pieribone et al., 1995, Vila-Porcile et al., 2009). The immunohistochemical analysis supports this view: in the three MCAo rats galanin-Li was weak in the LC versus strongly labeled neurons and processes in two of the three controls. This inhibition should be strengthened by the parallel up-regulation of GalR1 seen in our study and may serve to dampen LC activity. Such a decrease in activity may aim at evoking a ‘rest state’ for the neuron, perhaps improving/facilitating recovery and regeneration. This process could be supported by activation of GalR2, which is expressed in LC (Burazin et al., 2000, O’Donnell et al., 2003). This receptor has in other systems has been shown to mediate trophic effects, such as enhancing neurite outgrowth and neuroprotection, involving ERK/MAPK and Akt (Holmes et al., 2000, Mahoney et al., 2003, Hobson et al., 2008).

4.4. Aspects on methodology 

The reference gene used in qRT-PCR studies should be expressed in all cells at constant expression levels under all circumstances (Thellin et al., 1999). However, expression stability and ranking of reference genes vary both between tissues, including phases of the estrous cycle, and due to the effects of experimental ischemia (Harrison et al., 2000, Medhurst et al., 2000, Meldgaard et al., 2006, Tian et al., 2007). To compensate for gene expression differences, it has been suggested that multiple reference genes should be used for normalization of gene expression between samples from the same tissue (Vandesompele et al., 2002).

The most stable gene expression in rat mammary gland during phases of the estrous cycle has been shown to be Sdha, Tbp and Atp5b (Hvid et al., 2010), a relevant issue since we used female rats in the present study. Furthermore, a recent validation study showed that Sdha and Ywahz are optimal reference genes when studying in vivo models of cerebral ischemia using qRT-PCR (Gubern et al., 2009). We therefore used these two genes in the present study for normalization of gene expression between samples from the same tissue. We have used the sham rats as controls, because they minimize bias due to surgery.

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Disclosure/conflict of interest 

The authors have no conflict of interest to disclose. There is no duality of interest.

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Acknowledgements 

The study was financed by the County Council of Östergötland, Sweden, and supported by The Swedish Research Council and The Marianne and Marcus Wallenberg Foundation.

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References 

  1. Agoston DV, Komoly S, Palkovits M. Selective up-regulation of neuropeptide synthesis by blocking the neuronal activity: galanin expression in septohippocampal neurons. Exp. Neurol. 1994;126:247–255
  2. Beal MF, MacGarvey U, Swartz KJ. Galanin immunoreactivity is increased in the nucleus basalis of Meynert in Alzheimer’s disease. Ann. Neurol. 1990;28:157–161
  3. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986;17:472–476
  4. Ben-Ari Y, Lazdunski M. Galanin protects hippocampal neurons from the functional effects of anoxia. Eur. J. Pharmacol. 1989;165:331–332
  5. Benveniste H. Glutamate, microdialysis, and cerebral ischemia: lost in translation?. Anesthesiology. 2009;110:422–425
  6. Bond BC, Virley DJ, Cairns NJ, Hunter AJ, Moore GB, Moss SJ, et al. The quantification of gene expression in an animal model of brain ischaemia using TaqMan real-time RT-PCR. Brain Res. Mol. Brain Res. 2002;106:101
  7. Branchek TA, Smith KE, Gerald C, Walker MW. Galanin receptor subtypes. Trends Pharmacol. Sci. 2000;21:109–117
  8. Brecht S, Buschmann T, Grimm S, Zimmermann M, Herdegen T. Persisting expression of galanin in axotomized mamillary and septal neurons of adult rats labeled for c-Jun and NADPH-diaphorase. Brain Res. Mol. Brain Res. 1997;48:7–16
  9. Burazin TC, Larm JA, Ryan MC, Gundlach AL. Galanin-R1 and -R2 receptor mRNA expression during the development of rat brain suggests differential subtype involvement in synaptic transmission and plasticity. Eur. J. Neurosci. 2000;12:2901–2917
  10. Calza L, Giardino L, Hokfelt T. Thyroid hormone-dependent regulation of galanin synthesis in neurons and glial cells after colchicine administration. Neuroendocrinology. 1998;68:428–436
  11. Ch’ng JL, Christofides ND, Anand P, Gibson SJ, Allen YS, Su HC, et al. Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury. Neuroscience. 1985;16:343–354
  12. Chan-Palay V. Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J. Comp. Neurol. 1988;273:543–557
  13. Cortes R, Villar MJ, Verhofstad A, Hokfelt T. Effects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study. Proc. Natl. Acad. Sci. USA. 1990;87:7742–7746
  14. Crawley JN. Functional interactions of galanin and acetylcholine: relevance to memory and Alzheimer’s disease. Behav. Brain Res. 1993;57:133–141
  15. De Michele M, Sancesario G, Toni D, Ciuffoli A, Bernardi G, Sette G. Specific expression of galanin in the peri-infarct zone after permanent focal cerebral ischemia in the rat. Regul. Pept. 2006;134:38–45
  16. Ding X, MacTavish D, Kar S, Jhamandas JH. Galanin attenuates beta-amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons. Neurobiol. Dis. 2006;21:413–420
  17. Elliott-Hunt CR, Marsh B, Bacon A, Pope R, Vanderplank P, Wynick D. Galanin acts as a neuroprotective factor to the hippocampus. Proc. Natl. Acad. Sci. USA. 2004;101:5105–5110
  18. Elliott-Hunt CR, Pope RJ, Vanderplank P, Wynick D. Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage. J. Neurochem. 2007;100:780–789
  19. Gabriel SM, Knott PJ, Haroutunian V. Alterations in cerebral cortical galanin concentrations following neurotransmitter-specific subcortical lesions in the rat. J. Neurosci. 1995;15:5526–5534
  20. Gabriel SM, Koenig JI, Kaplan LM. Galanin-like immunoreactivity is influenced by estrogen in peripubertal and adult rats. Neuroendocrinology. 1990;51:168–173
  21. Goldlust EJ, Paczynski RP, He YY, Hsu CY, Goldberg MP. Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride-stained rat brains. Stroke. 1996;27:1657–1662
  22. Gubern C, Hurtado O, Rodriguez R, Morales JR, Romera VG, Moro MA, et al. Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia. BMC Mol. Biol. 2009;10:57
  23. Habert-Ortoli E, Amiranoff B, Loquet I, Laburthe M, Mayaux JF. Molecular cloning of a functional human galanin receptor. Proc. Natl. Acad. Sci. USA. 1994;91:9780–9783
  24. Harrison DC, Medhurst AD, Bond BC, Campbell CA, Davis RP, Philpott KL. The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia–caspase-3 as a case study. Brain Res. Mol. Brain Res. 2000;75:143–149
  25. Heilig M. The NPY system in stress, anxiety and depression. Neuropeptides. 2004;38:213–224
  26. Hobson SA, Bacon A, Elliot-Hunt CR, Holmes FE, Kerr NC, Pope R, et al. Galanin acts as a trophic factor to the central and peripheral nervous systems. Cell Mol. Life Sci. 2008;65:1806–1812
  27. Holm L, Theodorsson E, Hokfelt T, Theodorsson A. Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats. Neuropeptides. 2011;45:17–23
  28. Holmes FE, Mahoney S, King VR, Bacon A, Kerr NC, Pachnis V, et al. Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity. Proc. Natl. Acad. Sci. USA. 2000;97:11563–11568
  29. Holmes PV, Blanchard DC, Blanchard RJ, Brady LS, Crawley JN. Chronic social stress increases levels of preprogalanin mRNA in the rat locus coeruleus. Pharmacol. Biochem. Behav. 1995;50:655–660
  30. Holmes PV, Crawley JN. Olfactory bulbectomy increases prepro-galanin mRNA levels in the rat locus coeruleus. Brain Res. Mol. Brain Res. 1996;36:184–188
  31. Hwang IK, Yoo KY, Kim DS, Do SG, Oh YS, Kang TC, et al. Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils. Brain Res. 2004;1023:193–199
  32. Hvid H, Ekstrom CT, Vienberg S, Oleksiewicz MB, Klopfleisch R. Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues. Vet. J. 2010;190:103–108
  33. Hökfelt T, Wiesenfeld-Hallin Z, Villar M, Melander T. Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci. Lett. 1987;83:217–220
  34. Jacobowitz DM, Skofitsch G. Localization of galanin cell bodies in the brain by immunocytochemistry and in situ hybridization. In:  Hökfelt T,  Bartfai D,  Jacoboswitz D,  Ottoson D editor. Galanin – A new multifunctional peptide in the neuro-endocrine system. Wenner-Gren International Symposium Series 58. London: Macmillan Press Houndsmill; 1990;p. 69–92
  35. Johnson GD, Nogueira Araujo GM. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods. 1981;43:349–350
  36. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, et al. Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc. Natl. Acad. Sci. USA. 1988;85:7408–7412
  37. Kerr BJ, Cafferty WB, Gupta YK, Bacon A, Wynick D, McMahon SB, et al. Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur. J. Neurosci. 2000;12:793–802
  38. Kokaia M, Holmberg K, Nanobashvili A, Xu ZQ, Kokaia Z, Lendahl U, et al. Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin. Proc. Natl. Acad. Sci. USA. 2001;98:14006–14011
  39. Lee HY, Hwang IK, Kim DH, Kim JH, Kim CH, Lim BO, et al. Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils. Exp. Anim. 2005;54:21–27
  40. Lerner JT, Sankar R, Mazarati AM. Galanin and epilepsy. EXS. 2010;102:183–194
  41. Liu HX, Brumovsky P, Schmidt R, Brown W, Payza K, Hodzic L, et al. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc. Natl. Acad. Sci. USA. 2001;98:9960–9964
  42. Lu X, Lundstrom L, Bartfai T. Galanin (2–11) binds to GalR3 in transfected cell lines: limitations for pharmacological definition of receptor subtypes. Neuropeptides. 2005;39:165–167
  43. Ma X, Tong YG, Schmidt R, Brown W, Payza K, Hodzic L, et al. Effects of galanin receptor agonists on locus coeruleus neurons. Brain Res. 2001;919:169–174
  44. Mahoney SA, Hosking R, Farrant S, Holmes FE, Jacoby AS, Shine J, et al. The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons. J. Neurosci. 2003;23:416–421
  45. Mazarati A, Lu X, Kilk K, Langel U, Wasterlain C, Bartfai T. Galanin type 2 receptors regulate neuronal survival, susceptibility to seizures and seizure-induced neurogenesis in the dentate gyrus. Eur. J. Neurosci. 2004;19:3235–3244
  46. Mazarati AM, Hohmann JG, Bacon A, Liu H, Sankar R, Steiner RA, et al. Modulation of hippocampal excitability and seizures by galanin. J. Neurosci. 2000;20:6276–6281
  47. Mazarati AM, Liu H, Soomets U, Sankar R, Shin D, Katsumori H, et al. Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J. Neurosci. 1998;18:10070–10077
  48. McEwen BS. Sex, stress and the hippocampus: allostasis, allostatic load and the aging process. Neurobiol. Aging. 2002;23:921
  49. Medhurst AD, Harrison DC, Read SJ, Campbell CA, Robbins MJ, Pangalos MN. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J. Neurosci. Methods. 2000;98:9–20
  50. Melander T, Hokfelt T, Rokaeus A. Distribution of galaninlike immunoreactivity in the rat central nervous system. J. Comp. Neurol. 1986;248:475–517
  51. Melander T, Hokfelt T, Rokaeus A, Cuello AC, Oertel WH, Verhofstad A, et al. Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J. Neurosci. 1986;6:3640–3654
  52. Melander T, Staines WA, Rokaeus A. Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs. Neuroscience. 1986;19:223–240
  53. Meldgaard M, Fenger C, Lambertsen KL, Pedersen MD, Ladeby R, Finsen B. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology. J. Neurosci. Methods. 2006;156:101–110
  54. Mennicken F, Hoffert C, Pelletier M, Ahmad S, O’Donnell D. Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system. J. Chem. Neuroanat. 2002;24:257–268
  55. Mitsukawa K, Lu X, Bartfai T. Galanin, galanin receptors, and drug targets. EXS. 2010;102:7–23
  56. Mufson EJ, Cochran E, Benzing W, Kordower JH. Galaninergic innervation of the cholinergic vertical limb of the diagonal band (Ch2) and bed nucleus of the stria terminalis in aging, Alzheimer’s disease and Down’s syndrome. Dementia. 1993;4:237–250
  57. O’Donnell D, Ahmad S, Wahlestedt C, Walker P. Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1. J. Comp. Neurol. 1999;409:469–481
  58. O’Donnell, D., Mennicken, F., Hoffert, C., Hubatxch, D., Pelletier, M., Walker, P., Ahmad, S. Localization of galanin receptor subtypes in the rat CNS. Handbook of Chemical Neuroanatomy, 2003, pp. 195–244.
  59. O’Meara G, Coumis U, Ma SY, Kehr J, Mahoney S, Bacon A, et al. Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons. Proc. Natl. Acad. Sci. USA. 2000;97:11569–11574
  60. Pease, D.C., 1962. Buffered formaldehyde as a killing agent and primary fixative for electron microscopy. Anat. Rec. 142, 342.
  61. Pieribone VA, Xu ZQ, Zhang X, Grillner S, Bartfai T, Hokfelt T. Galanin induces a hyperpolarization of norepinephrine-containing locus coeruleus neurons in the brainstem slice. Neuroscience. 1995;64:861–874
  62. Pirondi S, Fernandez M, Schmidt R, Hokfelt T, Giardino L, Calza L. The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells. J. Neurochem. 2005;95:821–833
  63. Pirondi S, Giuliani A, Del Vecchio G, Giardino L, Hokfelt T, Calza L. The galanin receptor 2/3 agonist Gal2–11 protects the SN56 cells against beta-amyloid 25–35 toxicity. J. Neurosci. Res. 2010;88:1064–1073
  64. Platt JL, Michael AF. Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J. Histochem. Cytochem. Off. J. Histochem. Soc. 1983;31:840–842
  65. Raghavendra Rao VL, Bowen KK, Dhodda VK, Song G, Franklin JL, Gavva NR, et al. Gene expression analysis of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia. J. Neurochem. 2002;83:1072–1086
  66. Rokaeus A, Melander T, Hokfelt T, Lundberg JM, Tatemoto K, Carlquist M, et al. A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci. Lett. 1984;47:161–166
  67. Room P, Postema F, Korf J. Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a fluorescent double labeling technique. Brain Res. 1981;221:219–230
  68. Seutin V, Verbanck P, Massotte L, Dresse A. Galanin decreases the activity of locus coeruleus neurons in vitro. Eur. J. Pharmacol. 1989;164:373–376
  69. Sevcik J, Finta EP, Illes P. Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with mu-opioid receptors. Eur. J. Pharmacol. 1993;230:223–230
  70. Shen PJ, Larm JA, Gundlach AL. Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression. Eur. J. Neurosci. 2003;18:1362–1376
  71. Skofitsch G, Jacobowitz DM. Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides. 1985;6:509–546
  72. Skofitsch G, Jacobowitz DM. Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides. 1986;7:609–613
  73. Sweerts BW, Jarrott B, Lawrence AJ. Expression of preprogalanin mRNA following acute and chronic restraint stress in brains of normotensive and hypertensive rats. Brain Res. Mol. Brain Res. 1999;69:113–123
  74. Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1981;1:53–60
  75. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V. Galanin – a novel biologically active peptide from porcine intestine. FEBS Lett. 1983;164:124–128
  76. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, et al. Housekeeping genes as internal standards: use and limits. J. Biotechnol. 1999;75:291–295
  77. Theodorsson A, Holm L, Theodorsson E. Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats. Brain Res. Brain Res. Protoc. 2005;14:181–190
  78. Theodorsson A, Holm L, Theodorsson E. Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain. Neuropeptides. 2008;42:79–87
  79. Theodorsson A, Theodorsson E. Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle cerebral artery in rats: no effect of ischemia on galanin in the stroke area but decreased levels in the hippocampus. Peptides. 2005;26:2257–2264
  80. Theodorsson E, Rugarn O. Radioimmunoassay for rat galanin: immunochemical and chromatographic characterization of immunoreactivity in tissue extracts. Scand. J. Clin. Lab. Invest. 2000;60:411–418
  81. Tian YF, Zhang PB, Xiao XL, Zhang JS, Zhao JJ, Kang QY, et al. The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR. Acta Anaesthesiol. Scand. 2007;51:158–164
  82. Valentino RJ, Van Bockstaele E. Convergent regulation of locus coeruleus activity as an adaptive response to stress. Eur. J. Pharmacol. 2008;583:194–203
  83. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.
  84. Wang S, Gustafson EL. Galanin receptor subtypes. Drug News Perspect. 1998;11:458–468
  85. Waters SM, Krause JE. Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues. Neuroscience. 2000;95:265–271
  86. Vila-Porcile E, Xu ZQ, Mailly P, Nagy F, Calas A, Hokfelt T, et al. Dendritic synthesis and release of the neuropeptide galanin: morphological evidence from studies on rat locus coeruleus neurons. J. Comp. Neurol. 2009;516:199–212
  87. Villar MJ, Cortes R, Theodorsson E, Wiesenfeld-Hallin Z, Schalling M, Fahrenkrug J, et al. Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience. 1989;33:587–604
  88. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG. Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J. Biol. Chem. 1987;262:16755–16758
  89. Xu Z, Cortes R, Villar M, Morino P, Castel MN, Hokfelt T. Evidence for upregulation of galanin synthesis in rat glial cells in vivo after colchicine treatment. Neurosci. Lett. 1992;145:185–188
  90. Xu ZQ, Bartfai T, Langel U, Hokfelt T. Effects of three galanin analogs on the outward current evoked by galanin in locus coeruleus. Ann. NY Acad. Sci. 1998;863:459–465
  91. Xu ZQ, Shi TJ, Hokfelt T. Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors. J. Comp. Neurol. 1998;392:227–251
  92. Zamboni I, De Martino C. Buffered picric acid formaldehyde. A new rapid fixative for electron microscopy. J. Cell. Biol. 1967;35:148A
  93. Zigmond, R.E., 2001. Can galanin also be considered as growth-associated protein 3.2? Trends Neurosci. 24, 494–496 (discussion 496).
  94. Zigmond RE, Hyatt-Sachs H, Mohney RP, Schreiber RC, Shadiack AM, Sun Y, et al. Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor. Perspect. Dev. Neurobiol. 1996;4:75–90

PII: S0143-4179(11)00103-X

doi:10.1016/j.npep.2011.11.001

Neuropeptides
Volume 46, Issue 1 , Pages 19-27, February 2012

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