Jou nal o Chemical Neu oana omy 128 (2023) 102226
A ailable online 23 Decembe 2022
0891-0618/© 2022 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
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Neu oana omical cha ac e iza ion o he G p o ein-coupled ecep o
ac i i y e oked by galanin- ela ed ligands
G. Ba eda-G´
omez
a
, I. Manuel
a
,
b
,
*
, R. Rod íguez-Pue as
a
,
b
a
Depa men o Pha macology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain
b
Neu odegene a i e diseases, Bioc uces Bizkaia Heal h Resea ch Ins i u e, Ba akaldo, Spain
ARTICLE INFO
Keywo ds:
Galanin
G p o ein
[
35
S]GTPγS
Au o adiog aphy
Ligand
Pep ide
ABSTRACT
Galanin neu opep ide is dis ibu ed h oughou he mammalian ne ous sys em modula ing a ple ho a o di e se
physiological unc ions, including nocicep ion, cogni ion and neu oendoc ine egula ion. The egula ion o he
galanine gic sys em is an in e es ing app oach o he ea men o di e en diseases associa ed o hose sys ems.
Ne e heless, he pha macological selec i i y and ac i i ies o some galanin ecep o (GalR) ligands a e s ill in
discussion and seem o depend on he dose, he ecep o sub ype and he second messenge s o which hey a e
coupled a di e en b ain a eas. The ac i i y o di e en GalR ligands on G
i/o
p o eins, was e alua ed by he
guanosine 5′-(γ-[
35
S] hio) iphospha e ([
35
S]GTPγS) au o adiog aphy in i o assay applied o a b ain issue
slices in he p esence o galanin, M15, M35, M40, gal(2−11) o galnon. The enhancemen o he [
35
S]GTPγS
binding induced by he chime ical pep ides M15, M35 and M40 was simila o ha p oduced by Gal in hose
b ain a eas showing he highes s imula ions, such as do sal pa o he ol ac o y nucleus and en al subiculum.
In con as o hese pep ides, using gal(2−11) no e ec was measu ed on G
i/o
p o ein coupling in a eas o he a
b ain wi h high GalR
1
densi y such as pos e io hypo halamic nucleus and amygdala, indica ing low selec i i y
o GalR
1
ecep o s. The e ec s e oked by he non-pep ide ligand, galnon, we e di e en om hose induced by
galanin, beha ing as agonis o an agonis depending on he b ain a ea, bu he s imula ions we e always blocked
by M35. Thus, he ac i i y o mos used GalR ligands on G
i/o
p o ein media ed signalling is complex and depends
on he b ain a ea. Mo e selec i e and po en GalR ligands a e necessa y o de elop new ea men s aimed o
modula e he galanine gic sys em.
1. In oduc ion
Galanin is a neu opep ide dis ibu ed h oughou he mammalian
cen al ne ous sys em (CNS). Galanin is pa icula ly p esen in nucleus
basalis o Meyne , some a eas o he hypo halamus, hippocampus and
amygdala, and is also exp essed in he aphe and locus coe uleus nuclei
(Melande e al., 1985; Gen leman e al., 1989; Sko i sch e al., 1986).
This neu opep ide exe s i s physiological e ec s by he in e ac ion wi h
G p o ein-coupled ecep o s (GPCR). To da e, h ee di e en galanin
ecep o (GalR) sub ypes (GalR
1
, GalR
2
and GalR
3
) ha e been iden i ied
and cloned ( o e iew B anchek e al., 2000). All o hem inhibi ade-
nylyl cyclase ac i i y by he coupling o G
i/o
p o eins (Wang e al.,
1998), e en i GalR
2
can be also coupled o G
q/11
p omo ing he ac i-
a ion o phospholipase C (Duan e al., 2022; Smi h e al., 1997; Wang
e al., 1998). Galanin is no he unique endogenous ligand o GalR; a
p esen , h ee o he pep ides belonging o he galanin amily ha e been
desc ibed: galanin message-associa ed p o ein (GMAP), he galanin-like
pep ide (GALP) and ala in, al hough bo h GMAP and ala in show e y
low a ini y o galanin ecep o s (Bough on e al., 2010; Lundk is e al.,
1995; Oh aki e al., 1999; San ic e al., 2006; Wang e al., 1997).
Recen ly, ano he endogenous ligand o GalR2 and GalR3, called
spexin, has been desc ibed by syn eny, examining he e olu iona y
connec ion o he physical co-localiza ion o SPX, GAL and KISS genes in
some e eb a e ch omosomes (Kim e al., 2014).
Galanin in in ol ed in he modula ion o many physiological p o-
cesses including nocicep ion, cogni ion, neu oendoc ine egula ion,
and, due o i exp ession wi h se o onin and no ad enaline, he possible
ole o he pep ide in mood egula ion (Bellido e al., 2002; Liu e al.,
2001; McDonald e al., 1998; Melande e al., 1987). Galanin has been
also in ol ed in he physiopa hology o di e en neu odegene a i e and
* Co espondence o: Depa men o Pha macology. Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa E-48940, Vizcaya,
Spain.
E-mail add ess: [email p o ec ed] (I. Manuel).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Chemical Neu oana omy
jou nal homepage: www.else ie .com/loca e/jchemneu
h ps://doi.o g/10.1016/j.jchemneu.2022.102226
Recei ed 14 Oc obe 2022; Recei ed in e ised o m 21 Decembe 2022; Accep ed 21 Decembe 2022
Jou nal o Chemical Neu oana omy 128 (2023) 102226
2
neu opsychia ic diseases, including Alzheime ’s disease o anxie y,
espec i ely. Regula ions on galanin syn hesis o he exp ession o his
neu opep ide i sel as well on GalR ha e been epo ed in hose neu o-
logical diseases (Chan-Palay, 1988; M¨
olle e al., 1999; Zhao e al.,
2013). Thus, he syn hesis o selec i e and po en d ugs ha ac on GalR
could help o de elop new he apeu ic app oaches. Howe e , mos o
ligands showing a ini y o GalR sub ypes ha e a low po ency and
selec i i y. Mainly, GalR ligands a e agmen s o pep ides de i ed om
galanin, owing o he ac ha he i s 16 amino acids o galanin show
high a ini y o GalR and a e c i ical o he binding o his neu opep ide
o i s ecep o s (Langel and Ba ai, 1998). Indeed, di e en galanin
agmen s ha e been used as GalR agonis s, al hough hei pha maco-
logical p o ile di e om ha p oduced by galanin; e.g., Gal (1−15)
seems o ha e s onge e ec s han obse ed wi h galanin in beha io al
es s ela ed o anxie y-like beha io s (Mill´
on e al., 2014). In his sense,
Gal(1−15) has been assayed o possible he apeu ic applica ions as
an idep essan in an augmen a ion s a egy in combina ion wi h
SSRI- ype compounds (Flo es-Bu gess e al., 2017, 2019, 2022). On he
o he hand, Gal (1−16) agmen shows a di e en pha macological
p o ile om ha p oduced by galanin in do sal hippocampus (Fisone
e al., 1989; Gi o i e al., 1993; Gi ens e al., 1992). These disc epancies
indica e ha he galanin N- e minal agmen is pa icula ly ele an o
he GalR sub ypes selec i i y (Hedlund e al., 1994). Ano he galanin
agmen p oposed as GalR
2
agonis s is gal(2−11), also named as
AR-M1896, bu i has been de ec ed ha gal(2−11) also ecognizes
GalR
3
sub ype wi h a simila a ini y (Liu e al., 2001; Lu e al., 2005).
Chime ical pep ides de i ed om galanin N- e minal agmen ha e
also been syn hesized. Fo example: M15 [gal(1−13)-subs ance P
(5−11)] (Ba ai e al., 1991), M32 [gal(1−13)-neu opep ide Y(25−36)]
(A idsson e al., 1993); M35 [gal(1−13)-b adykinin(2−9)] (Og en
e al., 1992), C7 [gal(1−13)-span ide I] (C awley e al., 1993), galpa an
[gal(1−13)-mas opa an] (Langel e al., 1996), gal(1−13)-[Ala
10,11
]ET-I
(6−21)-NH
2
(Ruczynski e al., 2005), M38 [gal(1−13)-(Ala-Leu)
3
-A-
la-NH
2
] (Xu e al., 1995) and M40 [gal(1−13)-P o-P o-(Ala-Leu)
3
-A-
la-NH
2
] (Leibowi z and Kim, 1992). Se e al s udies ha e showed GalR
an agonis -like p ope ies o hese molecules, bu expe imen al e i-
dences seem o poin ou ha some o hese ligands could beha e also as
agonis s depending on he dose, ype o issue o GalR sub ype (Ba ai
e al., 1993; Fa hi e al., 1997; Ko olkiewicz e al., 2002; Wang e al.,
1999). Thus, M35 ac s as an an agonis in many expe imen al models
such as he lexo e lex and ch onic cons ic ion inju y o he scia ic
ne e in a , bu in galanin knockou (KO) mice, his pep ide would ha e
an agonis e ec enhancing neu i e ou g ow h om cul u ed adul
do sal oo ganglia neu ons (Mahoney e al., 2003; Xu e al., 1997).
O he wo GalR ligands a e he M617 [gal(1−13)-Gln
14
-b adykinin
(2−9)-NH
2
], p oposed as a GalR
1
agonis , and he M871 [gal(2−13)-
Glu-His-(P o)
3
-(Ala-Leu)
2
-Ala-NH
2
], conside ed as a GalR
2
an agonis
(Sollenbe g e al., 2006 and, 2010). Howe e , he e is no in o ma ion
a ailable abou hei selec i i y o GalR
3
sub ype.
Mo e ecen ly, o he pep ide gic galanin analogues ha e been syn-
hesized, like he agonis Gal-B2 wi h sligh selec i i y o he GalR
2
han
o GalR
1
(Robe son e al., 2010); J18, a non-speci ic agonis (Saa
e al., 2013); and Ala
5
-galanin (2−11), a ull agonis o GalR
2
(Webling
e al., 2016). In addi ion, some non-pep idical GalR ligands ha e been
syn he ized, such as spi ocuma anon (Sch 202596) (Min e al., 1997),
[2,3-dihyd o-2-(4-me hylphenyl)−1,4-di hiepine-1,1,4,4- e oxide]
(Sco e al., 2000), galnon [(7-((9- luo enyl-me hoxyca bonyl) ciclo-
hexylalanyllysyl) amino-4-me hylcouma in)] (Saa e al., 2002) and
galmic (Ba ai e al., 2004). These molecules display low-a ini y o
GalR and hey do no disc imina e sub ypes. Howe e , wo compound
based on he s uc u e o 3-a ylimino-2-indolones, ha e been p oposed
as GalR
3
speci ic an agonis s: SNAP 37889 [1-phenyl-3-[[3-(-
i luo ome hyl)pheny-l]imino]−1 H-indol-2-one], SNAP 398299
[1-[3-(2-py o-lidinyle hoxy)phenyl]−3-{[3- i luo ome hyl)phenyl]
aza-me hylene}benzo[d]azolin-2-one] (Swanson e al., 2005) and [3-(3,
4-diclo ophenylimino)−1-(6-me hoxypy idin-3-yl)indolin-2-one (Ba
e al., 2006). Finally, a no el GalR
1
-speci ic agonis has ecen ly been
desc ibed, a me hyllan hionine-s abilized GalR agonis , called M89b
(Kuipe s e al., 2021).
The esul s o he p esen s udy u he con ibu e o elucida e he
ac i i y igge ed by some o he mos used GalR ligands: M15, M35,
M40, gal(2−11) and galnon, by mapping he unc ional coupling o G
i/o
p o eins a di e en a eas o he a b ain. Fo his pu pose, hese
compounds ha e been assayed, alone o in combina ion, on a b ain
sec ions using he guanosine 5′-(γ-[
35
S] hio) iphospha e ([
35
S]GTPγS)
au o adiog aphy me hod.
2. Ma e ial and me hods
2.1. Chemicals
[
35
S]GTPγS (1250 Ci/mmol) was pu chased om Pe kinElme
(Bos on, MA, USA). Ra galanin, M15 (galan ide) and gal(2−11) we e
acqui ed om Ame ican Pep ide Company (Sunny ale, CA, USA). Gal-
non, M35 and M40 we e ob ained om Bachem (Weil am Rhein, Ge -
many). All o he chemicals we e ob ained om s anda d sou ces and
we e o he highes pu i y comme cially a ailable.
2.2. Ra issue
Eigh -week-old male Sp ague-Dawley a s (Ha lan, Spain) weigh ing
250–275 g we e used. A e being anaes he ised, hei b ains we e
apidly emo ed and ozen a –80 ºC. Then, sec ions o 20 µm we e cu
a −20 ◦C in a c yos a , moun ed on gela ine-coa ed slides and s o ed a
−20 ◦C un il assay.
The p esen s udy co e s a la ge numbe o a eas o he en i e b ain.
Thus, he mos ep esen a i e b ain le els ha e been used o measu e
each o he a eas unde s udy. As an example, he 2.70 mm le el om
B egma has been used o measu e he in abulba pa o he an e io
commissu e and he mino o ceps o he co pus callosum. On he o he
hand, he sagi al slices we e ob ained a 1.90 mm la e al, ollowing he
Paxinos and Wa son a b ain a las s e eo axic coo dina es (Paxinos and
Wa son, 1998).
All p ocedu es we e pe o med in acco dance wi h Eu opean animal
esea ch laws (Di ec i e 2010/63/EU) and he Spanish Na ional
Guidelines o Animal Expe imen a ion (RD 53/2013, Law 32/2007).
All expe imen al p o ocols we e app o ed by he Local E hics Commi -
ee o Animal Resea ch o he Uni e si y o he Basque Coun y (UPV/
EHU) (CEEA 388/2014).
2.3. [
35
S]GTPγS au o adiog aphy
Tissue sec ions we e ai -d ied o 15 min and hen incuba ed in a
bu e con aining 50 mM T is-HCl, 3 mM MgCl
2
, 0.2 mM EGTA, 100 mM
NaCl and 3 mU/ml adenosine deaminase (pH 7.4) o 20 min a oom
empe a u e. Then, a second incuba ion was ca ied ou wi h he same
bu e bu supplemen ed wi h 2 mM GDP and 1 mM DTT o 20 min.
Finally, a hi d incuba ion was pe o med in [
35
S]GTPγS (0.04 nM) o 2
h a 30ºC in absence o ligands ( o calcula e he basal binding). The a -
ini ies o he pep ide compounds used di e s in e ms o pKi in up o
ou o de s o magni ude wi h ha desc ibed o galnon (galanin pKi 9.5;
M15 pKi 9.6; M35 pKi 10; M40 pKi 8.6 s galnon pKi 4.9) (IUPHAR/BPS
Guide o Pha macology, 2022). The e o e, o achie e he op imal
s imula ions o he GALR
1
ecep o he ollowing concen a ions we e
used: galanin (1
μ
M), gal(2−11) (1
μ
M), M15 (1
μ
M), M35 (1
μ
M), M40
(1
μ
M) and galnon (100
μ
M), as well as in he p esence o he gal-
non/M35 and galnon/M40 mix u es, a he same mola i ies when used
by sepa a e. The non-speci ic binding was de e mined in he p esence o
GTPγS (10
μ
M). Slides we e washed wice in a bu e 50 mM T is-HCl,
d ied unde a cold ai low and exposed o β adia ion-sensi i e ilms
(Kodak Biomax MR) wi h a se o s anda ds, [
14
C]-mic oscales (Ame -
sham, UK).
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
3
2.4. Quan i a i e image analysis o au o adiog ams
The ilms we e de eloped, scanned and quan i ied by ans o ming
he op ical densi ies in o nCi/g issue equi alen (nCi/g .e.) (Image J-
FIJI so wa e, Be hesda, MA, USA). The slide backg ound and non-
speci ic densi ies we e sub ac ed. The pe cen ages o s imula ion
we e calcula ed om he basal and ligand-s imula ed [
35
S]GTPγS
binding densi ies acco ding o he o mula (s imula ed x 100/basal) –
100. Da a we e exp essed as mean alues ±SEM o 7 animals. S a is ical
analyses we e ca ied ou using a one-way ANOVA o epea ed mea-
su es. The c i e ion o s a is ical signi icance was p <0.05 in all s a-
is ical e alua ions.
The compa a i e s udy be ween di e en ligands o he GalR
unc ional coupling o G
i/o
p o eins was analysed using Pea son linea
co ela ions, when he da a ollowed a no mal co ela ion o Spea man’s
when hey did no . The co ela ion coe icien s ob ained in hese ana-
lyses indica e he di ec ion and magni ude o he ela ionship be ween
he a iables ( ; in e al be ween −1 and +1), de e mining he alues o
s a is ical p obabili y.
3. Resul s
3.1. Galanin ecep o ligands ac i i y
The highes alues o [
35
S]GTPγS binding s imula ion induced by
galanin, M15, M35 and M40 (1
μ
M) we e ob ained in he do sal pa o
Fig. 1. [
35
S]GTPγS au o adiog aphic images o sagi al issue sec ions om he a b ain in absence o ligand (A) and in he p esence o 1
μ
M galanin (B), 1
μ
M gal
(2−11) (C), 1
μ
M M15 (D), 1
μ
M M35 (E), 1
μ
M M40 (F), 100
μ
M galnon (G), 100
μ
M galnon and 1
μ
M M40 (H), 100
μ
M galnon and 1
μ
M M35 (I). The non-speci ic
binding was de e mined in he p esence o 10
μ
M GTPγS (J). AOD: an e io ol ac o y nucleus; Sp5: spinal igeminal ac . Scale ba =5 mm.
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
4
he an e io ol ac o y nucleus (Fig. 1). Howe e , nei he gal(2−11) (1
μ
M) no galnon (100
μ
M) we e able o mimic his e ec o e he basal
binding (Table 1, Fig. 1).
In he en al subiculum and in he la e al ol ac o y ac , whe e
galanin inc eased he [
35
S]GTPγS binding in a 40% (p =0.04 and
p=0.003, espec i ely) o e he basal, he chime ical pep ides M15 and
M35 s imula ed abou a 30% (p =0.05 an p =0.001, espec i ely)
(Table 1). Ne e heless, he M40 pep ide only enhanced he basal
binding in a ele an way in he la e al ol ac o y ac (12%, p <0.05).
A simila e ec was also obse ed in he pos e io hypo halamic nucleus,
whe e galanin induced a s imula ion o 44% (p =0.026), whe e hese
chime ical pep ides did no exceed he 15% (Table 2).
A mode a e s imula ion was p oduced by galanin in he in e nal
capsule (26%, p =0.02), do sal ho n laye s I and II o he spinal co d
(22%, p =0.018), medial (13%, p =0.003) and cen al amygdaloid
nucleus (20%, p =0.034) and he spinal igeminal ac (12%,
p=0.033). The M35 pep ide induced a simila s imula ion o he [
35
S]
GTPγS binding in mos o hose a eas wi h he excep ion o amygdala
(Tables 1 and 2). Howe e , M15 only sligh ly enhanced he coupling o
GalR o G
i/o
p o eins in he spinal igeminal ac (10%, p =0.005),
while M40 and gal(2−11) induced a non-signi ican s imula ion o 12%
in he laye s I and II o he spinal co d (Fig. 1; Table 2). In addi ion, gal
(2−11) weakly inc eased he [
35
S]GTPγS binding in whi e ma e a eas
such as in e nal capsule (10%, p <0.05).
On he con a y, galnon e oked a 71% (p =0.004) o s imula ion no
only in he in e nal capsule bu also in o he whi e ma e a eas mainly
cons i u ed o ibe ac s, such as py amidal ac , co pus callosum,
longi udinal asciculus o he pons, an e io pa o he an e io
commissu e and sub o nical o gan. Howe e , galanin ailed o s imula e
he [
35
S]GTPγS binding in hose whi e ma e a eas. A simila e ec was
obse ed wi h M35 in he soli a y ac nucleus, whe e his ligand only
sligh ly enhanced he [
35
S]GTPγS binding (14%, p =0.05), al hough
galanin did no inc eased i signi ican ly (Table 2).
On he o he hand, gal(2−11), M15, M35, M40 and galnon induced
an s a is ically signi ican educ ion o he [
35
S]GTPγS basal binding
(in e se agonis e ec ) in he do sal pa o he den a e gy us, in he
cen omedial halamic nucleus and in he medial hypo halamic nucleus,
whe e galanin did no . In addi ion, he chime ical pep ide M15 also
beha ed as an in e se agonis in he nucleus o he soli a y ac and in
he ol ac o y ube cle. M40 and galnon showed he same e ec in his
a ea, as well as in he s ia um. Fu he mo e, M40 e oked a mode a e
educ ion o he basal binding in he co pus callosum, while gal(2−11)
dec eased i in he en al subiculum, la e al pa ab achial nucleus and
amygdaloid nuclei (Tables 1 and 2).
Galnon p oduced a simila ac ion in he pa ab achial nucleus and in
he amygdaloid nuclei. In ac , his molecule ac ed as an in e se agonis
in mos o he analysed b ain a eas, being his e ec mo e ele an in he
accumbens nucleus and cen al g ay. Ne e heless, he galnon and M35
mix u e enhanced he in e se agonism e ec o bo h ligands in all
analysed a eas, wi h he excep ion o basal pa o he an e io ol ac o y
nucleus, I and II laye s o he spinal co d and pos e io hypo halamic
nucleus. Mo eo e , M35 educed he galnon s imula ion in hese a eas,
e en dec easing he [
35
S]GTPγS binding unde he alues o he basal
binding (in e se agonism) (Fig. 2). By con as , galnon and M40 mix u e
did no each a high dec ease in he [
35
S]GTPγS basal binding. Al hough
M40 pa ially blocked he s imula ion induced by galnon in g ey ma e s
a eas, in whi e ma e a eas such as in e nal capsule (50%, p =0.008),
co pus callosum (28%, p =0.004) and an e io commissu e (39%,
p=0.037), a signi ican s imula ion was obse ed (Tables 1 and 2).
3.2. Compa a i e co ela ion s udy o he ac i i y elici ed by GalR ligands
The s imula ion o he [
35
S]GTPγS binding induced by he di e en
GalR ligands was compa ed be ween hem, using co ela ion analysis.
The ollowing signi ican co ela ions we e obse ed be ween he
s imula ion e oked by galanin and ha p oduced by gal(2−11)
( =0.3903; p =0.0186), M15 ( =0.5039; p =0.0017), M35
( =0.8090; p <0.0001), M40 ( =0.5278; p =0.0009) and he
mix u e o galnon/M35 ( =0.4998; p =0.0019) and galnon/M40
( =0.4050; p =0.0143), bu no wi h he galnon s imula ion (Figs. 3
and 4). In addi ion, he M40 s imula ion was co ela ed wi h he M15
( =0.6787; p <0.0001), M35 ( =0.6906; p <0.0001), galnon/M35
( =0.6614; p <0.0001) and galnon/M40 ( =0.5116; p =0.0014)
s imula ions.
The ac i i y induced by M35 showed a co ela ion wi h ha p o-
duced by M15 ( =0.5934; p <0.0001) and he mix u es o galnon/
M35 ( =0.5686; p =0.0003) and galnon/M40 ( =0.5306;
p=0.0009). Howe e , he M15 s imula ion was mo e simila o he
mix u es o galnon/M35 ( =0.5245; p =0.0010) and galnon/M40
( =0.6264; p <0.0001).
Finally, he ac i i y induced by galnon (100
μ
M) only co ela ed
wi h ha obse ed in he p esence o his molecule wi h o he pep ides:
galnon/M35 ( =0.4242; p =0.0111; excluding he an e io ol ac o y
nucleus) and galnon/M40 ( =0.8022; p <0.0001; excluding he
an e io ol ac o y nucleus) (Fig. 4). In ac , galnon/M35 mix u e showed
a posi i e co ela ion wi h galnon/M40 mix u e ( =0.7402;
p<0.0001).
Table 1
Au o adiog aphic densi ies o he speci ic binding o [
35
S]GTPγS induced by galanin, M15, M35, M40, gal(2–11) (1
μ
M), galnon (100
μ
M), and galnon/M35 and
galnon/M40 combina ions in he inencephalon and elencephalon o he a .
B ain egion Pe cen ages o s imula ion o e [
35
S]GTPγS basal binding (%)
galanin M15 M35 M40 gal (2–11) galnon galnon+M35 galnon+M40
Rinencephalon
An e io ol ac o y nucleus, do sal pa 156 ±34 ** 110 ±27 136 ±46 108 ±39 9 ±4 0 ±56 69 ±60 151 ±20 *
La e al ol ac o y ac nucleus 41 ±10 ** 35 ±8 ** 30 ±6 * 12 ±6 * 1 ±2 -21 ±8 -25 ±7 * -7 ±9
Ol ac o y ube cle -1 ±12 -14 ±4 1 ±8 -22 ±4 -8 ±4 -36 ±11 * -53 ±6 -38 ±9
Telencephalon
S ia um -4 ±12 -11 ±5 -15 ±9 -19 ±4 * -11 ±6 -30 ±11 * -55 ±2 ** -48 ±4 **
Accumbens nucleus 5 ±12 -13 ±6 -0 ±12 -1 ±8 -7 ±8 -44 ±13 * -49 ±2 ** -45 ±6 **
An e i o commissu e, an e io pa 6 ±12 1 ±1 -2 ±3 -4 ±1 -4 ±2 51 ±18 -45 ±3 ** 39 ±12 *
Co pus callosum 1 ±9 -6 ±7 -9 ±6 -13 ±2 * * -2 ±2 67 ±10 ** -43 ±5 ** 28 ±12 *
Cen al amygdaloid nucleus 20 ±7 * 2 ±8 6 ±9 3 ±4 -25 ±9 * -34 ±2 * -40 ±5 ** -32 ±4 **
Medial amygdaloid nucleus 13 ±3 ** -5 ±6 6 ±7 0 ±3 -28 ±10 * -34 ±2 * -33 ±8 ** -33 ±7 *
Sub o nical o gan 0 ±4 -4 ±8 -10 ±6 -6 ±3 -3 ±6 31 ±8 * -39 ±3 ** -15 ±8
Ven al subiculum 41 ±12 * 36 ±13 * 25 ±4 *** 6 ±14 -30 ±4 -12 ±9 -21 ±6 -3 ±14
G anula laye o he do sal den a e gy us -14 ±8 -29 ±4 ** -29 ±12 * -27 ±7 * -14 ±14 -34 ±5 ** -43 ±4 ** -28 ±4 **
Molecula laye o he do sal den a e gy us -12 ±6 -14 ±8 ** -26 ±7 * -20 ±6 * -18 ±8 -37 ±6 ** -48 ±1 *** -40 ±3 **
Da a a e mean ±SEM alues o ou o se en animals. ANOVA ollowed by Bon e oni’s es . * p <0.05; ** p <0.01; *** p <0.001
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
5
4. Discussion
In his s udy, we ha e analyzed he di e ences in he [
35
S]GTPγS
binding e oked by some o he mos used galanin ecep o s ligands. Fo
his pu pose, an exhaus i e ana omical analysis o he ac i a ion o
galanin GPCRs was pe o med in a b ain slices, since his is a alid
animal model o he s udy o he ac i a ion o galanin ecep o s by
unc ional au o adiog aphy (Ba eda-G´
omez e al., 2014). Ini ially, he
s imula ion o GalRs e oked by galanin i sel was analyzed and, as we
p e iously desc ibed, he highes s imula ions induced by his neu o-
pep ide we e obse ed in he do sal pa o he an e io ol ac o y nu-
cleus and in he la e al ol ac o y ac (Ba eda-G´
omez e al., 2005). This
e ec was also mimicked by M15, M35 and M40, bu no by gal(2−11).
Howe e , he non-pep ide ligand, galnon educed he [
35
S]GTPγS basal
binding in he la e al ol ac o y ac bu i did no modi y he s imula ion
induced by M35 and M40 in hese ol ac o y nuclei. The e o e, galnon
does no seem o ecognize GalR
1
, since bo h a eas showed a high
exp ession o his GalR sub ype (O´Donnell e al., 1999). In his con ex ,
he low exp ession o GalR
2
could explain he absence o esponse o gal
(2−11) in hese nuclei, because his ligand is conside ed as an agonis o
GalR
2
and GalR
3
(O´Donnell e al., 1999; Lu e al., 2005).
Ea ly s udies analysing he dis ibu ion o galanin in he CNS al eady
sugges ed ha his neu opep ide could play an impo an ole in he
con ol o memo y, a en ion and lea ning, when i was shown ha
ce ain choline gic cells in he basal o eb ain o he a exp essed gal-
anin (R¨
okaeus e al., 1984). This g oup o galanin/choline ace yl-
ans e ase (ChAT)-posi i e neu ons oge he wi h ano he choline gic
neu ons p ojec o he hippocampus (Melande e al., 1985). Fu he
expe imen al e idence o he in e ac ion be ween he choline gic sys em
and galanin a e ha his neu opep ide inhibi s scopolamine-induced
ace ylcholine elease in bo h he ce eb al co ex (Wang e al., 1999)
and he en al hippocampus (Fisone e al., 1987). Acco dingly, he high
GalR
1
exp ession in he subiculum o he en al hippocampus
(O´Donnell e al., 1999), could i wi h he obse ed high ac i i y elici ed
by galanin, M15, M35 and M40, bu no by galnon, and gal(2−11) ha
beha ed as in e se agonis s in his a ea. This in e se agonis beha iou
o gal(2−11) was also obse ed in di e se b ain nuclei, such as amyg-
dala, medial hypo halamic, medial mammilla y and la e al pa a-
b achial, and in di e en laye s o he do sal den a e gy us. Indeed, all o
es ed ligands, included galanin, educed he basal binding in his hip-
pocampal egion. A possible explana ion o his e ec could be he
esul o p omo ing he coupling o GalR
2
o G
q/11
, conside ing ha his
sub ype is highly exp essed in he den a e gy us o he a (O´Donnell
e al., 1999). In his con ex , A. Maza a i and co-wo ke s obse ed
di e en e ec o GalR
2
depending on he G p o ein sub ype ac i a ion
and hey p oposed ha he coupling o GalR
2
o G
i/o
p o eins in he
hippocampus induced an iepilep ogenic ac ions, whils he coupling o
G
q/11
p o eins ac i a ed he p ocon ulsan pa hway (Maza a i e al.,
2006). Rega ding he en al hippocampus, a b ain egion mainly
in ol ed in s ess, emo ions and a ec i e cha ac e is ics o cogni ion;
he egula ion o he galanin esponse on choline gic neu o ansmission
has been s udied (Yoshi ake e al., 2011), bu also in o he ela ed a eas
such as he bed nucleus o s ia e minalis (Mille e al., 1997). This nu-
cleus whose neu ons co-exp ess galanin and GalR
1
, p ojec s o he sep al
complex and en al hippocampus. Bed nucleus o s ia e minalis, as
well as di e se elencephalic a eas, such as sep al nuclei, diagonal band,
cen al amygdaloid nucleus and s ia e minalis, showed a low, bu
s a is ically signi ican , galanin s imula ions. This weak s imula ion
ag ees wi h he mode a e [
125
I]-galanin binding epo ed in p e ious
s udies and p obably is due o he ac i a ion o GalR
1
, which is mainly
exp essed in hese a eas (Melande e al., 1988; Mennicken e al., 2002;
O´Donnell e al., 1999; Sko i sch e al., 1986).
Ano he a ea wi h high p esence o galanine gic neu ons is he
amygdala (Sko i sh e al., 1986). This nucleus is in ima ely ela ed o
s ess- ela ed diso de s (Ba nabas e al., 2016). Ne e heless, no ele-
an inc ease in he [
35
S]GTPγS basal binding was obse ed by any o
he analysed GalR ligands in he amygdaloid nucleus, despi e he high
densi y o GalR in his a ea. Mo eo e , galnon, as well as gal(2−11),
educed signi ican ly he basal ac i i y, wi hou being al e ed by he
p esence o M35 and M40. Thus, a leas in hese nuclei, galnon and gal
(2−11) seem o ac o e he same ype o ecep o s, which a e no ec-
ognised by he chime ical pep ides M35 and M40. The amygdala and he
en al hippocampus a e wo egions in ol ed in he ol ac o y signalling
pa hway and pa icipa e in he p ocesses o es ablishing and ecalling
ol ac o y memo y, as well as in he associa ion o ol ac o y memo y wi h
o he e en s. In a , hese egions ha e a high densi y o [
125
I]-galanin
binding si es and immuno eac i e ib es o his neu opep ide (Sko i sch
e al., 1986, Melande e al., 1986, 1988, Jacobowi z e al. 2004). In
addi ion, bo h he amygdala and he subiculum and CA1 a ea o he
en al hippocampus, ha e high GalR exp ession, especially o he GalR
1
sub ype ha couples o G p o eins o he G
i/o
amily (O’Donnell e al.,
1999; Wa e s and K ause, 2000). Conside ing ha o he a eas o he
ol ac o y sys em, such as he basal nucleus o he s ia e minalis, ol ac-
o y bulb and ac , diagonal band nucleus, pi i o m co ex and en o-
hinal co ex, a e also ich in galanin and i s ecep o s, we can assume
ha his neu opep ide is ac i ely in ol ed in he egula ion o ol ac o y
signalling pa hways in he CNS (Jacobowi z e al., 2004; Melande e al.,
1988;).
Table 2
Au o adiog aphic densi ies o he speci ic binding o [
35
S]GTPγS induced by galanin, M15, M35, M40, gal(2–11) (1
μ
M), galnon (100
μ
M), and galnon/M35 and
galnon/M40 combina ions in he diencephalons, mesencephalon, hombencephalon and spinal co d o he a .
B ain egion Pe cen ages o s imula ion o e [
35
S]GTPγS basal binding (%)
galanin M15 M35 M40 gal (2–11) galnon galnon+M35 galnon+M40
Diencephalon
Pa a en icula halamic nucleus, an e io pa 4 ±7 -4 ±9 4 ±4 1 ±11 -9 ±7 -27 ±8 -48 ±7 * -33 ±6 *
Cen omedial halamic nucleus -7 ±7 -15 ±6 -22 ±4 * * -10 ±7 -5 ±1 -30 ±8 * -48 ±3 ** -46 ±4 **
Medial hypo halamic nucleus -1 ±6 -14 ±4 * -16 ±3 * -12 ±7 -27 ±6 * * -32 ±9 * -43 ±9 * -41 ±9 *
Pos e io hypo halamic nucleus 44 ±14 * 14 ±9 10 ±8 7 ±7 5 ±5 -22 ±7 -6 ±4 -6 ±10
Medial mammilla y nucleus, mediola e al pa 4 ±2 -30 ±14 -4 ±8 -18 ±14 -29 ±13 -29 ±4 -44 ±4 -39 ±3
Mesencephalon
Cen al g ay 2 ±5 -6 ±2 2 ±4 3 ±6 -11 ±6 * * -37 ±2 -45 ±4 ** -39 ±3 **
In e nal capsule 26 ±8 * 5 ±10 20 ±5 -3 ±4 10 ±6 * 71 ±12 ** -24 ±7 * 50 ±10 **
Rhombencephalon and spinal co d
Longi udinal asciculus o he pons -6 ±7 5 ±8 0 ±8 21 ±8 -15 ±6 55 ±13 * -30 ±5 ** 40 ±9 *
La e al pa ab achial nucleus -6 ±8 -11 ±7 0 ±11 5 ±11 -23 ±6 -34 ±4 ** -35 ±7 ** -32 ±4 **
Py amidal ac -1 ±7 0 ±9 -3 ±2 2 ±8 -13 ±1 104 ±23 * -21 ±3 113 ±28 **
Soli a y ac nucleus 8 ±4 -20 ±4 14 ±5 * 8 ±5 -10 ±8 -16 ±9 ** -30 ±5 * * -13 ±8
Spinal igeminal ac 12 ±4 10 ±1 * * 12 ±1 7 ±5 3 ±7 -18 ±7 ** -22 ±4 * -101 ±3
Laye s I and II o he spinal co d 22 ±4 * 1 ±10 17 ±8 11 ±8 14 ±7 -24 ±1 ** -13 ±11 -14 ±6
Da a a e mean ±SEM alues o ou o se en animals. ANOVA ollowed by Bon e oni’s es . * p <0.05; ** p <0.01; *** p <0.001.
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
6
In he cen omedial and pa a en icula halamic nuclei, galanin did
no s imula e he GalR ac i i y. This absence o e ec con as s wi h he
ele a ed exp ession o GalR
1
and wi h he high densi y o [
125
I]-galanin-
binding si es obse ed in bo h nuclei (O´Donnell e al., 1999; Melande
e al., 1988). In a simila way, in o he halamic and epi halamic egions,
such as euniens nucleus and la e al habenula, no s imula ion was p o-
duced by galanin o by any o he assayed compounds (O´Donnell e al.,
1999; Melande e al., 1988). The low s imula ion o G
i/o
p o eins ac-
i i y obse ed in he pi i o m co ex and do sal hippocampus could
indica e ha a compensa o y mechanism occu ed in hese b ain e-
gions, so ha he high densi y o GalR is accoun ing o he low e i-
ciency in he coupling o G
i/o
p o eins. Ne e heless, he possibili y exis
ha he GalR would be coupled o G
q/11
p o eins, which canno be
quan i ied using his echnique.
Hypo halamus is ano he egion wi h a high densi y o GalR coupled
o G
i/o
p o eins, mainly he magnocellula p eop ic, pos e io hypo ha-
lamic and a cua e nuclei (Ba eda-G´
omez e al., 2005). Acco ding o his
obse a ion, an ele a ed s imula ion o he [
35
S]GTPγS binding was
quan i ied in he p esence o galanin in he pos e io hypo halamic
nucleus, while he es o analysed pep ides gene a ed lowe
s imula ions. This high s imula ion by galanin in he hypo halamic nu-
cleus is ema kable since his a ea is closely ela ed o anxious-like
beha iou and ood in ake, being highly exp essed in se e al hypo ha-
lamic nuclei in ol ed in appe i e con ol such as he pa a en icula
nucleus (PVN) o he a cua e nucleus (Gundlach e al., 2001). Galanin
has o exigenic e ec s, as bo h i.c. . and di ec adminis a ion in o he
PVN causes an inc ease in ood in ake, al hough his esponse is o lowe
in ensi y and du a ion han ha p oduced by neu opep ide Y (Leibo-
wi z, 2005). In addi ion, high- a die s inc ease galanin le els in he
PVN, in addi ion o inc easing lep in, iglyce ides, non-es e i ied a y
acids and glucose, while dec easing insulin and co icos e one le els
(Leibowi z e al., 2004). Fu he mo e, bo h insulin and lep in ha e been
shown o inhibi galanin and neu opep ide Y exp ession in he hypo-
halamus (Sahu, 1998; Wang and Leibowi z, 1997), as well as o inhibi
also insulin elease in he panc eas, hus es ablishing a eedback
mechanism (Lindskog, Ah ´
en 1991; Wang and Leibowi z, 1997). The
ac ha he blockade o a y acid oxida ion leads o a dec ease in
galanin mRNA and an inc ease in GalR
1
exp ession in PVN suppo s he
impo ance o galanin in modula ing lipid me abolism (Go ba yuk,
H¨
ok el 1998; Tang e al., 1997). Con a y o o he galanin ligands
Fig. 2. [
35
S]GTPγS au o adiog aphic images o co onal issue sec ions om he a b ain in absence o ligand (A) and in he p esence o 100
μ
M galnon (B), 100
μ
M
galnon and 1
μ
M M35 (C), 100
μ
M galnon and 1
μ
M M40 (D). aci: an e io commissu e, in abulba pa ; mi: o ceps mino o he co pus callosum. Scale
ba =2 mm.
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
7
s udied, galnon was able o dec ease he [
35
S]GTPγS basal binding.
Howe e , his in e se agonism e ec was blocked by M35 and M40.
Thus, galnon and he chime ic pep ides M35 and M40 seem o ac o e
he same ecep o s in he pos e io hypo halamic nucleus bu wi h
di e en pha macological p ope ies.
In he hombencephalon he highes s imula ions we e ob ained in
he spinal igeminal nucleus and ac , as well as in he mesencephalic,
e icula , la e al pa ab achial and soli a y ac nuclei. M35 and M40
enhanced he [
35
S]GTPγS binding in he same p opo ion as galanin in
he spinal igeminal ac and soli a y ac nucleus. On he con a y,
galnon ac ed as in e se agonis in bo h nuclei. This e ec induced by
galnon was also obse ed in laye s I and II o he spinal co d, whe e
galanin inc eased he GalR coupling o G
i/o
p o eins. In ac , M35, M40
and gal(2−11) sligh ly s imula ed he [
35
S]GTPγS binding in his a ea.
These esul s ag ee wi h he high densi y o [
125
I]-galanin binding si es
and wi h he ele a ed exp ession o GalR
1
desc ibed in hese laye s o
he spinal co d, al hough he p esence o o he GalR sub ypes has also
been epo ed (Mennicken e al., 2002; O´Donnell e al., 1999; Sko i sch
e al., 1986). Expe imen al e idence sugges s ha GalR
1
is ound on
glu ama e gic in e neu ons in he pos e io ho n o he spinal co d,
which would suppo he hypo hesis ha he an inocicep i e e ec s
exe ed by galanin, ia GalR
1
, may be media ed by a enua ion o
exci a o y one in he spinal co d (Land y e al., 2006). Howe e , his
neu opep ide a low doses also exhibi s p onocicep i e ac ions, which
a e p obably media ed by GalR
2
(Liu e al., 2001; Jimenez-And ade
e al., 2004). In line wi h his hypo hesis, Jim´
enez-And ade desc ibed
ha he GalR
2/3
agonis AR-M1896, inc eases capsaicin-induced
neu ogenic in lamma ion (Jimenez-And ade e al., 2006). Mo eo e ,
he spinal co d and spinal ganglia ha e high le els o galanin du ing
de elopmen (Xu e al., 1996) and a e pe iphe al ne e damage in
adul s (H¨
ok el e al., 1987). This pa e n o exp ession in bo h he
neu opep ide and i s ecep o s sugges s ha galanin plays an impo an
ole in he con ol o pain, pa icula ly neu opa hic pain o pain
ollowing ne e inju y, al hough i s unc ion is no ye clea .
The s udy o he co ela ions be ween he s imula ions o he
di e en d ugs p o ided in o ma ion on he pha macological p o ile o
he es ed molecules. When he s imula ions induced by se e al ligands
co ela e, i is in e ed ha his phenomenon is a consequence on ac ing
on he same binding si es. In addi ion, he ac o he absence o
connec ion indica es ha hey can ac on di e en ecep o s and igge
di e en esponses. Thus, M15, M35 and M40 induced a simila s im-
ula ion o basal binding o [
35
S]GTPγS han galanin, indica ing ha
hese molecules a e agonis s o GalRs, bu he ac ha hey show
di e en deg ees o co ela ion be ween hem could indica e ha hey
show selec i i y o some o hei sub ypes, suppo ing p e ious s udies
( o e iew see F eimann e al., 2015). In addi ion, galanin induced
ac i a ion o G
i/o
p o eins does no co ela e o ha p oduced by gal
(2−11), p obably due o he ac ha GalR
2
is mainly coupled o G
q/11
p o eins in b ain issue. Finally, galnon educed he basal ac i i y in
mos o he analysed a eas and inc eased he [
35
S]GTPγS binding in
di e se whi e ma e egions, whe e galanin does no induce any e ec ,
sugges ing ha his non-pep ide molecule ac s as agonis o in e se
agonis o GPCRs di e en om GalR. Mo eo e , GPCR exp essed in
whi e ma e a eas show a ini y o M35 and M40, compounds ha
an agonize he s imula ion gene a ed by galnon. The e o e, M35 and
M40 would be agonis s o GalR, bu also an agonis o hose non-GalR
ecognised by galnon.
In conclusion, his ana omical s udy analysed he in i o ac i i y o
d ugs ha ha e a ini y o galanine gic ecep o s. Some galanin-like
d ugs usually used in expe imen al p ocedu es show dispa a e e icacy
and selec i i y o he di e en galanin ecep o sub ypes. The p esen
s udy using he [
35
S]GTPγS echnique, indica es di e en ac i i ies
e oked by hese ligands on G
i/o
p o ein coupled ecep o s, om agonis
o an agonis e ec s depending on he d ug used. Mo eo e , hese di -
e ences seem o be speci ic o disc e e b ain a eas o nucleus. Thus, he
epo ed esul s con ibu e o he pha macological cha ac e iza ion o
Fig. 3. Co ela ion be ween he s imula ion o he [
35
S]GTPγS binding induced
by 1
μ
M galanin and he s imula ion e oked by 1
μ
M M15 (A), 1
μ
M M35 (B)
and 1
μ
M M40 (C) in di e en a eas o he a CNS. The discon inuous line is a
ep esen a i e co ela ion o 1:1. No e ha hese chime ical pep ides p esen an
agonis e ec simila o ha showed by galanin.
G. Ba eda-G´
omez e al.
Jou nal o Chemical Neu oana omy 128 (2023) 102226
8
GalR ligands o u he imp o e he design o mo e speci ic d ugs o
modula e he galanin media ed e ec s in selec ed b ain a eas, ying o
a oid side e ec s.
CRediT au ho ship con ibu ion s a emen
Gab iel Ba eda-G´
omez: Concep ualiza ion, Me hodology, Fo mal
analysis, W i ing – o iginal d a . I ´
an Manuel: Da a cu a ion, W i ing –
o iginal d a , W i ing – e iew & edi ing. Ra ael Rod íguez-Pue as:
Concep ualiza ion, Resou ces, Funding acquisi ion, P ojec adminis a-
ion. All au ho s ha e ead and ag eed o he published e sion o he
manusc ip .
Acknowledgemen s
Suppo ed by g an s om he egional Basque Go e nmen
IT1454–22 awa ded o he "Neu ochemis y and Neu odegene a ion"
consolida ed esea ch g oup and ISCIII Spanish Minis y o Heal h
PI20/00153 and co- unded by he Eu opean Union (ERDF "A way o
make Eu ope"). Technical and human suppo p o ided by Gene al
Resea ch Se ices SGIke [Uni e si y o he Basque Coun y (UPV/
EHU)].
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