B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

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Research Report

Muscarinic receptor changes in the gerbil thalamusduring aging

Fuencisla Pilar-Cuéllara, Miguel Ángel Paniaguab, Rebeca Díez-Alarciac,Severiano Dos_Anjosb, Sheyla Montorib, Carlos Cesar Pérezd, Arsenio Fernández-Lópezb,⁎aDpto. Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander 39011, SpainbArea Biología Celular, Facultad de Biología y Ciencias Ambients, Universidad de León, Campus de Vegazana s/n. 24071, León, SpaincDpto. Farmacología, Facultad de Medicina, Universidad del País Vasco, Leioa 48940, Bizkaia, SpaindArea Medicina Veterinaria, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n. 24071, León, Spain

A R T I C L E I N F O

⁎ Corresponding author. Departamento BiolUniversidad de León, Campus de Vegazana s

E-mail address: arsenio.fernandez@unileoAbbreviations: ACh, acetylcholine; ADA,

dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6nervous system; [35S]GTPγS, [35S]guanylyl-5scopolamine; OXO, oxotremorine; PVT, parave

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.09.038

A B S T R A C T

Article history:Accepted 13 September 2008Available online 23 September 2008

Here we studied muscarinic receptors in the gerbil thalamus at 8 different ages — from 6 to36 months — using receptor and functional autoradiography. The pharmacological profileinhibiting [3H]N-methyl scopolamine ([3H]NMS) binding with 50 and 200 nM pirenzepine,30 nM pFHHSiD and 100 nM AF-DX 116 revealed the predominance of the M2 muscarinicsubtype in the thalamic nuclei studied, mainly in the anteroventral, anteromedial andparaventricular thalamic nuclei. These data correlated with the highest [35S]guanylyl-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) binding induced in these nuclei by themuscarinic agonistoxotremorine in functional autoradiographic assays. Significant aging-dependent increasesin the functional response in these three nuclei were observed, but only the anteroventraland anteromedial thalamic nuclei showed aging-dependent increases in [3H]NMS binding.Since these nuclei exert relevant functions, in which cholinergic pathways are involved andacetylcholine release is reported to decrease during aging, we suggest that the anteroventraland anteromedial thalamic nuclei would play critical roles in the cholinergic transmissionthat require compensatory mechanisms during the aging process and that are not observedin other thalamic nuclei.

© 2008 Elsevier B.V. All rights reserved.

Keywords:RodentAutoradiography[3H]NMS[35S]GTPγSOxotremorineAF-DX 116

1. Introduction

Muscarinic receptors bind selectively to the neurotransmitteracetylcholine (ACh) and belong to the superfamily of seventransmembrane-spanning domain receptors, coupled to Gproteins. Five muscarinic receptor subtypes have been

ogía Molecular (Area Bio/n. 24071, León, Spain. Fan.es (A. Fernández-Lópezadenosine deaminase; A-one; AM, anteromedial t′-O-(γ-thio)-triphosphatentricular thalamic nucleu

er B.V. All rights reserved

described: M1–M5. The M1, M3 and M5 subtypes are coupled topertussis toxin-insensitive G-proteins (Gq/11), which stimulatephospholipases A2, C and D (Conklin et al., 1988; Peralta et al.,1988; Sandmann et al., 1991). The M2 and M4 subtypes arecoupled to pertussis toxin-sensitive G-proteins (Gi/o), thusinhibiting adenylyl cyclase activity (Peralta et al., 1988). These

logía Celular), Facultad de Ciencias Biológicas y Ambientales,x: +34 987 29 19 17.).F-DX 116, 11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-halamic nucleus; AV, anteroventral thalamic nucleus; CNS, central; LDTg, laterodorsal tegmental nucleus; [3H]NMS, [3H]N-methyls; Pz, pirenzepine; pFHHSiD, para-fluoro-hexahydro-sila-diphenidol

.

39B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

muscarinic subtypes bind to different Gi/o protein subpopula-tions, theM2 receptorbindspreferentially toGiα−1,Giα−2 andGiα−3,andGoα, whileM4 couples toGiα−2 andGoα (Migeonet al., 1995). Inthe central nervous system (CNS), muscarinic receptors havebeen described to be involved in different functions such asmotor control, body temperature regulation, cardiovascularregulation, learning andmemory (Caulfield and Birdsall, 1998).

The aging process is related to a number of encephalicchanges, such as loss of functionality of neural connections, areduction in blood flow, changes in the receptor numbers ofthe different neurotransmitter systems, etc. Among them, the“cholinergic hypothesis of aging”, referred to the decrementsin the basal forebrain ACh signaling postulated in the early80s, indicates that the impairment of the cholinergic functionin the CNS would contribute to the cognitive declineassociated with the aging process and neurodegenerativediseases such as the Alzheimer disease (AD) (Bartus et al.,1982; Bartus, 2000; Terry and Buccafusco, 2003). Other changessuch as a 75% decrease in the biosynthesis of ACh, followed byamore outstanding impairment of the ability for the release ofneurotransmitter following potassium stimulation were alsodescribed (Gibson and Peterson, 1981; Wu et al., 1988; Terryand Buccafusco, 2003).

The Mongolian gerbil (Meriones unguiculatus) is known tohave genetically determined susceptibility to seizures(Loskota et al., 1974), which can be elicited by simple externalstimuli (Ludvig et al., 1991) and we have previously describedthat this species presents a seizure-refractory period after asingle stimulation, and inhibition of seizures after repetitivestimulation (Revilla et al., 1999). The gerbil mean lifespan hasbeen reported to be between 32 and 36 months for males,although ages 48–54 months have been reported (Cheal, 1986;Spangler et al., 1997), and studies on the aging on this specieshave been reported using animals of up to 36 months old

Table 1 – [3H]NMS binding values for labeling of the total numbethe gerbil brain

6 months 16 months 20 months

Medial geniculatenucleus,dorsal part

Total 148±8 144±7 147±6AF 100 nM 98±5 95±3 94±3

Medial geniculatenucleus,medial part

Total 110±5 94±6 100±5AF 100 nM 70±4 60±4 54±2

Medial geniculatenucleus,ventral part

Total 148±7 137±12 147±7AF 100 nM 102±4 99±5 91±4

Reticularthalamic nucleus

Total 115±3 112±6 128±2AF 100 nM 72±3 56±5 61±7

Paraventricularthalamic nucleus

Total 187±3 169±9 179±12AF 100 nM 131±3 103±6 a,b 113±5

Anteroventralthalamic nucleus

Total 263±7 242±9 c 262±7AF 100 nM 179±3 148±10 156±11

Anteromedialthalamic nucleus

Total 177±4 162±13 a 192±4AF 100 nM 134±5 103±10 109±11

One-way ANOVA analysis, followed by a Student–Newman–Keuls test, wabinding values in the presence of 100 nM AF-DX 116 in the different agsignificant differences (P<0.05) between them (n=5). Total binding valueexpressed in fmol/mg tissue as mean±S.E.M.

(Arkin et al., 2003). However, studies carried out onmuscarinicreceptors in the aging gerbil brain only use individuals of up to24 months old and do not reveal any clear pattern ofmodification. Thus, in some studies a decrease in the numberof muscarinic receptors has been reported in some encephalicregions, such as the cerebral cortex, CA1 and the dentate gyrusof the hippocampus and striatum comparing 3, 11 and21 month-old animals (Hara et al., 1992), while other studieshave described an increase in the hippocampus and nomodifications in the cerebral cortex although comparing 1and 16 month-old animals (Araki et al., 1993). Furthermore,these studies on aging used only two or three age groupswhilecurrent knowledge suggests the use of a range of ages todetermine the pattern of age-related changes (Coleman et al.,2004). Thus, herewe report a study using animals of 8 differentages: 6 to 36 months old.

The aim of the study is to characterize the muscarinicsystem, the number and functionality of muscarinic receptorsin different thalamic nuclei of the gerbil brain, mainly theanterior thalamic nuclei, which show one of the highest levelsof the M2 muscarinic subtype. In fact, this is the mostabundant subtype in the thalamus, accounting for about 50%of the total muscarinic receptor population (Levey et al., 1991;Levey, 1993; Piggott et al., 2002). The anterior thalamic nucleireceive input from themammillary body of the hypothalamus,and are connected with the limbic cortices. Thus, this limbiccircuit is associated with the learning process concerningdiscriminative avoidance behavior, spatial and/or performingworkingmemory, and the spatial processing system related tonavigation (Aggleton et al., 1996; Byatt and Dalrymple-Alford,1996; Oda et al., 2001), all of them functions that decline duringthe aging process. In particular, the anteroventral thalamicnucleus (AV) receives its main cholinergic innervation fromthe laterodorsal tegmental nucleus (LDTg) (Oda et al., 2003),

r of muscarinic receptors in different thalamic structures of

22 months 26 months 28 months 31 months 36 months

155±7 154±5 138±4 164±8 151±8111±3 109±3 106±6 111±4 108±4

106±6 122±3 94±3 98±8 98±764±2 65±5 65±6 65±6 58±4

165±9 132±3 132±4 160±8 142±5115±3 103±3 104±5 104±4 102±6

124±4 a 135±6 b 99±6 a,b,c 135±8 c 95±1274±3 63±2 70±5 59±2 57±5

196±6 177±16 158±8 231±6 168±12140±5 a 120±4 116±6 142±8 b 122±13279±9 a 285±7 236±9 a,b 307±6 b,c 282±9172±5 168±7 161±7 163±7 168±11201±5 197±7 178±10 b 224±5 a,b 234±20132±5 128±11 130±4 120±6 136±5

s performed to compare the [3H]NMS binding values, and the [3H]NMSes studied. Groups labeled with the same letter (a, b, or c) presenteds and binding values in the presence of 100 nM AF-DX 116 (AF) are

40 B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

and this cholinergic innervation modulates the neuronalactivity of the AV via muscarinic receptors (Gonzalo-Ruizand Lieberman, 1995). Theseneural circuits havebeen reportedto be involved in the learning process concerning discrimina-tive avoidance behavior, spatial and/or performing workingmemory, and the spatial processing system related to naviga-tion (Aggleton et al., 1996; Byatt and Dalrymple-Alford, 1996;Oda et al., 2001), functions that asmentioneddeclineduring theaging process. This is also related to an increase of the numberof AChbinding sites in theAVduring the learning process (Vogtet al., 1991; Mitchell et al., 2002). The M2 subtype is localizedpostsynaptically in theAV in proximal regions of glutamatergicdendrites (Oda et al., 2001). Cholinergic terminals are closelyrelated to excitatory glutamatergic afferents, and hence theascending cholinergic afferents from the LDTg control AVneuronal activity by modulating excitatory inputs (Oda et al.,2003) via postsynaptic M2 receptors (Oda et al., 2001).

In this work we employed both classical receptor auto-radiography and the more recent functional autoradiography,which is used to identify changes in G protein activation by aspecific receptor (Sim et al., 1995). It uses the radioligand [35S]guanylyl-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) to determinethe activation of Gi/o proteins (Sim et al., 1995), allowing thereceptor stimulation induced by the M2 and/or M4 muscarinicreceptor subtypes to be determined. This method is based on

Fig. 1 – Autoradiographic images of [3H]NMS (A and B) and [35S]Gthalamic nuclei. Total [3H]NMS binding (A); [3H]NMS binding in th(C); and OXO-induced [35S]GTPγS binding (D). All images showed

the use of [35S]GTPγS, a poorly hydrolysable GTP analogue,which replaces GDP when the receptor is activated (Sim et al.,1995, 1996, 1997a,b). Different approaches to reducing [35S]GTPγS binding in the absence of agonists (basal binding) havebeen used (Laitinen, 1999), and recently other authors haveincluded adenosine deaminase (ADA) in their protocol as aroutineway of eliminating the effect of endogenous adenosine(Moore et al., 2000), which strongly increases basal binding.

2. Results

2.1. Receptor autoradiographic studies

Table 1 shows the [3H]NMS binding values obtained withreceptor autoradiography in the thalamus of the animals ofdifferent ages. The highest total [3H]NMS binding values(Fig. 1A) were found in the AV in all ages studied, rangingbetween 240 and 300 fmol/mg protein. According to one-wayANOVA analysis (Table 1), some nuclei showed significantdifferences among the different ages. However, when thebinding values of each nucleus at different ages were studiedusing linear regression data, we only found a significantincrease in the [3H]NMS binding values linked to the agingprocess in the anteromedial thalamic nucleus (AM) (Fig. 2, A).

TPγS (C and D) binding in the gerbil brain at the level of thee presence of 100 nM AF-DX 116 (B); basal [35S]GTPγS bindingat same magnification. Bar: 2 mm.

41B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

Incubation in the presence of 100 nM AF-DX 116 (Fig. 1B)results in inhibition of the [3H]NMS binding values, rangingfrom 43–79% of the total binding (Table 1), where the reticularthalamic nucleus from 31-month old animals displayed themaximal inhibition. M2 muscarinic binding values wereobtained by subtracting the [3H]NMS binding values in thepresence of AF-DX 116 (i.e. the M2 muscarinic subtypeinhibited) from their corresponding total [3H]NMS bindingvalues (i.e. total muscarinic binding) for each nucleus and agestudied. Linear regression of the M2 muscarinic binding datarevealed a significant age-dependent increase in inhibitionmediated by AF-DX 116 in the AV and AM (Fig. 2, B).

Incubation in the presence of 50 nM and 200 nM pirenze-pine resulted in non-significant inhibition (less than 10%) of[3H]NMS binding in all the nuclei and ages studied. Thepresence of 30 nM pFHHSiD in the incubation media alsoresulted in non-significant inhibition (less than 15%) of theradioligand binding in all the nuclei and ages studied.

Fig. 2 – Total [3H]NMS binding and [3H]NMS bindingdisplaced by 100 nM AF-DX 116. These plots represent thethalamic structures that showed significant modifications in[3H]NMS binding (binding data are expressed as fmol/mgtissue) among the different ages studied (P<0.05). (A) total[3H]NMS binding; the anteromedial thalamic nucleus (▪)showed a significant increase (P=0.032). (B) [3H]NMS bindingdisplaced by 100 nM AF-DX 116; the anteromedial (▪) andanteroventral (z) thalamic nuclei showed a significantincrease (P=0.026 and P=0.005, respectively).

2.2. Functional autoradiographic studies

Table 2 shows the [35S]GTPγS binding values obtained byfunctional autoradiography in the thalamus along thedifferent ages studied. The AV (Fig. 1D) had the higheststimulation values following stimulation with OXO. Thepresence of 3 μM OXO in the incubation medium resulted innet-stimulation values ranging from 318 to 563 nCi/g eq. tissuein the different ages (7- to 10-fold binding values with respectto basal binding values). The AM, the paraventricular thalamicnucleus (PVT), and the medial geniculate nucleus, dorsal andmedial parts, in the different ages displayed approximatelyhalf the net-stimulation values of the AVwhen incubatedwithOXO, while the reticular thalamic nucleus and the medialgeniculate nucleus, ventral part, displayed roughly onequarter of the stimulation values observed in the AV in thedifferent ages.

When the data were analyzed following the linearregression between the basal binding values of each nucleusand the different ages we failed to find significant age-dependent changes in any of the nuclei studied.

Analysis of the linear regression data between the net OXO-induced stimulation values of each nucleus and the differentages revealed a significant age-dependent increase in the AV,AMandPVT forboth the total andnet-stimulationvalues (Fig. 3).

3. Discussion

Pharmacological analysis of the gerbil thalamic nuclei bymeans of autoradiography indicates the predominance of theM2 muscarinic subtype, as can be concluded from the differentligands used to inhibit [3H]NMS binding. Thus, Pz is amuscarinic agent considered to be a preferential antagonistfor theM1 subtype, although to a smaller extent also for theM4

subtype, with a pKIM1 = 7.8–8.5 and a pKIM4 = 7.1–8.1respectively (Alexander et al., 2007). Accordingly, the [3H]NMS occupancy of M1 receptors with the amount of Pz used inthis study (50 nM) can be estimated at 6% following theequation of Cheng and Prusoff (Cheng and Prusoff, 1973), whilethe occupancy of M1 and M4 receptors using 200 nM Pz can beestimated at 20%. This study reveals that [3H]NMS binding isnot inhibited by Pz (less than 10%), and it may thus beconcluded that the M1 and M4 subtypes are very scarce orabsent in the gerbil thalamic nuclei studied, as previouslyreported for the mouse (Piggott et al., 2002; Oki et al., 2005), rat(Zeeberg, 1999) and rhesus monkey (Flynn and Mash, 1993).

Regarding the muscarinic antagonist pFHHSiD, considereda preferential M3 muscarinic antagonist with a pKIM3=7.8–7.9(Ladinsky et al., 1990), the occupancy of M3 receptors using30 nM pFHHSiD can be estimated at 6%, following Cheng andPrusoff equation (Cheng and Prusoff, 1973). The low [3H]NMSbinding inhibition (less than 15%) obtained with this ligandindicates that M3 muscarinic receptors are very scarce in thegerbil thalamicnuclei studied, as also reported for the rat (Wanget al., 1989;Odaetal., 2001) and rhesusmonkey (FlynnandMash,1993). However, the presence of the M3 muscarinic subtype hasmainly been described in distal dendritic portions in the AV,ventrolateral part, related to cholinergic afferents from thelaterodorsal tegmental nucleus (LDTg) (Oda et al., 2001).

Table 2 – Basal [35S]GTPγS binding values and net stimulated [35S]GTPγS binding values for the muscarinic agonist OXO indifferent thalamic structures during the aging process

6 months 16 months 20 months 22 months 26 months 28 months 31 months 36 months

Medial geniculatenucleus, dorsal part

Basal 61±2 74±8 88±8 70±4 61±3 88±4 102±10 89±5OXO net 173 104 143 111 159 132 155 178

Medial geniculatenucleus,medial part

Basal 45±2 44±2 73±2 45±2 48±3 65±2 76±11 71±4OXO net 108 78 104 91 99 111 113 108

Medial geniculatenucleus,ventral part

Basal 52±4 58±6 68±4 61±3 48±3 72±4 80±5 70±4OXO net 34 23 27 34 26 35 12 22

Reticular thalamicnucleus

Basal 36±2 43±3 66±3 41±2 41±3 58±2 56±1 56±1OXO net 47 44 54 49 47 68 49 61

Paraventricularthalamic nucleus

Basal 75±6 83±7 117±11 84±3 67±6 115±9 102±6 101±6OXO net 165 154 223 198 205 244 230 306

Anteroventralthalamic nucleus

Basal 57±4 62±5 95±2 55±2 53±4 80±4 80±4 80±4OXO net 362 318 a 472 365 b 434 473 494 563 a,b

Anteromedialthalamic nucleus

Basal 63±4 69±5 100±7 64±4 61±5 84±5 90±8 67±7OXO net 197 154 244 222 253 276 229 280

One-way ANOVA analysis, followed by a Student–Newman–Keuls test, was performed to compare the [35S]GTPγS binding values in basal andOXO-induced conditions in the different ages studied. Groups labeled with the same letter (a or b) presented significant differences (P<0.05)between them (n=5). Values are expressed in nCi/g equivalent tissue, as mean±S.E.M.

42 B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

The muscarinic antagonist AF-DX 116 is considered apreferential M2 muscarinic antagonist with a pKIM2=7.1–7.2(Buckley et al., 1989; Dorje et al., 1991; Dong et al., 1995). Incontrast to other muscarinic subtypes, the occupancy of M2

receptors using 100 nM AF-DX 116 can be estimated at 33%.Thus, the [3H]NMS binding inhibition obtainedwith this ligandsupports the notion that M2 muscarinic receptors wouldaccount for the main or even exclusive muscarinic subtypein the thalamic nuclei, confirming in the gerbil the datareported for other mammals, such as themouse (Piggott et al.,2002; Oki et al., 2005), rat (Wang et al., 1989; Oda et al., 2001) orprimates (Flynn and Mash, 1993). Our data are also inagreement with studies on the M2 muscarinic subtype basedon immunocytochemical studies (Sikes and Vogt, 1987;Amadeo et al., 1995), in which this subtype has been reportedto appear mainly in the AV, where these receptors would bepostsynaptic and would be located in the proximal region ofdendrites (Oda et al., 2001; Oda et al., 2003). Furthermore, M2-positive cells of this nucleus have been reported to be thalamicrelay neurons, since the AV is devoid of GABAergic inter-neurons (Oda et al., 2001).

Our functional autoradiography data using the muscarinicagonist oxotremorine (OXO) indicate that some thalamicstructures such as the AV, AM and PVT show higher [35S]GTPγS binding than other thalamic nuclei, confirming aprevious report (Pilar-Cuellar et al., 2005). The functional andreceptor autoradiography data correlate, indicating that nucleiwith higher M2/M4 receptor densities show higher functionalresponses. Since receptor autoradiography revealed the scantpresence of the M4 muscarinic subtype, we assume that thefunctional response would mainly be mediated by the M2

subtype, which is confirmed in the AV — the thalamicstructure with the highest M2 subtype density —, as observedin this study and previous reports (Oda et al., 2001). Followingthe terminology of Sim et al. (1995), the AV, AM and PVT can besaid to show a high catalytic amplification factor (netfunctional response/M2 receptor number) (2.9 to 4.6) as

compared to other thalamic nuclei, such as the medialgeniculate nucleus, ventral part, and the reticular thalamicnucleus, where the amplification factor is low (0.7 to 1.1).

How do muscarinic receptors behave during aging in thegerbil thalamus? Our receptor autoradiographic study onlyrevealed a significant age-dependent muscarinic receptorincrease in nuclei with high muscarinic levels, mainly in theAM. AF-DX 116 appeared to be the main inhibitor of [3H]NMSbinding, supporting the idea that theM2muscarinic receptor isthe main subtype in the AM and AV. Thus, the data onfunctional and receptor autoradiography support an increasein the M2 muscarinic subtype in the AV and AM during theaging process. The simplest explanation for the aging-dependent increases observed in [35S]GTPγS binding wouldbe an increase in the number of the receptors. Nevertheless,changes in the stimulation efficiency of Gi/o proteins orchanges in the Gi/o protein population cannot be discarded.In this regard, we only observed an age-dependent increase inthe [35S]GTPγS binding response but not in [3H]NMS binding inthe PVT. Accordingly, mechanisms other than an increase inthe receptor number should be invoked to account for theaging-dependent increased response to OXO stimulation.Despite this, since we failed to find significant increases inbasal binding, we cannot correlate the functional increasewith an increased Gi/o population, as has been reported for Gαi

in rat heart associated with the aging process (Kilts et al.,2002). The increase in the number of M2 muscarinic receptorsin the AV and AM could be explained in terms of a response tothe decrease in ACh release produced during the aging process(Gibson and Peterson, 1981; Auld et al., 2002; Terry andBuccafusco, 2003) because the M2 subtype has been reportedto be postsynaptic, at least in the AV (Oda et al., 2001; 2003).These data contrast with the aging-associated decreasesobserved in cerebral cortex, CA1, dentate gyrus and striatum(Hara et al. 1992). However, the decrease in AChwould supportthis different response since M2 muscarinic receptors havebeen reported to be presynaptic in neocortex, hippocampus

Fig. 3 – Linear regression of the thalamic structures showingsignificantmodifications (P<0.05) of net stimulated [35S]GTPγSbinding expressed as nCi/g equivalent tissue in the differentages studied. (A) net OXO-stimulated [35S]GTPγS binding; theparaventricular thalamic nucleus (▪) showed a significantincrease (P=0.007). (B) net OXO-stimulated [35S]GTPγS binding;the anteromedial thalamic nucleus (◊) showed a significantincrease (P=0.040). (C), net OXO-stimulated [35S]GTPγSbinding; the anteroventral thalamic nucleus (E) showed asignificant increase (P=0.015).

43B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

and striatum (Rouse et al., 1997) and postsynaptic in the AV.Other possible explanation of this increase in AV, is that as theM2 muscarinic receptors are located in glutamatergic afferentneurons (Oda et al., 2001), it would produce an inhibition of thefiring or the glutamatergic input, thus trying to reduce the

glutamate induced toxicity, which is increased during theaging process (Liu et al., 1996; Brewer, 1998; Segovia et al.,2001). An additional possibility is that higher functionalresponse might be the consequence of increased agonistbinding. In this regard, an increased response to OXO in agedrats has been proposed to be dependent upon a change inconformation of cholinergic receptors, suggesting theexistence of two or more states of the receptor with differentaffinities for OXO (Birdsall et al., 1978; Pedigo et al., 1984; Lippaet al., 1985; Raskovsky et al., 1988). Thus, in aged rats thenumber of muscarinic receptors in the high-OXO affinityconformation would be augmented, resulting in a strongereffect of the agonist (Espinola et al., 1999). Since the presenceof high-affinity M2 homodimers, and also M1/M2 and M2/M3

heterodimers, has been reported using recombinant receptors(Goin and Nathanson, 2006), the age-dependent increase inM2

muscarinic numbers could result in an increase in dimeriza-tion for M2 muscarinic receptors; this could at least in partexplain the differences in OXO affinity.

In conclusion, a few thalamic nuclei display aging-dependent increases in muscarinic radioligand binding andG protein agonist-induced stimulation, in contrast to most ofthe thalamus. Our data suggest that these nuclei would have acompensatory mechanism to preserve the functionality of thecholinergic system during aging that could be related tolearning andmemory functions, in which the AV, for example,has been reported to play an important role.

4. Experimental procedure

All animals used in this study were treated in accordance withthe European Communities Council Directive of 24 November1986 (86/609/EEC) on the care and use of animals in scientificresearch and the NIH Office of Animal Care and Use (OACU)guidelines (http://oacu.od.nih.gov/). The experimental proto-col was approved by the University of León's EthicsCommittee.

To avoid possible variability induced in females by changesin hormonal levels in the different stages of their oestral cycle(Lovel, 1986), five male gerbils, Meriones unguiculatus, of eachstage, 6, 16, 20, 22, 26, 28, 31 and 36months old, were used. Theanimals were housed individually at 20 °C under standardconditions — 12 h light and 55% humidity — and fed with theA04 (UAR, Epiny sur Orge, France) maintenance rat diet andwater ad libitum. The animals were decapitated between 11and 13 a.m. on two consecutive days to avoid possibledifferences due to circadian rhythms. Their brains wererapidly removed, frozen in liquid nitrogen, and stored at−80 °C until used.

4.1. Receptor autoradiographic studies

Coronal 10 μm-thick brain sections were obtained with acryostat, mounted on silanized slides, and stored at −80 °Cuntil used. The protocol used for the autoradiographiclocalization of muscarinic receptors was that described byCortes et al., (1984), with minimal modifications. Thus, slideswere incubated in 0.5 nM [3H]N-methyl scopolamine ([3H]NMS)(79 Ci/mmol, Amersham Pharmacia Biotech) in 80 mM Na/K

44 B R A I N R E S E A R C H 1 2 4 3 ( 2 0 0 8 ) 3 8 – 4 6

phosphate buffer, pH 7.4, for 60 min at room temperature todetermine total binding. To determine the different muscari-nic subtypes, a series of ligands was used: 50 nM Pz for M1

subtype, 200 nM Pz for M1+M4 subtypes, 100 nM AF-DX 116 forM2 subtype and 30 nM pFHHSiD for M3 subtype. Non-specificbinding was determined as that persisting in the presence of1 μM atropine. After the incubation period, slides were rinsedin the samebuffer at 4 °C,washed twice for 5min in cold buffer,rinsed in cold distilledwater and dried under a cold air stream.Autoradiographs were generated by exposing the slides withthe labeled tissue sections to tritium-sensitive films ([3H]Hyperfilm, Amersham) for 15 days together with the appro-priate radioactive standards, after which they were developedusing standard procedures. The autoradiographs were finallyanalyzed and quantified using a computer-assisted imageanalysis system (Scion Image, Scion Co) tomeasure filmopticaldensities, and densitometric readings were converted intovalues of tissue-bound radioligand expressed in fmol radioli-gand/mg tissueusing the Prism4.00 software (GraphPadPrism).

We calculated the percentage of receptor occupancy foreach of the antagonists assayed, using the following equation(Limbird, 1986):

% of occupancy by the inhibitor = I½ �= I½ � + EC50ð Þð Þ � 100

The EC50 values were calculated following Cheng andPrusoff equation (Cheng and Prusoff, 1973):

KDI = EC50= 1 + L⁎½ �=KDLð Þð Þ

4.2. Functional autoradiographic studies

Consecutive sections to those used in receptor autoradio-graphy were used for functional autoradiography. It should benoted that this technique requires that sections be stored forno longer than 15 days. The autoradiographs corresponding tomuscarinic agonist-stimulated [35S]GTPγS binding sites wereobtained following the initial functional autoradiographyprotocol (Sim et al., 1995), modified to include adenosinedeaminase (ADA) to reduce basal binding (Moore et al., 2000).Thus, slides were incubated for 30min at room temperature in50mMTris–HCl buffer, pH 7.7, containing 3mMMgCl2, 0.2 mMEGTA, 100 mM NaCl, 1 mM DTT and 2 mM GDP. Then, theslides were incubated for 120 min at room temperature in thesame buffer containing 3 mU/ml ADA and 0.05 nM [35S]GTPγS(1250 Ci/mmol, New England Nuclear) to determine basalactivity (basal binding). A concentration of 3 μM OXO (apreferential M2 muscarinic agonist) was used to characterizeM2 muscarinic receptor-coupled G protein activation (OXO-stimulated binding) (Pilar-Cuellar et al., 2005).

The specificity of the muscarinic effect was assessed byinhibiting agonist stimulation with 1 μM atropine, a non-selective muscarinic antagonist. Non-specific binding wasdefined by incubating consecutive sections under the sameconditions in the presence of 10 μM unlabelled GTPγS.

After incubation, the slides were washed twice in ice-cold50 mM Tris–HCl buffer, pH 7.4, and once in ice-cold distilledwater. They were then dried in a cold air stream and exposedto Kodak Biomax-MR film (Amersham) together with 14Cradioactive microscales for 4 days to generate the autoradio-

graphs. The optical densities of the autoradiographs weremeasured using specific image analysis software (ScionImage, Scion Co) and converted to nCi/g tissue using asecond-order polynomial equation whose parameters wereobtained from a non-linear regression correlating nCi/g tissuefrom the 14C radioactive microscales (provided by the manu-facturer) with the corresponding optical densities (measuredunder the same conditions as the autoradiograph). Netstimulation values (stimulated binding–basal binding values)were analyzed for the different ages studied.

Receptor and functional autoradiographic data wereanalyzed using one-way ANOVA followed by a Student–New-man–Keuls test (P<0.05) comparing the different age groups.Also, to determine themodifications in [3H]NMSand [35S]GTPγS(basal and net-stimulated) binding in the different structuresduring aging, the data were fitted by linear regression usingGraphPad Prism, version 4.00, for Windows. The correlationbetween age and number of receptors or functional responsewas assumed only when the slope was significantly differentfrom zero (P<0.05), for both positive and negative slopes.

Acknowledgments

This work was supported by grants from the SAF 2002-00292.We wish to thank Dr. Julen Susperregui, Dpt. MatemáticaAplicada for the support in statistics and Mrs. Marta Fernán-dez Caso and María Rehberger (Dpt. Biología Molecular) fortheir technical support.

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