Scholarly article on topic 'Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus'

Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus Academic research paper on "Biological sciences"

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Neuropeptides
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{Galanin / "Galanin receptor subtype selective ligands" / GAL1R / GAL2R / "Label-free real-time technology" / XCELLigence / Excitotoxicity / M1154}

Abstract of research paper on Biological sciences, author of scientific article — Kristin Webling, Jessica L. Groves-Chapman, Johan Runesson, Indrek Saar, Andreas Lang, et al.

Abstract The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1–3R. Throughout the last 20years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GAL1R and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL1R selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats. The peptide ligands were evaluated in vitro for their binding preference in a competitive 125I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL1R in the prevention of neuronal cell death.

Academic research paper on topic "Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus"

Pharmacological stimulation of GALiR but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus

Kristin Webling b'*'1, Jessica L. Groves-Chapman a1, Johan Runesson b, Indrek Saarc, Andreas Lang d, Rannar Sillard b, Erik Jakovenko b, Barbara Kofler d, Philip V. Holmes a, Ülo Langelb,c

a Neuroscience Program, Biomedical and Health Science Institute, Department of Psychology, The University of Georgia, Athens, GA, USA b Department of Neurochemistry, Stockholm University, Svante Arrheniusv. 16B, SE-10691, Stockholm, Sweden c Institute of technology, University of Tartu, Nooruse 1,50411, Tartu, Estonia

d Research Program for Receptorbiochemistry and Tumormetabolism, Laura Bassi Centre of Expertise THERAPEP, Department of Pediatrics/University Hospital Salzburg, Paracelsus Medical University, Müllner Hauptstr. 48, 5020, Salzburg, Austria

ABSTRACT

The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1-3R. Throughout the last 20 years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GALiR and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL^ selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats.

The peptide ligands were evaluated in vitro for their binding preference in a competitive 125I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL^ in the prevention of neuronal cell death.

© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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ARTICLE INFO

Article history:

Received 5 October 2015

Received in revised form 23 November 2015

Accepted 7 December 2015

Available online 11 December 2015

Keywords: Galanin

Galanin receptor subtype selective ligands

Label-free real-time technology

XCELLigence

Excitotoxicity

1. Introduction

Excitotoxicity is involved in a variety of acute and chronic neurodegenerative conditions in the central nervous system (CNS) such as hypoxia-ischemia, status epilepticus, Alzheimer's disease, Parkinsons disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Neuronal damage by glutamate excitotoxicity was identified as early as 1969 (Olney, 1969) and has been extensively studied since then. Despite more than 50 years of research, there are currently no pharmacological interventions in the clinical settings of acute neuro-degenerative conditions.

One of the most well documented excitotoxins is kainic acid (KA), an analog of glutamic acid, which induces the pathological changes

Abbreviations: CHO cells, Chinese hamster ovary cells; DMEM, Dulbecco's modified essential medium; FBS, fetal bovine serum; GAL1R, galanin receptor subtype 1; GAL2R, galanin receptor subtype 2; GAL3R, galanin receptor subtype 3; HEK cells, human embryonic kidney cells; KA, kainic acid; TFA, trifluoroacetic acid.

* Corresponding author.

E-mail address: kristin.webling@neurochem.su.se (K. Webling).

1 Shared first author.

partially mimicking neurodegeneration. Thus, KA-induced murine neurodegeneration has been used as a model for exploring relevant pharmacological treatment of excitotoxicity in neurodegenerative disorders. Furthermore, central administration of KA has been shown to produce convulsions through activation of the excitatory amino acid receptors. Therefore, administration of KA has also been used as a model for the study of epilepsy (Ben-Ari and Cossart, 2000). Centrally administered KA induces secondary damage in a number of structures and areas associated with the epileptic seizure, i.e. the cell damage is not caused by a direct KA-induced excitotoxic mechanism (Jarrard, 2002). It can therefore be difficult to dissociate direct and indirect neuroprotec-tive effects in paradigms where seizures are induced, since an indirect neuroprotection through anticonvulsant properties of any pharmaceutical might be interpreted as a direct neuroprotective action of the drug (Mazarati, 2004). Subsequently, in the present study, KA was administered intracerebroventricular (i.c.v.) in a dose that was earlier optimized to ensure neuronal cell death with a direct excitotoxic mechanism (Reiss et al., 2009), thus allowing us to study the neuroprotective components of the ligands used.

There are numerous compounds that have been proposed to have either or both neuroprotective and anticonvulsant activities. One of

http://dx.doi.org/10.1016/j.npep.2015.12.009

0143-4179/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

these is the neuropeptide galanin. Galanin is a 29 amino acid (30 in humans) bioactive peptide distributed in brain, spinal cord and gut, which has been ascribed involvement in a diversity of physiological actions (Lang et al., 2007) such as food intake (Saar et al., 2011), mood (Kuteeva et al., 2008), nociception (Jimenez-Andrade et al., 2006), modulating the effects of drug abuse (Jackson et al., 2011) and recently in regulation of hair growth (Holub et al., 2012). The expression of galanin is highly plastic and markedly up-regulated in many areas under certain physiological conditions. Galanin exerts its effects via three members (GAL1-3R) of the G-protein-coupled receptor (GPCR) superfamily. The receptors have distinct distribution patterns, as well as different signaling pathways (Langetal., 2015). GALiRandGAL3R has been reported to predominantly signal through Gi/o, leading to reduced cAMP-levels, whereas GAL2R mainly signal through Gq/11, leading to inositol phosphate accumulation and an increase in intracellular [Ca2+] (Branchek et al., 2000).

It has been shown that galanin plays a developmental survival role and functions as a trophic factor to adult neurons (Abbosh et al., 2011). By utilizing several transgenic mouse lines and the GAL2/3R selective ligand galanin (2-11), Wynick and colleagues have previously demonstrated that GAL2R mediates this effect (Hobson et al., 2008).

Several lines of evidence reveal that galanin functions as an endogenous neuroprotective factor for hippocampal neurons. A recombinant adeno-associated viral (AAV) system that overexpresses galanin together with the fibronectin secretory signal sequence attenuates the neuronal death of centrally administered KA (Haberman et al., 2003). Furthermore, transgenic galanin knock-out mice display a greater cell death than wildtype littermates when KA is administered i.p., and in concordance with this a reduction of cell death is seen in galanin overexpressing (OE) mice (Elliott-Hunt et al., 2004). In vitro studies in hippocampal cultures from these transgenic mice have confirmed that endogenous galanin diminishes excitotoxicity and apoptosis (Elliott-Hunt et al., 2004). The authors proposed that this neuroprotection is mediated primarily through the regulation of hippocampal excitability. A recent study showed that administration of M15, a non-selective galanin receptor antagonist significantly increases cell death in several hippocampal areas after systemic administration of KA (Schauwecker, 2010).

There are conflicting data regarding which receptor subtype mediates this neuroprotective role of galanin. Studies utilizing organotypic and dispersed primary hippocampal cultures have shown the importance ofGAL2R in galanin-mediated neuroprotection, when cell cultures are exposed to staurosporine (Elliott-Hunt et al., 2004), glutamate (Pirondi et al., 2005) and amyloid-p (Ding et al., 2006). In contrast, Elyse Schauwecker and colleagues have shown in a series of publications that there is a correlation between the expression level of GAL^ and neuronal cell death after excitotoxic assaults by systemic administration of KA (Kong et al., 2008; Schauwecker, 2010). Furthermore, it has been reported that GAL1R knock-out mice have an enhanced susceptibility to excitotoxin-induced neuronal injury (Mazarati et al., 2004; Mazarati, 2004). In order to delineate the contribution of the different galanin receptors, galanin receptor subtype selective ligands are needed, however to date only a few galanin receptor subtype selective ligands are available (Lang et al., 2015; Webling et al., 2012).

Recently, label-free real-time technologies have emerged as a powerful tool to study GPCR signaling. These techniques have numerous advantages including that neither the ligand nor the receptor require labeling; simplifying assay design and minimizing artifacts or liabilities created by the labeling process (Minor, 2008; Nayler et al., 2010; Scott and Peters, 2010). Cellular impedance based technology detects small changes in the contact area between cells and electrodes. These changes can be due to alterations in cell numbers and/or cell morphology, which is both affected by GPCR signaling (Scott and Peters, 2010). With this single detection system the responses from several GPCRs, known to couple to different signaling pathways can be quantified. This gives a huge advantage particularly when screening for subtype selective li-gands of galanin receptors, since the galanin receptor subtypes have been shown to signal through different G-proteins.

In this study, we present a novel peptide with GALV2R selective binding, namely M1154 (for sequence see Table 1). Furthermore, we introduce a single protocol for screening new ligands for subtype selective receptor signaling for all three galanin receptor subtypes, which is likely to promote the research effort to develop galanin receptor subtype specific ligands. M1154 together with previously published galanin receptor subtype selective ligands M617 and M1145 were evaluated in an excitotoxic assay using i.c.v. administrated KA in rats.

2. Materials and methods

2.1. Peptide sequence design

The N-terminal part of galanin, residue 1-14, is highly conserved among species and is vital for receptor interaction and biological activity. Exemplifying the importance of the N-terminal portion of galanin for receptor binding, galanin(1-16), despite lacking half the galanin sequence, retains high affinity binding (Table 2). All three receptors display high affinity for galanin but are distinguishable by the fact that GAL^ does not tolerate N-terminal deletions of the galanin peptide in comparison to the other two receptors. This difference has successfully been used in the design of several peptides, including the M871 (Sollenberg et al., 2006, 2010) and the M1145 peptide (Runesson et al., 2009), which both bind less to GAL^ compared to GAL2R. An important advance in the field was the publication ofthe galanin fragment, galanin (2-11), as a non-GAL^ ligand (Liuetal., 2001; Luetal.,2005). Two GAL^ preferential ligands have also been developed, the M617 and the Gal-B2, with a modest, 25-fold and 15-fold selectivity for GAL^ compared to GAL2R respectively (Bulaj et al., 2008; Lundstrom et al., 2005). The GAL3R interaction for M617 has also been characterized, showing a 200-fold difference when compared with GAL^ (Sollenberg et al., 2010).

The novel peptide M1154 (for sequence see Table 1) was designed to minimize affinity to GAL3R, as a mixed GAL1/2R ligand has been implicated as a putative therapeutic in several cases (Mitsukawa et al., 2008). For the design of M1154, particular interest was turned to the M617 peptide and its high affinity for GALiR. Deletion of Gly12 in several galanin analogous severely affects the interaction with GAL3R relative to GAL1R and GAL2R (Runesson et al., unpublished data), possibly due to the narrow binding pocket in GAL3R (Runesson et al., 2010) which does not tolerate the relative movement of the Pro13 induced kink in these galanin analogous. Furthermore, we have shown earlier that the Ala21Arg mutation in M617 reduces the GAL3R affinity six-fold (Sollenberg et al., 2010). M1154 combines these two modifications known to reduce GAL3R affinity relative to the other two galanin receptor subtypes, creating a GAL3R non-interacting galanin receptor ligand.

2.2. Cell culture

Bowes human melanoma cells (American type Culture Collection CRL-9607) expressing human GAL1R were cultured in Eagle's minimal

Table 1

Amino acid sequences of peptides used or discussed in this study.

Name Sequence Reference

Galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-amide Vrontakis et al.

(1-29), rat (1987)

Galanin GWTLNSAGYLLGPHAI-amide Land et al.

(1-16) (1991)

Galanin WTLNSAGYLL-amide Liu et al.

(2-11) (2001)

M617 GWTLNSAGYLLGPQPGFSPFR-amide Lundstrom et al.

(2005)

M1145 RGRGNWTLNSAGYLLGPVLPPPALALA-amide Runesson et al.

(2009)

M1154 GWTLNSAGYLLPQPGFSPFA-amide

Table 2

Experimental K determined by displacement studies with Porcine-[125I]-Galanin on Bowes Melanoma Cells expressing human GA^R, CHO cells stably transfected with human GAL2R or Flp-In T-REX 293 cells with tetracycline induced expression of human GAL3R.

Ki (nM)

Name --Ki GAL1R/K GAL2R Ki GALsR/Ki GAL2R

GAL1R GAL2R GAL3R

Galanin 1.75 ± 1.7a 2.98 ± 1.4a 4.49 ± 0.8a 0.6 1.5

Galanin(1-16) 0.78 ± 0.26 2.44 ± 0.57 8.98 ± 3.8 0.3 3.7

M617 0.23 ± 0.1b 5.71 ± 1.3b 49 ± 9.4c 25 8.6

M1145 587 ± 250a 6.55 ± 2.7a 497 ± 150a 90 76

M1154 11.7 ± 7.2 14.4 ± 4.1 >15,000 0.8 >1000

a Runesson et al. (2009). b Lundstrom et al. (2005). c Sollenbergetal. (2010).

essential medium with Glutamax-1 supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% non-essential amino acids, 100 U ml-1 penicillin and 100 pg ml-1 streptomycin. Chinese Hamster Ovary (CHO) K1 cells stably expressing human GAL2R (a kind gift from Kathryn A. Jones and Tiina P. Iismaa, Sydney, Australia) were cultured in Dulbecco's modified essential medium (DMEM) F-12 with Glutamax supplemented with 10% FBS, 2 mML-glutamine, 100 U ml-1 penicillin and 100 pg ml-1 streptomycin. Human embryonic kidney (HEK) 293 cells stably expressing rat GAL2R (a kind gift from Xiaoying Lu and Tamas Bartfai, La Jolla, USA) were cultured in DMEM supplemented with 10% FBS, 100 U ml-1 penicillin and 100 pg ml-1 streptomycin (Lu et al., 2010). SH-SY5Y cells with inducible expression of GALiR or GAL2R (Berger et al., 2004) were grown in minimum essential media supplemented with 10% FBS, 1% sodium puruvate, 1% non-essential amino acids and 100 U ml-1 penicillin and 100 pg ml-1 streptomycin. Flp-In T-REx 293 GAL3R cell line (a kind gift from F. Hoffman-La Roche Ltd., Basel, Switzerland) (Runesson et al., 2010) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U ml-1 penicillin, 100 pg ml-1 streptomycin, 15 pg/ml blasticidin S and 150 pg/ml Hygromycin B (Runesson et al., 2009). Cell cultures were all grown at 37 °C in a 5% CO2 incubator.

2.3. Peptide synthesis

Peptides were synthesized in a stepwise manner using small scale (0.1 mmol) 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy on p-methylbenzylhydrylamine (MBHA) resin on an automated Syro multiple peptide synthesizer (MultiSynTech GmbH, Witten, Germany). Fmoc-L-amino acids (Iris Biotech GmbH, Marktredwitz, Germany) were coupled as hydroxybenzotriazole (HOBt) esters. The peptides were finally cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane and 2.5% H2O solution for 3 h. All peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Discovery® C-18 Supelco® column (Sigma-Aldrich, Stockholm, Sweden) using a gradient of acetonitrile/water containing 0.1% TFA. The identity of the purified products was verified by Perkin Elmer prOTOF™ 2000 matrix-assisted laser desorption ionization time-of-flight mass-spectrometer (Perkin Elmer, Upplands Vasby, Sweden). The mass-spectra were acquired in positive ion reflector mode using a-cyano-4-hydroxycinnamic acid as a matrix (Sigma-Al-drich, Stockholm, Sweden) (10 mg/ml, 7:3 acetonitrile:water, 0.1% TFA).

2.4. Galanin receptor binding studies

Cells for 125I-galanin-receptor displacement studies were seeded in 150 mm cell culture dishes and cultured 2-4 days until confluent. The GAL3R inducible cell line was treated with tetracycline (1 pg/mL) 24 h prior to cell harvesting for inducing expression of GAL3R The dishes were washed thrice and cells scraped off into phosphate-buffered saline (PBS) and centrifuged twice at 4 °C, 3000 xg for 5 min. The pellet was resuspended in assay buffer (20 mM HEPES, 5 mM MgCl2, pH 7.4) supplemented with EDTA (5 mM) and incubated on ice for 45 min before

centrifugation at 4 °C, 8500 xg for 15 min. After washing the pellet in assay buffer and repeated centrifugation the obtained pellet was resuspended in assay buffer supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and stored at - 80 °C until use. The protein concentration was determined according to Bradford (BioRad, Stockholm, Sweden). Displacement studies on cell membranes were performed in a final volume of 200 pl, containing 0.1-0.12 nM porcine-[125I]-galanin (2200 Ci/mmol, Perkin-Elmer Life Science, Boston, MA, USA), 30 pg cell membrane, and various concentrations of peptide (10-5-10-11 M). Peptides were diluted in assay buffer supplemented with 0.3% BSA using silanized (dichlorodimethylsilane, Sigma-Aldrich, St. Louis, MO, USA) tubes, 96-well plates and pipette tips. Samples were incubated at 37 °C for 30 min while shaking. After that the samples were transferred and filtered through a MultiScreen-FB filter plate (Millipore, Billerica, MA, USA) pre-soaked in 0.3% polyethyl-enimine solution (Sigma-Aldrich, St. Louis, MO, USA) and removed using vacuum. The filters were washed thrice with HM-buffer and the retained radioactivity was determined in a (3-counter (Tri-Carb Liquid Sqintillation Analyzer, model 2500 TR, Packard Instrument Company, Meriden, CT, USA) using OptiPhase Supermix Cocktail (Perkin-Elmer Life Science, Boston, MA, USA) as scintillation fluid. IC50 values for the peptides were calculated using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) and converted into Ki values using the equation of Cheng-Prusoff (Cheng and Prusoff, 1973).

2.5. Galanin receptor signaling studies — cAMP measurements

SH-SY5Y cells stably expressing GAL2R were seeded in a 48 well plate and grown to confluency. Media was then changed to DMEM containing [3H]adenine (3 uCi/ml) purchased from Perkin Elmer (Waltham, MA, USA) for 2 h. The cells were washed twice with Hank's balanced salt solution (HBSS) before treated with 20 pM forskolin and/or M1154 for 30 min in the presence of 1 mM 3-isobutylmethylxanthine (IMBX), BAY 60-7550 (1 pM) and rolipram (10 pM) or with HBSS alone. The buffer were then removed and cells lysed with 5% (w/v) trichloroacetic acid followed by 250 pL trichloroacetic acid supplemented with cAMP (0.1 mM) and ATP (0.1 mM) for 20 min at 4 °C. The lysates were stored at - 80 °C until applied to Dowex 50 W-X4 columns (200-400 mesh) and washed with water and subsequently placed over alumina columns. The alumina columns were washed with water before placed over scintillation vials and eluted with 6 mL imidazole (0.1 M). To each vial 10 mL scintillation liquid (Ultima Gold XR) were added and the samples were analyzed in a liquid scintillation counter (Tri-Carb model 1600 TR; Packard Instrument Company).

2.6. Galanin receptor signaling studies — label-free real time technology

The xCELLigence system (Roche Diagnostics, Sweden) is based on the ACEA RT-CES cell sensor electrodes, which allow monitoring and analysis of the kinetic aspects of cellular behavior. The technology is described in detailed elsewhere (Peters and Scott, 2009; Yu et al., 2006), (Scott and Peters, 2010; Solly et al., 2004). Briefly, this assay is based

on the principle that activated GPCRs will cause a change in cell morphology, regardless of which G-protein signaling cascade used. The change in cell morphology influences the contact area between cells and the electrodes on which the cells are grown and this small change is measured in real time as impedance. Experiments were performed using the EVIEW-Plates™ from ACEA (San Diego, CA, USA). One day before the experiment, 100 pl of medium was added to each well and background recorded. Following background measurement, 100 pl of media containing the cell suspension was seeded on the EVIEW-Plate™, incubated at room temperature for 30 min and then placed on the device station, and hosted in an incubator at 37 °C with 5% CO2. The cells were allowed to equilibrate for 24 h (48 h for the Flp-In T-REX 293 cell line where 1 pg/ml tetracycline was added 24 h prior to ligand addition to induce GAL3R expression) and impedance were constantly monitored every minute throughout the whole experiment. To evaluate the effect of cell density on impedance responses and to identify a better signal-to-noise ratio, we tested cell densities ranging from 5000 to 40,000 cells/well. Optimal density was 40,000 cells/well for Bowes human melanoma cells stably expressing GAL1R, 30,000 cells/ well for HEK 293 cells expressing GAL2R and 20,000 cells/well for the in-ducible Flp-in T-REX 293 GAL3R cells. Prior to the experiment, media were replaced by 180 pl low-FBS (1%) containing cell culture media, according to the manufactures instruction, and incubated for 1 h. Then, 20 pl of the compounds diluted in PBS, at 10x the desired concentration, were gently added. In experiments aimed at evaluating antagonism, both compound and galanin were added simultaneously. Data were analyzed using the integrated software package expressing changes in cell electrode impedance as changes in cell index, CI (Yu et al., 2006) and normalized to the cell index at the time of ligand addition. Concentration effect curves were generated by calculation of the area under curve (AUC) between 0 and 3600 s and plotted against ligand concentration. The subsequently calculation of EC50 values was determined by non-linear regression using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).

2.7. Behavioral experiments and cell counts in the hippocampus

Adult, male Sprague-Dawley rats (total number of 41, weighing 250-300 g) were obtained from Harlan Inc. (Indianapolis, IN, USA) and housed individually throughout the experiment in 42 L x 22 W x 20 H cm polycarbonate cages. Rats were maintained in a temperature and humidity-controlled environment on a 12-h light/dark schedule. Food and water were available ad libitum. Rats were allowed a one week adaption to the animal facility prior to cannulation surgeries and were randomly assigned to one of six groups (www.randomizer.org): M1145 (10 pg; 20 pg; n = 6 respectively n = 8); M1154 (20 pg, n = 9); M617 (20 pg, n = 7); Saline Control (n = 8); Naive Control (n = 3). All procedures were conducted in accordance with NIH Guide for Care and Use of Laboratory Animals and were approved by the University of Georgia Animal Care and Use Committee. Rats were anesthetized with a 1 -3% isoflorane/oxygen mixture delivered through a vaporizer and nose cone and mounted in a stereotactic frame. A longitudinal incision was made along the scalp and overlaying connective tissue and periosteum was scraped away from the scalp. Cannulae (1 cm) were surgically implanted into the right lateral ventricle at the following coordinates (from Bregma): posterior 1.0 mm, lateral 1.5 mm, and ventral 3 mm according to the rat atlas of Paxinos & Watson (Paxinos and Watson, 1986). Cannulae were secured to the skull using 3 stainless steel screws and epoxy. All rats received 1 mg/kg meloxicam post-surgery. Cannulae placement was verified at the end of all procedures by injecting 2 mg/ml of fast green dye following euthanasia and verifying its presence in the ventricles. One week following surgeries, rats were injected with one of five compounds plus 0.2 pg of kainic acid. Doses of compounds were as follows: low dose M1145 10 pg, high dose M1145 20 pg, M1154 - 20 pg, M617 - 20 pg, and Saline - 20 pg. All drugs were dissolved in deionized water and injected in a volume

of 5 pl. Naïve animals did not undergo cannulation surgery or receive any drugs. Seizure behavior was scored live by a rater blind to group assignment for 30 min post microinjections and video recorded for additional ratings. The rating scale was adapted from the Racine scale (Racine, 1972; Reiss et al., 2009) and averages of seizure rating scores were calculated for analysis. Animals were perfused transcardially with PBS followed by a 4% formalin solution 48 h post-i.c.v. injections and seizure rating. Brains were extracted, fixed in 4% formalin solution, and kept frozen at — 20 °C until sectioned. Sections from both the dorsal and ventral hippocampus (20 pm) were thaw mounted on slides. One slide per region, dorsal and ventral hippocampus, was nissl-stained and cells of the CA3 region were counted under a microscope and recorded. A two-way contingency table analysis of high and low seizure scores was conducted to assess effect of treatment on seizure ratings. A Kruskal-Wallis test was conducted to evaluate differences among the treatment conditions; pairwise comparisons were conducted to evaluate differences between treatment groups and saline group. These statistical analyses were conducted using PASW Statistics Windows version 18.0 (IBM Corporation, Armonk, NY, USA).

3. Results

3.1. Galanin receptor binding studies

125l-galanin-receptor displacement experiments with galanin, galanin (1-16) and M1154 were performed on cell membranes from human Bowes melanoma cells endogenously expressing GAL-^, CHO K1 cells stably expressing GAL2R and Flp-In T-REX 293 cells with inducible expression of GAL3R. Displacement of 125l-galanin by M1154 at GAL^ showed a relatively high affinity with a K of 11.7 ± 7.2 nM, whereas galanin (1-16) had a K of 0.78 ± 0.26 nM in the same experiments. When tested on GAL2R, M1154 had a K of 14.4 ± 4.1 nM whereas galanin (1-16) had a K of 2.44 ± 0.57 nM on the same receptor and cell line. When tested on GAL3R M1154 showed no binding up to 10 000 nM, whereas galanin (1-16) had a K of 8.98 ± 3.8 nM see (Table 2; Fig. 1b). These results suggest that M1154 is selective for GAL1/2R and the difference towards GAL3R is greater than 1000 times (Table 2; Fig. 1a).

3.2. Galanin receptor signaling studies using cAMP measurements

GAL^ and GAL2R expressing SH-SY5Y cells were treated with M1154 at different concentrations (13 nM-10 pM) together with 20 pM forskolin. The GAL1/2R-selective agonist M1154 decreases forskolin stimulated cyclic adenosine-monophosphate production significantly in a dose-dependent manner in GAL1R expressing cells with an EC50 of 159.6 nM (See Table 3, Fig 2A). In contrast, in SH-SY5Y cells expressing GAL2R M1154 increases the cAMP production dose-dependently with an EC50 of 1.53 pM (See Table 3, Fig 2B).

3.3. Galanin receptor signaling studies using a real-time label-free technique based on impedance

To assess if galanin receptor stimulation could be detected by using an impedance based technology, xCELLigence, cells were challenged with the full-length galanin. Compound addition induced a fast concentration-dependent response in normalized cell index (NCI) with different concentration dependent profiles for each receptor subtype (Fig 3A-D). Two different GAL2R cell lines were characterized, CHO cells expressing human GAL2R and HEK cells expressing rat GAL2R. These cell lines displayed similar profiles for galanin (Fig 3B-C), although the signal to noise ratio was significantly higher in the HEK cells and this cell line was therefore continuously used. Because the instrument monitors the impedance changes elicited by receptor activation in real time, it is possible to generate concentration-activity curves in a variety of ways, dependent on the time point or period of

Fig. 1. Galanin receptor binding studies. Displacement of porcine-[125I]-galanin from membranes by peptide M1154 (A) and the galanin fragment, galanin(1-16) (B). Membranes were from human Bowes melanoma cells expressing GAL1R (open circle), CHO cells expressing GAL2R (closed square) and Flp-In T-REx 293 cells expressing GAL3R (closed triangle). The data is from three representative experiments performed in duplicates, presented as mean ± SEM. Calculated K values are summarized in Table 2.

time chosen and if peak values or AUC is used. The aim was to generate a robust protocol that could be used independent of the type of activated G-protein and eliminate the need for a clear peak maximum, since that is not often seen in the literature (Schroeder et al., 2010; Scott and Peters, 2010). Qualification of normalized cell index, NCI, signals for concentration effect curves and the subsequently calculation of EC50 was therefore performed by calculation of the AUC between 0 and 3600 s. Galanin showed similar EC50 values to GAL^ and GAL2R, 1.1 ± 0.11 nM and 8.26 ± 1.9 nM, respectively (Table 3, Fig 3), while the EC50 towards GAL3R was 412 ± 38 nM. In the present study, M617 displays EC50-values that are considerable lower at GAL^ and GAL2R, 11.4 ± 0.67 nM and 24.6 ± 3.8 nM, respectively (Table 3, Fig 3), although the relative three time preference for GAL^ over GAL2R was observed in both studies. In the present setup, M617 displays an EC50-value of 2840 ± 1090 nM for GAL3R which results in a signaling profile for the galanin receptor subtypes more in concordance with the binding profile (Table 2 and Table 3). We have previously shown the agonistic properties at GAL2R of M1145 in an IP accumulation assay, with an EC50 value of 38 nM (Runesson et al., 2009). In this study, a slightly lower EC50-value of 16 ± 4.7 nM was obtained with the xCELLigence system. Here, we also characterize for the first time the ability of M1145 to activate GALiR and GAL3R. M1145 acts as an agonist at all receptor subtypes, although the potency varies significantly, with a more than 70 times difference in the calculated EC50-values (Table 4, Fig 4). The novel peptide, M1154, displayed a slightly lower potency than galanin at GAL^ and GAL2R with an EC50-value of 124 ± 47 nM and 26 ± 1.1 nM, respectively. M1154 had no effect at any concentration tested on GAL3R (Table 4, Fig 4). To address the efficacy of M1154, the maximal response induced by M1154 were compared to the maximal response (Emax normalized to 100 percentage) induced

Table 3

Obtained EC50-values for rat galanin and galanin receptor ligands in signaling studies utilizing traditional end-point assays, cAMP (GAL1R), cAMP or IP-production (GAL2R) and GTPyS (GAL3R). Cells used were Bowes Melanoma Cells expressing GAL1R, CHO, SH-SY5Y or HEK cells stably transfected with GAL2R or Flp-In T-REX 293 cells expressing GAL3R.

Name EC50 (nM)

GAL1R gal2r GAL3R

RT-CES Galanin(1-29) 1.1 8.3 412

Galanin(1-29) 31.6a 173a 530b

M1145 n.t 38b n.t

M617 104a 304a 121c

M1154 159 1530 n.t

n. t not tested a Lundström et al. (2005). b Runesson etal. (2009). c Sollenbergetal. (2010).

Fig. 2. Galanin receptor signaling studies utilizing cAMP measurements of M1154 on SH-SY5Y cells expressing GAL1R (A) and GAL2R (B). Data presented are mean values of at least triplicates ± SEM. * p-value <0.05, **p-value <0.01 and *** p-value <0.001.

Fig. 3. Galanin receptor signaling studies utilizing the Impedance based (RT-CES) system. Concentration dependent profiles of galanin or the galanin fragment, galanin (1-16), in (A) Bowes melanoma cells expressing human GALiR, (B) CHO K1 cells expressing human GAL2R (C) HEK cells expressing rat GAL2R (D) Flp-In T-REX 293 cells with tetracycline induced expression of human GAL3R

by galanin. M1154 behaved as a full agonist, with a similar Emax value as galanin (data not shown). Application of M1154 (10 nM) induced a shift to the left of the concentration activity curve of galanin with a consistent modification of the EC50 (EC50 = 21 nM), confirming a M1154-mediated agonistic effect (Fig 5). Application of galanin (1 or 10 nM) induced a similar shift to the left of the concentration activity curve of M1154 (EC50 = 10-90 nM) (Fig 3B), confirming again the both galanin and M1154-mediated an agonist effect.

3.4. Behavioral experiments and hippocampal cell counts

KA (0.2 pg) administered i.c.v. induced seizures-typical behaviors to a similar extent as reported in previous studies (Reiss et al., 2009). A Pearson chi square test revealed no significant changes in the seizure rating scores for any of the administered galanin receptor ligands, X2(4, N = 38) = 1.839, p = 0.765 (Fig 6). Administration of KA resulted in a dramatic cell loss in the CA3 region (Fig 7) for both dorsal and ventral hippocampus. When the cell number was quantified, a trend towards a reduction ofcell death was seen for all co-administrated galanin receptor ligands (Fig 7). The results from an independent samples Kruskal-Wallis tests for cell count differences among treatments indicated significance after KA administration for both the dorsal hippocampus, x2(5, N = 41) = 18.248, p = 0.003; and the ventral hippocampus, X2(5, N = 38) = 15.702, p = 0.008. Follow-up pairwise comparisons indicated that cell counts in the dorsal hippocampus were significantly different from the vehicle treatment for two groups, M1154 + KA (p = 0.038) and M617 + KA (p = 0.001). Additionally, pairwise comparisons indicated that cell counts in the ventral hippocampus were

significantly different from the vehicle treatment in the M617 + KA group (p < 0.001) (Figs. 6 and 8).

4. Discussion

In the present study, we investigated the pharmaceutical potential of galanin receptor subtypes to block KA-induced neurodegeneration, as a model for excitotoxicity. This study was motivated by the fact that galanin has been shown to modulate excitability in the hippocampus and administration ofgalanin has been shown to affect neurodegeneration in several paradigms, including different epilepsy models (Mazarati et al., 2006). Several attempts have been made to characterize the contribution of each galanin receptor subtype to different aspects of neuroprotection; and both GAL1R and GAL2R have been shown to exert neuroprotective effects (Elliott-Hunt et al., 2007; Mazarati and Lu, 2005; Schauwecker, 2010), while very few studies have addressed the contribution of GAL3R in galanin-mediated neuroprotection.

To address the contribution of the individual receptor subtypes, we designed and characterized a novel GAL1/2R agonist, namely the M1154 peptide. The novel M1154 peptide was then compared with two previously developed galanin receptor subtype selective ligands, M617 which is GAL1R selective, and M1145 which is GAL2R selective. To further improve the development of selective galanin receptor ligands, we here present a robust protocol for receptor signaling, utilizing a label-free technique, to be able to test the receptor activation signature of our receptor ligands on all three galanin receptor subtypes using a single methodology. This is highly motivated since each galanin receptor subtype has its own unique capacity to activate the different

Table 4

Potency of galanin receptor ligands in the impedance (RT-CES) system. Cells used where Bowes Melanoma Cells expressing human GALiR, HEK cells stably transfected with rat GAL2R or Flp-In T-REX 293 cells with tetracycline induced expression of human GAL3R

EC50 (nM)

EC50 GAL1R/EC50 GAL2R

ec50 gal3r/ec50 gal2r

Galanin(1-29) M617 M1145 M1154

1.1 ±0.11 11.4 ±0.67 1260 ± 119 124 ±47

8.26 ± 1.9 24.6 ± 3.8 16 ±4.7 26 ± 1.1

412 ±38 2840 ± 1090 2670 ± 502 >31,600

0.13 0.46 79 4.8

50 120 170 >1210

Fig. 4. Galanin receptor signaling studies. Potency of galanin receptor subtype selective ligands in the xCELLigence impedance based (RT-CES) system; Galanin (A-C), M617 (D-F), M1145 (G-I) and M1154 (J-L). Cells used where Bowes Melanoma Cells expressing human GAL1R, HEK cells stably transfected with rat GAL2R or Flp-In T-REX 293 cells with inducible expression of human GAL3R. The data is from three representative experiments performed in at least duplicates, presented as mean ± SEM. Calculated EC50 values are summarized in Table 4.

G-protein subtypes which makes it difficult to address receptor subtype selectivity for novel ligands. The xCELLigence system has similar or improved sensitivity when compared to traditional endpoint assays performed in the same laboratory (see Table 3). The relative low EC50 for galanin at GAL3R could reflect intrinsic receptor properties or might be related to the utilized cell clone. We have earlier shown a similar EC50-value for galanin, 530 nM (Table 3), in the commonly used

Fig. 5. Galanin receptor signaling studies at HEK cells stably transfected with rat GAL2R. Galanin dose response curve (closed circle) with an EC50 value of 8.2 nM was shifted to the right by 1 nM (open square) M1154 given an EC50 value of 6.0 nM, 10 nM (closed triangle) given an EC50 value of 5.8 nM, 100 nM (closed diamond) given an EC50 value of 0.56 nM and 1000 nM (closed square) M1154 given an EC50 value of 0.045 nM. Experiments were performed in duplicates and presented as mean ± SEM.

GTP^-assay (Runesson et al., 2009). It has been hypothesized in other studies that transfection of cell lines with GAL3R yield very low and variable receptor expression, which affect the possibility to detect the signal efficacy at this receptor (Ohtaki et al., 1999) (Berger et al., 2004;

Fig. 6. Average seizure rating following i.c.v. administration of either saline, M617, M1145 or Ml 154 followed by i.c.v. injection of KA Data presented as means ± SEM. No significant differences in seizure rating between treatments were found using a two-way contingency table analysis.

Fig. 7. Cell count from both dorsal (open bars) and ventral (closed bars) hippocampus (region CA3) after i.c.v. administration of either saline, M617, M1145 or M1154 followed by i.c.v. injection of KA Data are presented as means ± SEM. # p < 0.05 ### p < 0.001 as compared to ventral hippocampus in vehicle exposed animals, ** p < 0.01 as compared to dorsal hippocampus in vehicle exposed animals; Kruskal-Wallis pairwise comparisons test.

Lang et al., 2005); it may account for the low EC50 for galanin seen at the GAL3R cell line, although, we have earlier shown that the GAL3R cell clone used in this study has similar receptor expression as the GAL-iR and GAL2R cell lines (Runesson et al., 2009). Unfortunately, signaling properties of the GAL3R and the pharmacological profiles of common galaninergic ligands are still ill-defined and more studies are needed to characterize the properties of GAL3R (Lang et al., 2015).

Lundstrom and colleagues showed that M617 has agonistic properties at both GAL1R and GAL2R, evaluated through cAMP and IP assays, with an EC50-value of 104 and 304 nM, respectively (Lundstrom et al., 2005). A later publication ascribed the M617 peptide the ability to mediate activation of GAL3R, measured as the ability to inhibit forskolin produced cAMP, at a similar concentration as for GAL1R and GAL2R, with an EC50-value of 121 ± 48 nM (Sollenberg et al., 2010).

A recent publication reports a second wave of G-protein signaling from internalized receptors in vesicles (Irannejad et al., 2013), which might explain a persistent effect of added ligands to GPCRs that easily can be detected using the real-time impedance based technology and

relevant time sections can be determined for analysis. Furthermore, very few studies address the contribution of GAL3R, mostly because we still lack reliable pharmacological signatures at GAL3R for commonly used galaninergic tools. Recently, multiple endoplasmic reticulum retention motifs have been identified in the GAL3R, which can explain a low cell surface expression in recombinant systems (Robinson et al., 2013). Robinson and co-workers present a new cell line were modifications from GAL^ have been inserted to GAL3R in order to investigate the intracellular trafficking and function of GAL3R (Robinson et al., 2013).

In the present study, we demonstrate the generation of a novel galanin analog, M1154, with binding affinities very similar to galanin and the galanin fragment, galanin (1-16), towards GAL^ and GAL2R. However, M1154 displays no ability to displace galanin at GAL3R in binding studies up to 10 pM (Fig 1, Table 2). In addition, galanin and galanin (1-16) were tested for comparison. The galanin fragment, galanin (1-16), displays a 10 times lower Kj in this study compared to an earlier publication (Smith et al., 1998), now more in concurrence with the binding affinity of the full-length peptide towards GAL3R, giving a similar binding profile for the galanin and the galanin (1-16) peptide on all three receptor subtypes (Table 2). We found that M1154, in concurrence with galanin, to produce a clear stimulation through both GAL^ and GAL2R in the xCELLigence system and in concordance with the binding results, no activity was seen when tested at GAL3R (Fig 4). Consistent with previous studies, the evaluation of the signal characteristics of M617 and M1145 in the present study revealed these ligands to also be GAL^and GAL2R selective, respectively (Lundstrom et al., 2005; Runesson et al., 2009; Sollenberg et al., 2010).

Administration of KA has been utilized as a model of both neurodegeneration and status epilepticus (Ben-Ari and Cossart, 2000; Wang et al., 2005). We and others have shown that CA3 pyramidal cells are most vulnerable to KA treatment (Ben-Ari and Cossart, 2000; Reiss et al., 2009). Thus, KA provides the opportunity to test the neural circuits that protect CA3 neurons from hyperexcitability. Even so, there are other obvious effects of i.c.v. KA-administration, such as induction of seizure-typical behaviors. In the present study, i.c.v. administration was utilized to insure a robust excitotoxic effect within the CA3 region, which is mediated through a direct excitotoxic effect of KA. Therefore, compounds that effectively reduce the KA-induced cell death in CA3 in this model most likely reflect neuroprotective effects not involved in anticonvulsant activity. We previously reported that i.c.v. administration of a galanin receptor antagonist had no effect on seizure-typical

Fig. 8. Nissl stain images of dorsal hippocampus (CA3 region): (A) Naive control, (B) Saline + KA, (C) M1154 + KA, (D) M617 + KA.

behaviors induced by i.c.v. administrated KA (Reiss et al., 2009). In the present study none of the i.c.v. administrated galanin receptor agonists at the chosen doses had an effect on the seizure-typical behaviors induced by KA, depicted in Fig. 6. Doses of 20 pg has been shown to be at the high end of the dose-range for i.c.v. administration of galanin peptide analogs in previously reported behavioral studies (Lang et al., 2007; Saar et al., 2011; Kuteeva et al., 2008). At 20 pg the GALiR selective ligand M617 was neuroprotective, in contrast to the GAL2R selective ligand M1145. The novel GAL1/2R selective ligand M1154 were also neuroprotective and did not significantly differ from M617, further supporting GAL1R activation as neuroprotective. Testing higher doses would exceed the range normally used for behavioral studies. Subsequently, with lower doses it would be difficult to reach statistical significant differences to vehicle due to the small effect size. However, it should be noted that the commonly used technique involving a single dose of KA is not optimal for measuring drug effects on acute behavioral seizures because of the high variability observed between subjects (Reddy and Kuruba, 2013). Thus, it can be speculated that the seizure-typical behaviors induced by KA are galanin-independent and most likely due to overflow of KA and hyperexcitability in the motor cortex (Sperk, 1994). Therefore, we cannot exclude that these ligands have anticonvulsant properties.

I.c.v. administration of KA induces rigorous neuronal cell death in the CA3 region of both ventral and dorsal hippocampus (Fig 7). Animals treated with the GAL2R selective ligand, M1145, were equally affected in the CA3 region by the i.c.v. KA-administration compared to vehicle treated animals. In contrast, administration of the mixed GAL1/2R agonist M1154 reduced cell loss significantly. Furthermore, animals treated with the GAL1R preferential ligand displayed further reductions in cell death. These results indicate that the galanin-mediated neuroprotective effect occurs through GAL1R and not GAL2R. This is in concordance with earlier studies on inbred mice where a lower expression of GAL1R correlated with a larger cell loss than wildtype littermates in the CA3 region when exposed to KA (Kong et al., 2008; Schauwecker, 2010). Similar to this study, these animals had no alteration in seizure-typical behaviors, separating neuroprotective and anticonvulsant effects of the gala-ninergic system (Schauwecker, 2010). The galanin ligands used in this study were not chemically modified to increase stability or penetrate the blood-brain barrier and are therefore expected to be degradated within a few minutes in serum (Bulaj et al., 2008) and i.c.v. administration was the administration route used in the animal studies. For possible pharmaceutical evaluation, chemically stabilized galanin analogs would be preferable. Professor H. Steve White and colleagues have presented both GAL1R selective and GAL2R selective analogs that are sys-temically active (Bulaj et al., 2008; Jequier Gygax et al., 2014; Robertson et al., 2012; Robertson et al., 2010; White et al., 2009), as well as peripherally active galanin ligands (Metcalf et al., 2015). Based on the modification presented by Bulaj et al. (Bulaj et al., 2008), novel GAL2R selective ligands and one GAL2R specific ligand were generated by Saar and colleagues (Saar et al., 2013).

5. Summary

A new protocol for evaluating novel GPCR ligands using real-time impedance based technology showed similar or slightly lower EC50 values when compared to previously published galanin ligands EC50 values received from cAMP signaling in the same laboratory. M1154, a novel GAL1/2R selective ligand was designed and its ability to significantly reduce the excitotoxicity of i.c.v. administered KA was evaluated. M1154 was shown to be an agonist for both GAL1R and GAL2R. Our data indicate, that M617 and M1154, but not M1145, significantly reduced neuronal cell death, in the KA-excitoxicity model. These findings suggest that the neuroprotective effect of pharmacological stimulation of galanin receptors in vivo after i.c.v. administration of KA in the CA3 region is mediated through GAL1R. This suggests that a GAL1R agonist could potentially be used for treatment of exposure to excitotoxic compounds.

Acknowledgments

We thank Kathryn A. Jones and Tiina P. Iismaa (Neurobiology Program, Garvan Institute of Medical Research, Sydney, Australia) for CHO cells stably transfected with human GAL2, Xiaoying Lu and Tamas Bartfai (Scripps Research Institute, LaJolla, USA) for HEK293 cells stably transfected with rat GAL2, Linda Lundström and Silvia Gatti-McArthur (F. Hoffmann-La Roche AG, Basel, Switzerland) for Flp-In T-REx 293 cell line stably transfected with the human GAL3. This work was supported by grants from the Olle Engkvist Byggmästares Foundation, Helge Ax:son Johnsons foundation and Sven & Dagmar Salens foundation (JR), Swedish Science Foundation (VR-Med) (ÜL) (521-2011-2461) and the Austrian Research Promotion Agency (822782/THERAPEP) (BK).

References

Abbosh, C., Lawkowski, A., Zaben, M., Gray, W., 2011. GalR2/3 mediates proliferative and trophic effects of galanin on postnatal hippocampal precursors. J. Neurochem. 117, 425-436. http://dx.doi.org/10.1111/j.1471-4159.2011.07204.x. Ben-Ari, Y., Cossart, R., 2000. Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci. 23, 580-587. http://dx.doi.org/10.1016/S0166-2236(00)01659-3.

Berger, A., Lang, R., Moritz, K., Santic, R., Hermann, A., Sperl, W., Kofler, B., 2004. Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells. Endocrinology 145 (2), 500-507. http://dx.doi.org/10.1210/en.2003-0649. Branchek, T.A., Smith, K.E., Gerald, C., Walker, M.W., 2000. Galanin receptor subtypes. Trends Pharmacol. Sci. 21 (3), 109-117. http://dx.doi.org/10.1016/S0165-6147(00)01446-2.

Bulaj, G., Green, B.R., Lee, H.-K., Robertson, C.R., White, K., Zhang, L., Sochanska, M., Flynn, S.P., Scholl, E.A., Pruess, T.H., Smith, M.D., White, H.S., 2008. Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities. J. Med. Chem. 51 (24), 8038-8047. http://dx.doi.org/10. 1021/jm801088x.

Cheng, Y., Prusoff, W.H., 1973. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (150) of an enzymatic reaction. Biochem. Pharmacol. 22 ( 23), 3099-3108. Ding, X., MacTavish, D., Kar, S., Jhamandas, J.H., 2006. Galanin attenuates ß-amyloid (Aß) toxicity in rat cholinergic basal forebrain neurons. Neurobiol. Dis. 21 (2), 413-420. http://dx.doi.org/10.1016/j.nbd.2005.08.016. Elliott-Hunt, C.R., Marsh, B., Bacon, A., Pope, R., Vanderplank, P., Wynick, D., 2004. Galanin acts as a neuroprotective factor to the hippocampus. Proc. Natl. Acad. Sci. 101 (14), 5105-5110. http://dx.doi.org/10.1073/pnas.0304823101. Elliott-Hunt, C.R., Pope, R.J.P., Vanderplank P., Wynick D., 2007. Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage. J. Neurochem. 100 (3), 780-789. http://dx.doi.org/10.1111/j.1471-4159.2006.04239.x. Haberman, R.P., Samulski, R.J., McCown, T.J., 2003. Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion. Nat. Med. 9 (8), 1076-1080. http://dx.doi.org/10.1038/nm901. Hobson, S.A., Bacon, A., Elliot-Hunt, C.R., Holmes, F.E., Kerr, N.C.H., Pope, R., Vanderplank, P., Wynick, D., 2008. Galanin acts as a trophic factor to the central and peripheral nervous systems. Cell. Mol. Life Sci. 65,1806-1812. http://dx.doi.org/10.1007/s00018-008-8154-7.

Holub, B.S., Kloepper, J.E., Toth, B.I., Biro, T., Kofler, B., Paus, R., 2012. The neuropeptide galanin is a novel inhibitor of human hair growth. Br. J. Dermatol. 167 (1), 10-16. http://dx.doi.org/10.1111/j.1365-2133.2012.10890.x. Irannejad, R., Tomshine, J.C., Tomshine, J.R., Chevalier, M., Mahoney, J.P., Steyaert, J., Ras-mussen, S.G.F., Sunahara, R.K., El-Samad, H., Huang, B., Zastrow, V.,.M., 2013. Confor-mational biosensors reveal GPCR signalling from endosomes. Nature 495 (7442), 534-538. http://dx.doi.org/10.1038/nature12000. Jackson, K.J., Chen, X., Miles, M.F., Harenza, J., Damaj, M.I., 2011. The neuropeptide galanin and variants in the GalR1 gene are associated with nicotine dependence. Neuropsychopharmacology 36,2339-2348. http://dx.doi.org/10.1038/npp.2011.123. Jarrard, L.E., 2002. Use of excitotoxins to lesion the hippocampus: update. Hippocampus

12,405-414. http://dx.doi.org/10.1002/hipo.10054. Jequier Gygax, M., Klein, B.D., White, H.S., Kim, M., Galanopoulou, A.S., 2014. Efficacy and tolerability of the galanin analog NAX 5055 in the multiple-hit rat model of symptomatic infantile spasms. Epilepsy Res. 108, 98-108. http://dx.doi.org/10.1016/j. eplepsyres.2013.10.015. Jimenez-Andrade, J.M., Lundström, L., Sollenberg, U.E., Langel, Ü., Castaneda-Hernandez, G., Carlton, S., 2006. Activation of peripheral galanin receptors: differential effects on nociception. Pharmacol. Biochem. Behav. 85, 273-280. http://dx.doi.org/10. 1016/j.pbb.2006.08.008. Kong, S., Lorenzana, A., Deng, Q., McNeill, T.H., Schauwecker, P.E., 2008. Variation in Galr1 expression determines susceptibility to excitotoxin-induced cell death in mice. Genes Brain Behav. 7 (5), 587-598. http://dx.doi.org/10.1111/j.1601-183X.2008.00395.x. Kuteeva, E., Wardi, T., Lundström, L., Sollenberg, U., Langel, Ü., Hökfelt, T., Ögren, S.O., 2008. Differential role of galanin receptors in the regulation of depression-like behavior and monoamine/stress-related genes at the cell body level. Neuropsychopharmacology 33, 2573-2585. http://dx.doi.org/10.1038/sj.npp. 1301660.

Land, T., Langel, Ü., Löw, M., Berthold, M., Unden, A., Bartfai, T., 1991. Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor. Int. J. Pept. Protein Res. 38 (3), 267-272. http://dx.doi.org/10.1111/j.1399-3011. 1991.tb01438.x.

Lang, R., Berger, A., Santic, R., Geisberger, R., Hermann, A., Herzog, H., Kofler, B., 2005. Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists. Neuropeptides 39 (3), 179-184. http://dx.doi. org/10.1016/j.npep.2004.12.015.

Lang, R., Gundlach, A.L., Kofler, B., 2007. The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol. Ther. 115,177-207. http://dx.doi.org/10.1016/j.pharmthera.2007.05.009.

Lang, R., Gundlach, A.L., Holmes, F.E., Hobson, S.A., Wynick, D., Hökfelt, T., Kofler, B., 2015. Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity. Pharmacol. Rev. 67,118-175. http://dx.doi.org/10.1124/ pr.112.006536.

Liu, H.X., Brumovsky, P., Schmidt, R., Brown, W., Payza, K., Hodzic, L., Pou, C., Godbout, C., Hökfelt, T., 2001. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc. Natl. Acad. Sci. 98 (17), 9960-9964. http://dx.doi.org/10.1073/pnas.161293598.

Lu, X., Lundström, L., Bartfai, T., 2005. Galanin (2-11) binds to GalR3 in transfected cell lines: limitations for pharmacological definition of receptor subtypes. Neuropeptides 39 (3), 165-167. http://dx.doi.org/10.1016/j.npep.2004.12.013.

Lu, X., Roberts, E., Xia, F., Sanchez-Alavez, M., Liu, T., Baldwin, R., Wu, S., Chang, J., Wasterlain, C.G., Bartfai, T., 2010. GalR2-positive allosteric modulator exhibits anti-convulsant effects in animal models. Proc. Natl. Acad. Sci. U. S. A. 107, 15229-15234. http://dx.doi.org/10.1073/pnas.1008986107.

Lundström, L., Sollenberg, U., Brewer, A., Kouya, P.F., Zheng, K., Xu, X.-J., Sheng, X., Robinson, J.K., Wiesenfeld-Hallin, Z., Xu, Z.-Q., Hökfelt, T., Bartfai, T., Langel, Ü., 2005. A Galanin Receptor Subtype 1 Specific Agonist. 11 (1), 17-27 (doi:10.1007/ s10989-004-1717-z).

Mazarati, A.M., 2004. Galanin and galanin receptors in epilepsy. Neuropeptides 38, 331-343. http://dx.doi.org/10.1016/j.npep.2004.07.006.

Mazarati, A., Lu, X., 2005. Regulation of limbic status epilepticus by hippocampal galanin type 1 and type 2 receptors. Neuropeptides 39 (3), 277-280. http://dx.doi.org/10. 1016/j.npep.2004.12.003.

Mazarati, A., Lu, X., Shinmei, S., Badie-Mahdavi, H., Bartfai, T., 2004. Patterns of seizures, hippocampal injury and neurogenesis in three models ofstatus epilepticus in galanin receptor type 1 (GalR1) knockout mice. Neuroscience 128 (2), 431-441. http://dx. doi.org/10.1016/j.neuroscience.2004.06.052.

Mazarati, A., Lundström, L., Sollenberg, U., Shin, D., Langel, Ü., Sankar, R., 2006. Regulation of kindling epileptogenesis by hippocampal galanin type 1 and type 2 receptors: the effects of subtype-selective agonists and the role of G-protein-mediated signaling. J. Pharmacol. Exp. Ther. 318 (2), 700-708. http://dx.doi.org/10.1124/jpet.106.104703.

Metcalf, C.S., Klein, B.D., McDougle, D.R., Zhang, L., Smith, M.D., Bulaj, G., White, H.S., 2015. Analgesic properties of a peripherally acting and GalR2 receptor-preferring galanin analog in inflammatory, neuropathic, and acute pain models. J. Pharmacol. Exp. Ther. http://dx.doi.org/10.1124/jpet.114.219063.

Minor, L.K., 2008. Label-free cell-based functional assays. CCHTS 11 (7), 573-580. http:// dx.doi.org/10.2174/138620708785204072.

Mitsukawa, K., Lu, X., Bartfai, T., 2008. Galanin — 25 years with a multitalented neuropeptide. Cell. Mol. Life Sci. 65 (12), 1796-1805. http://dx.doi.org/10.1007/s00018-008-8153-8.

Nayler, O., Birker-Robaczewska, M., Gatfield, J., 2010. Integration of label-free detection methods in GPCR drug discovery. Gilchrist A. GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions.John Wiley & Sons. Inc. http://dx.doi.org/10.1002/9780470627327.ch11

Ohtaki, T., Kumano, S., Ishibashi, Y., Ogi, K., Matsui, H., Harada, M., Kitada, C., Kurokawa, T., Onda, H., Fujino, M., 1999. Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus. J. Biol. Chem. 274 (52), 37041-37045. http://dx. doi.org/10.1074/jbc.274.52.37041.

Olney, J.W., 1969. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science 164, 719-721. http://dx.doi.org/10.1126/science. 164.3880.719.

Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. sixth ed. Academic Press 9780125476126 ISBN.

Peters, M.F., Scott, C.W., 2009. Evaluating cellular impedance assays for detection ofGPCR pleiotropic signaling and functional selectivity. J. Biomol. Screen. 14 (3), 246-255. http://dx.doi.org/10.1177/1087057108330115.

Pirondi, S., Fernandez, M., Schmidt, R., Hökfelt, T., Giardino, L., Calza, L., 2005. The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells. J. Neurochem. 95 (3), 821-833. http://dx.doi.org/10.1111/j.1471-4159.2005. 03437.x.

Racine, R.J., 1972. Modification of seizure activity by electrical stimulation .1. Afterdischarge threshold. Electroencephalogr. Clin. Neurophysiol. 32, 269-279.

Reddy, D., Kuruba, R., 2013. Experimental models of status epilepticus and neuronal injury for evaluation of therapeutic interventions. IJMS 14 ( 9), 18284-18318. http://dx. doi.org/10.3390/ijms140918284.

Reiss, J.I., Dishman, R.K., Boyd, H.E., Robinson, J.K., Holmes, P.V., 2009. Chronic activity wheel running reduces the severity of kainic acid-induced seizures in the rat: possible role of galanin. Brain Res. 1266,54-63. http://dx.doi.org/10.1016/j.brainres.2009. 02.030.

Robertson, C.R., Scholl, E.A., Pruess, T.H., Green, B.R., White, H.S., Bulaj, G., 2010. Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery. J. Med. Chem. 53 (4), 1871-1875. http://dx.doi.org/10.1021 /jm9018349.

Robertson, C.R., Pruess, T.H., Grussendorf, E., White, H.S., Bulaj, G., 2012. Generating orally active galanin analogues with analgesic activities. ChemMedChem 7, 903-909. http:// dx.doi.org/10.1002/cmdc.201100574.

Robinson, J., Smith, A., Sturchler, E., Tabrizifard, S., Kamenecka, T., McDonald, P., 2013. Development of a high-throughput screening-compatible cell-based functional assay to identify small molecule probes of the galanin 3 receptor (GalR3). Assay Drug Dev. Technol. 11 (8), 468-477. http://dx.doi.org/10.1089/adt.2013.526.

Runesson, J., Saar, I., Lundström, L., Järv, J., Langel, Ü., 2009. A novel GalR2-specific peptide agonist. Neuropeptides 43 (3), 187-192. http://dx.doi.org/10.1016/j.npep.2009.04. 004.

Runesson, J., Sollenberg, U.E., Jurkowski, W., Yazdi, S., Eriksson, E.E., Elofsson, A., Langel, Ü., 2010. Determining receptor-ligand interaction of human galanin receptor type 3. Neurochem. Int. 57 (7), 804-811. http://dx.doi.org/10.1016/j.neuint.2010.08.018.

Saar, I., Runesson, J., McNamara, I., Järv, J., Robinson, J.K., Langel, Ü., 2011. Novel galanin receptor subtype specific ligands in feeding regulation. Neurochem. Int. 58, 714-720. http: //dx.doi.org/10.1016/j.neuint.2011.02.012.

Saar, I., Lahe, J., Langel, K., Runesson, J., Webling, K., Järv, J., Rytkönen, J., Närvänen, A., Bartfai, T., Kurrikoff, K., Langel, Ü., 2013. Novel systemically active galanin receptor 2 ligands in depression-like behavior. J. Neurochem. http://dx.doi.org/10.1111/jnc. 12274.

Schauwecker, P.E., 2010. Galanin receptor 1 deletion exacerbates hippocampal neuronal loss after systemic kainate administration in mice. PLoS One 5 (12), e15657. http:// dx.doi.org/10.1371 /journal.pone.0015657.

Schroeder, R., Janssen, N., Schmidt, J., Kebig, A., Merten, N., Hennen, S., Mueller, A., Blaettermann, S., Mohr-Andrae, M., Zahn, S., Wenzel, J., Smith, N.J., Gomeza, J., Drewke, C., Milligan, G., Mohr, K., Kostenis, E., 2010. Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 28 (9), 943-950. http://dx.doi.org/10.1038/nbt.1671.

Scott, C.W., Peters, M.F., 2010. Label-free whole-cell assays: expanding the scope of GPCR screening. Drug Discov. Today 15 (17-18), 704-716. http://dx.doi.org/10.1016/j. drudis.2010.06.008.

Smith, K.E., Walker, M.W., Artymyshyn, R., Bard, J., Borowsky, B., Tamm, J.A., Yao, W.J., Vaysse, P.J., Branchek T.A., Gerald, C., Jones, KA, 1998. Cloned human and rat galanin GALR3 receptors. Pharmacology and activation of G-protein inwardly rectifying K+ channels. J. Biol. Chem. 273 (36), 23321-23326. http://dx.doi.org/10.1074/jbc.273. 36.23321.

Sollenberg, U.E., Lundström, L., Bartfai, T., Langel, Ü., 2006. M871 —a novel peptide antagonist selectively recognizing the galanin receptor type 2. Int. J. Pept. Res. Ther. 12 (2), 115-119. http: //dx.doi.org/10.1007/s10989-005-9008-x.

Sollenberg, U.E., Runesson, J., Sillard, R., Langel, Ü., 2010. Binding of chimeric peptides M617 and M871 to galanin receptor type 3 reveals characteristics of galanin receptor-ligand interaction. 16 (1), 17-22. http://dx.doi.org/10.1007/s10989-009-9197-9.

Solly, K., Wang, X., Xu, X., Strulovici, B., Zheng, W., 2004. Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev. Technol. 2 (4), 363-372. http://dx.doi.org/10.1089/adt2004.2363.

Sperk G., 1994. Kainicacid seizures in the rat. Prog. Neurobiol. 42 (1), 1-32. http://dx.doi. org/10.1016/0301 -0082(94)90019-1.

Vrontakis, M.E., Peden, L.M., Duckworth, M.L., Friesen, H.G., 1987. Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J. Biol. Chem. 262 (35), 16755-16758.

Wang, Q., Yu, S., Simonyi, A., Sun, G.Y., Sun, A.Y., 2005. Kainic acid-mediated excitotoxicity as a model for neurodegeneration. Mol. Neurobiol. 31 (1-3), 3-16. http://dx.doi.org/ 10.1385/MN:31:1-3:003.

Webling, K.E.B., Runesson, J., Bartfai, T., Langel, Ü., 2012. Galanin receptors and ligands. Front. Endocrinol. http://dx.doi.org/10.3389/fendo.2012.00146 ((Lausanne) 3, article 146.).

White, H.S., Scholl, E.A., Klein, B.D., Flynn, S.P., Pruess, T.H., Green, B.R., Zhang, L., Bulaj, G., 2009. Developing novel antiepileptic drugs: characterization of NAX 5055, a system-ically-active galanin analog, in epilepsy models. NURT 6, 372-380. http://dx.doi.org/ 10.1016/j.nurt2009.01.001.

Yu, N., Atienza, J.M., Bernard, J., Blanc, S., Zhu, J., Wang, X., Xu, X., Abassi, Y.A., 2006. Realtime monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors. Anal. Chem. 78 (1), 35-43. http://dx.doi.org/10.1021/ac051695v.