ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID … · 1 2 ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID RECEPTORS 3 IN THE PRODROMAL AND ADVANCED STAGES IN THE TRIPLE 4 TRANSGENIC

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin and cannabinoid receptors in the prodromal and advanced stages in the

triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.012

NSC 17098 No. of Pages 10

21 May 2016

Neuroscience xxx (2016) xxx–xxx

ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID RECEPTORSIN THE PRODROMAL AND ADVANCED STAGES IN THE TRIPLETRANSGENIC MICE MODEL OF ALZHEIMER’S DISEASE

16

IVAN MANUEL, a LAURA LOMBARDERO, a

FRANK M. LAFERLA, c LYDIA GIMENEZ-LLORT b ANDRAFAEL RODRIGUEZ-PUERTAS a*

aDepartment of Pharmacology, Faculty of Medicine, University of

the Basque Country (UPV/EHU), B� Sarriena s/n, 48940 Leioa, Spain

b Institut de Neurosciencies & Department of Psychiatry and Forensic

Medicine, Faculty of Medicine, Universitat Autonoma de Barcelona,

Avgda Can Domenech s/n, 08193 Bellaterra, Spain

cDepartment of Neurobiology & Behavior, University of

California Irvine, Irvine, CA, USA

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

Abstract—Neurochemical alterations in Alzheimer’s disease

(AD) include cholinergic neuronal loss in the nucleus basa-

lis of Meynert (nbM) and a decrease in densities of the M2

muscarinic receptor subtype in areas related to learning

and memory. Neuromodulators present in the cholinergic

pathways, such as neuropeptides and neurolipids, control

these cognitive processes and have become targets of

research in order to understand and treat the pathophysio-

logical and clinical stages of the disease. This is the case

of the endocannabinoid and galaninergic systems, which

have been found to be up-regulated in AD, and could there-

fore have a neuroprotective role. In the present study, the

functional coupling of G/o protein-coupled receptors to

GalR1, and the CB1 receptor subtype for endocannabinoids

were analyzed in the 3xTg-AD mice model of AD. In addition,

the activity mediated by Gi/o protein-coupled M2/4 muscarinic

receptor subtypes was also analyzed in brain areas involved

in anxiety and cognition. Thus, male mice were studied at 4

and 15 months of age (prodromal and advanced stages,

respectively) and compared to age-matched

non-transgenic (NTg) mice (adult and old, respectively). In

4-month-old 3xTg-AD mice, the [35S]GTPcS binding

stimulated by galanin was significantly increased in the

hypothalamus, but a decrease of functional M2/4 receptors

was observed in the posterior amygdala. The CB1

cannabinoid receptor activity was up-regulated in the ante-

rior thalamus at that age. In 15-month-old 3xTg-AD mice,

muscarinic receptor activity was found to be increased in

motor cortex, while CB1 activity was decreased in nbM. No

changes were found in GalR1-mediated activity at this age.

Our results provide further evidence of the relevance of lim-

bic areas in the prodromal stage of AD, the profile of which

is characterized by anxiety. The up-regulation of galaniner-

gic and endocannabinoid systems support the hypothesis

http://dx.doi.org/10.1016/j.neuroscience.2016.05.0120306-4522/� 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

*Corresponding author. Address: Department of Pharmacology,Faculty of Medicine, University of the Basque Country, E-48940Leioa, Vizcaya, Spain. Tel: +34-94-6012739; fax: +34-94-6013220.

E-mail address: [email protected] (R. Rodrıguez-Puertas).Abbreviations: bA, b-amyloid; AD, Alzheimer’s disease; EGTA,ethylene glycol tetraacetic acid; NTg, non-transgenic.

1

of their neuroprotective roles, and these are established

prior to the onset of clear clinical cognitive symptoms of

the disease. � 2016 IBRO. Published by Elsevier Ltd. All

rights reserved.

Key words: cholinergic, neuropeptides, neurolipids, Alzhei-

mer, G protein, autoradiography.

47

48

49

50

51

52

INTRODUCTION

Alterations in cholinergic neurotransmission seem to be

one of the most characteristic hallmarks of Alzheimer’s

disease (AD), as was firstly described, due to the

impairment of cholinergic neurons in the basal nucleus

of Meynert (nbM) (Whitehouse et al., 1982). Moreover,

a decrease in the density of M2 muscarinic receptors

has been described in areas related to memory and learn-

ing, such as the hippocampus and entorhinal cortex

(Rodrıguez-Puertas et al., 1997a). However, neurochem-

ical alterations also include the impairment of noradrener-

gic (Marcyniuk et al., 1986) and serotonergic (Palmer

et al., 1987) systems. Other neuromodulators, such as

neuropeptides and neurolipids, present in cholinergic

pathways, have become targets of research in order to

understand, prevent or treat the disease. This is the case

of the galaninergic and endocannabinoid systems, which

have been found to be up-regulated in AD and could have

a neuroprotective role (Manuel et al., 2014; Rodrıguez-

Puertas et al., 1997b). In fact, it has been described that

cholinergic cells of nbM are hyperinnervated by galanin

(Chan-Palay, 1988). Besides, an increase of 125I-galanin

binding was observed in AD brains in the hippocampus

and entorhinal cortex, the same areas in which M2 recep-

tor density was reduced (Rodrıguez-Puertas et al.,

1997b). In nbM and amygdala an increase in galanin125I-binding has also been observed (Mufson et al.,

2000; Perez et al., 2002). In the case of cannabinoid neu-

rotransmission, there is a reduction in CB1 receptors in

advanced Braak stages of the disease in different layers

of the hippocampus (Westlake et al., 1994). Furthermore,

there is an increase in both the activity and density of CB1

receptors in early and moderate stages (Manuel et al.,

2014). Nevertheless, cannabinoids are involved in

neuroprotective functions against excitotoxic damage

(Marsicano et al., 2003). In the same way, cannabinoids

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

2 I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx


21 May 2016

are also involved in the suppression of neuroinflammatory

processes in AD (Ehrhart et al., 2005).

Interestingly, pathophysiological processes underlying

AD, seem to occur almost a decade prior to obvious

clinical symptoms. The study of this long preclinical

phase is now considered to be of crucial importance,

together with classical research focused on the study of

more advanced stages of the disease (Sperling et al.,

2013). The identification of early pathophysiological alter-

ations is necessary in order to lead to an effective therapy

prior to the onset of the first clinical symptoms of AD. In

the prodromal stage of the disease, not only biological

markers specific for AD neuropathology and oxidative

stress, but also non-specific markers of neuronal degen-

eration and inflammation are explored as targets for early

drug therapies for AD (Bailey, 2007). As clearly stated by

Welsh-Bohmer (2008), in the very early stages, the neu-

ropsychological symptoms may not be apparent at all

(latent phase), but as the pathology worsens, the first

symptoms emerge (prodromal stage) followed by a full

manifestation of the clinical disease (dementia stage).

This growing concern for the initial stages of AD has lead

to basic research on the validity of animal models to

reproduce the wide spectrum of alterations found in AD

patients, ranging from the pathological processes to the

cognitive deficits and alterations in behavior (Gimenez-

Llort et al., 2007). Due to the genetic background of trans-

genic mice models of familial AD mutations, they may

prove to be a useful tool for the study of the pathophysio-

logical processes involved in AD, from the asymptomatic

stages to the prodromal and the advanced stages of the

disease. This is the case of the triple transgenic mice

model of AD (3xTg-AD) harboring bAPPSwe, PS1M146V

and tauP301L human transgenes, which mimics the devel-

opment of the main histopathological features of the dis-

ease (Oddo et al., 2003). These mice progressively

develop the neuropathological markers of AD in a tempo-

ral and regional-specific profile similar to that found in the

brain of AD patients. At 4 months of age, 3xTg-AD mice

develop intraneuronal b-amyloid (bA) deposits and this

age could be compared to the prodromal stages of the

disease. Indeed, we have consistently reported the pres-

ence of an anxious-like profile, together with some of the

first cognitive deficits (Billings et al., 2005; Gimenez-Llort

et al., 2006, 2007; Canete et al., 2015). The presence of

intracellular bA is initially detected in the hippocampal CA1

region, but at 6 months of age, it is also visible in the cor-

tex (Billings et al., 2005) and in the basolateral amygdala

(Espana et al., 2010), and is clearly related to cognitive

deficits and behavioral alterations. At 12 months of age

the appearance of extraneuronal bA is described and is

followed, at 15 months of age, by concomitant hyperphos-

phorylated tau protein in the hippocampus (Oddo et al.,

2003). At the neurochemical level, the basal forebrain

cholinergic system starts to be affected in 3xTg-AD mice,

early in the intracellular bA stage (Perez et al., 2011).

Histopathological alterations in the primary motor cortex

also appear soon after 3 months of age (Mastrangelo

and Bowers, 2008).

Within this context, the present work aims to describe

the activity of muscarinic, galanin and cannabinoid

Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin

triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http

receptors in 4- and 15-month-old 3xTg-AD mice. These

ages were chosen as those which mimic the above-

mentioned stages in the progress of the disease, that is,

the prodromal stage and the advanced stages,

respectively. Age-matched non-transgenic mice (NTg)

were used as controls. Therefore, the objective of the

study is to validate the neurochemical alterations in this

animal model at different stages, but also to contribute

to the understanding of the neuroanatomical substrates

which are involved in the prodromal and advanced

stages of the disease.

EXPERIMENTAL PROCEDURES

Animals

The group of Dr. Frank LaFerla (Dept. of Neurobiology

and Behavior, University of California Irvine, USA)

created 3xTg-AD transgenic mice as described by Oddo

et al. (2003). Brain samples used in this study were from

4- and 15-month-old homozygous male 3xTg-AD mice

and age-matched NTg mice (n= 6 in each group) of

the colonies established at Universitat Autonoma de Bar-

celona, Spain. All animals were born and bred in the ani-

mal housing facilities in the Medical Psychology Unit and

maintained under standard laboratory conditions in a 12-h

light/dark cycle, 22 ± 2 �C, 50–60% humidity, with food

and water ad libitum.

170

[35S]GTPcS binding assay

Brains were quickly removed and sectioned down the

midline into the right and left hemispheres. Then tissues

were frozen on dry ice, and kept at �80 �C. The brains

were cut in a Microm cryostat (HM 550, Thermo) to

obtain 20 lm sections that were mounted onto gelatin-

coated slides and these were stored at �20 �C until used.

The tissue sections were air-dried for 15 min and then

immersed in a Tris–HCl buffer 50 mM (pH 7.4) with

100 mM NaCl, 3 mM MgCl2 and 0.2 mM EGTA, and

preincubated in the previous mentioned Tris–HCl buffer

50 mM supplemented with 2 mM GDP and 1 mM

DL-dithiothreitol (DTT) and adenosine deaminase

(3 mU/ml). Later, sections were incubated for 2 h at

30 �C in the same buffer containing 0.04 nM [35S]GTPcS(1250 Ci/mmol; Perkin Elmer, Boston, MA, USA). The

agonist-stimulated binding was measured under the

same conditions in the presence of the specific GPCR

agonist: carbachol (100 lM) for M2/4 muscarinic

receptors, galanin (1 lM) for GalR1 receptors and WIN

55212,2 (100 lM) for CB1 receptors The receptor

subtype specificity was demonstrated by inhibiting the

[35S]GTPcS binding by co-incubation with atropine

(10 lM), M15 (1 lM) and SR141716 (10 lM), as

respective antagonists, together with carbachol, galanin

and WIN 55212,2. The chosen concentrations of

agonists and antagonists were similar to those used in

previous studies, and the criteria was based on the

potencies of the different compounds to produce a

receptor-mediated effect. Therefore, the concentrations

are more similar to physiological studies (EC50) than to

receptor binding analysis (affinity, Kd). Note that

and cannabinoid receptors in the prodromal and advanced stages in the

://dx.doi.org/10.1016/j.neuroscience.2016.05.012

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx 3


21 May 2016

antagonist concentrations were usually lower than those

of the agonists, since they bind to the G-protein

uncoupled conformation of the receptors that is favored

during the preincubation washing out of the endogenous

ligands and by the exogenous addition of GDP to the

incubation buffer. Non-specific binding was determined

in the presence of 10 lM of non-labeled GTPcS.Sections were washed twice in Tris–HCl buffer 50 mM

(pH 7.4), once in distilled water, and air-dried. Sections

were exposed to Kodak Biomax MR films with 14C

standards.

Quantitative image analysis of film autoradiograms

Films were scanned and quantified by transforming the

optical densities into nCi/g tissue equivalent (nCi/g t.e.)

using the 14C standards by means of NIH-IMAGE

analysis system (Bethesda, MA, USA). The percentages

of stimulation were calculated from the basal and

agonist stimulation of [35S]GTPcS. The slide

background and non-specific densities were subtracted.

Data were expressed as mean value ± SEM.

Differences between regions of genotypes were

analyzed by Student’s t test.

Thionine staining

The thionine staining was performed to facilitate the

identification of the different areas of the rat brain.

Tissues mounted onto gelatine-coated slides were

hydrated after thawing. The hydration was performed by

immersing tissues for 5 min in ethanol solutions in

descending order (100%, 96%, 70% and 50%). Then

sections were submerged in thionine solution for 5 min.

Later, tissues were washed with deionized water and

placed in ethanol solutions in ascending order to

dehydrate the tissue.

RESULTS

The results from the [35S]GTPcS autoradiography assays

were obtained as autoradiograms of the slices for each

mouse and for every experimental condition. A

representative example from one mouse is represented

in Fig. 1, where the basal activity is measured in the

autoradiogram obtained in the absence of agonist (A),

and the stimulation evoked by the muscarinic agonist

carbachol is observed in a consecutive section (B). The

muscarinic activity is antagonized by atropine (C), and

the non-specific activity is obtained from the incubation

in the presence of 10 lM GTPcS (D).

Cholinergic, endocannabinoid and galaninergicsystems at 4 months of age

Firstly, the basal [35S]GTPcS binding measured in 4-

month-old 3xTg-AD and NTg mice was quantified. The

presence of the three mutations did not modify the

basal activity measured in the absence of any

compound in any of the analyzed brain areas. Note that

the basal binding indicates the activity mediated by the

whole population of the different GPCR subtypes for any

neurotransmitter system coupled to Gi/o proteins in the



conditions of the present assay. The analysis of the

[35S]GTPcS binding mainly quantifies the activity

mediated by Gi/o proteins. The reason for this seems to

be that there is both a higher ratio of Glo/i proteins

compared to that of other families, and higher rates of

GTP exchange, resulting in a much stronger signal

which precludes the stimulation of the other types of G

proteins (Harrison and Traynor, 2003).

On the contrary, the cholinergic muscarinic receptor

activity induced by carbachol measured as Gi/o proteins

bound to [35S]GTPcS, was found to be decreased in

specific brain areas of 3xTg-AD mice. One of these

regions was the posterior portion of the amygdala in

which the percentage of carbachol-induced stimulation

was 60 ± 14% in NTg and 25 ± 8% (p< 0.05) in

3xTg-AD mice. In the nbM a reduction in the activity of

these receptors was also observed (NTg 118 ± 25%;

3xTg-AD 76 ± 17%), but this change was not

statistically significant. However, in the hippocampus, an

increase in the [35S]GTPcS binding induced by

carbachol was found in dentate gyrus (NTg 33 ± 16%;

3xTg-AD 67 ± 27%) (Fig. 2, Table 1).

The functional coupling of GalR1 to Gi/o proteins

induced by galanin was only modified in the

hypothalamic area in which there was an increase in the

[35S]GTPcS binding in the transgenic mice (NTg 14

± 15%; 3xTg-AD 47 ± 12%, p< 0.05). There was a

decrease in the nbM (NTg 35 ± 13%; 3xTg-AD 24

± 9%), although it was not of statistical significance.

[35S]GTPcS binding stimulated by the cannabinoid

agonist WIN55,212-2 was only different from that of,

NTg in the ventrolateral part of the anteroventral

thalamic nucleus (anterior thalamus) (NTg 66 ± 21%;

3xTg-AD 136 ± 15%; p< 0.05) (Fig. 2, Table 2).

In summary, in 4-month-old 3xTg-AD mice no

changes were observed in the basal GPCR-mediated

activity. MR activity was lower than that observed in

age-matched NTg mice in the basal forebrain, but

higher in hippocampus. GalR1 activity was up-regulated

in hypothalamus, and CB1 activity in the anterior

thalamus in comparison with controls.

Cholinergic, endocannabinoid and galaninergicsystems at 15 months of age

To examine the functionality of the GPCR in 3xTg-AD

mice in advanced neuropathological stages and age-

matched NTg mice, we also analyzed the basal activity.

At 15 months of age, the 3xTg-AD mice presented an

increase in the basal [35S]GTPcS binding compared to

NTg mice in different brain areas, such as the anterior

portion of the amygdala (NTg 639 ± 18 nCi/g t.e.; 3xTg-

AD 817 ± 31nCi/g t.e.; p< 0.01), the anteroventral

portion of the thalamus (NTg 578 ± 32 nCi/g t.e.; 3xTg-

AD 621 ± 18 nCi/g t.e.; p< 0.01), and the ventral

portion of this area (NTg 471 ± 29 nCi/g t.e.; 3xTg-AD

562 ± 15 nCi/g t.e.; p< 0.05). We also found an

increase in the basal [35S]GTPcS binding in the nbM

(NTg 607 ± 19 nCi/g t.e.; 3xTg-AD 752 ± 14 nCi/g t.e.;

p< 0.01). There was more activity in the striatum of

3xTg-AD mice than in that of NTg mice (NTg 665

± 24 nCi/g t.e.; 3xTg-AD 752 ± 16 nCi/g t.e.; p< 0.05);



286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

Fig. 1. Autoradiographic images of a representative NTg mouse of 4 months of age, showing [35S]GTPcS basal binding that was similar to that

observed in 3xTgAD mice (A), carbachol stimulation (100 lM) (B), carbachol stimulation antagonized with atropine (10 lM) (C) and non-specific

binding determined in the presence of 10 lM GTPcS (D).



21 May 2016

and the same was observed in globus pallidus (NTg 672

± 18 nCi/g t.e.; 3xTg-AD 741 ± 20 nCi/g t.e.; p< 0.05)

(Fig. 3, Table 3).

In both NTg and 3xTg-AD mice the activity mediated

by the three different GPCR analyzed was lower at

15 months than at 4 months of age. Although it has

been described that there is a general decrease with

age in the GPCR activity measured by [35S]GTPcSbinding, the different groups of age were analyzed in

different experiments at different times, and the inter-

experimental variations could also have contributed to

those differences (Gonzalez-Maeso et al., 2002). Further-

more, 15-month-old 3xTg-AD mice showed an increase in

the activity of Gi/o-coupled cholinergic muscarinic recep-

tors induced by carbachol in discrete areas of the cortex

such as the motor cortex in the 3xTg-AD mice (NTg 15

± 4%; 3xTg-AD 34 ± 5%; p< 0.05) (Fig. 4). No signifi-

cant changes were observed in the activity induced by

galanin and in fact, the areas with the highest

stimulations, corresponding to hippocampus CA1 (NTg

17 ± 7%; 3xTg-AD 21 ± 8%) and dentate gyrus (NTg

16 ± 7%; 3xTg-AD 12 ± 6%), were also low. However,

there were changes in CB1-mediated activity in the

15-month-old 3xTg-AD mice, in which a reduction in the

nbM was found (NTg 116 ± 10%; 3xTg-AD 57 ± 7%;

p< 0.05) (Fig. 5).

In summary, the basal activity was increased only in

the 15-month-old 3xTg-AD mice, but the regulation of

the three different GPCR analyzed was only statistically

significant for the increase in motor cortex of M2/4 and

for the decrease of CB1 in nbM.

350

351

352

353

354

355

DISCUSSION

The neurotransmission in AD is characterized by specific

and early alterations of the cholinergic system in the basal

forebrain pathways that innervate limbic structures such



as the hippocampus and cortical areas. However, other

systems, which modulate cholinergic pathways, are

hyperactive, such as the galaninergic system (Chan-

Palay, 1988; Rodrıguez-Puertas et al., 1997b). A further

finely tuned modulation seems to be controlled by other

systems using neurolipids, e.g. the endocannabinoid sys-

tem that responds in the earliest stages of AD by increas-

ing its activity (Manuel et al., 2014). The present study

uses 3xTg-AD mice to analyze these three systems by

measuring GPCR activity at ages corresponding to the

prodromal and advanced stages of the disease. The

results obtained indicate that the 3xTg-AD mouse model

mimics the neurochemical alterations observed in AD

patients in relation to muscarinic, galanin and cannabinoid

receptors in both early and late stages of the disease.

Basal binding

The presence of the three genetic alterations, bAPPSwe,

PS1M146V and tauP301L transgenes in the 4-month-old

3xTg-AD mice, did not produce any alteration in the

basal activity of GPCR. Recently, different studies have

demonstrated that at least part of this basal activity

accounts for endogenous ligands for GPCR that remain

present during the assay as a consequence of their

lipidic structure. It is very difficult to remove the

endogenous lipid ligands or neurolipids during the

preincubation stage of the [35S]GTPcS binding assay,

which is carried out in an aqueous buffer in adequate

physiologic pH and temperature conditions (Aaltonen

et al., 2008, 2012; Gonzalez de San Roman et al.,

2015).However, the observed up-regulation in the basal

activity of 15-month-old 3xTg-AD mice could be a com-

pensatory response to more severe synaptic impairments

that arise with aging and that are mediated by GPCR sub-

types, probably related to neurolipid signaling. In this

sense, at 4 months of age the bA is only detected inside



356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

Fig. 2. Autoradiographic images of sagittal tissue sections of [35S]GTPcS in 4-month-old NTg and 3xTg-AD mice showing stimulation by carbachol

(A and B), stimulation by galanin (C and D) and stimulation by WIN55,212-2 (E and F). Note that the carbachol stimulation in the posterior amygdala

is decreased in 3xTg-AD mice in comparison with NTg mice. On the contrary, stimulation by galanin is increased in the hypothalamus of 3xTg-AD

mice. The stimulation by WIN55,212-2, indicating mainly CB1-mediated cannabinoid receptor activity in the CNS (Llorente et al., 2014), was only

modified in the ventrolateral part of the anteroventral thalamic nucleus. Scale bar = 2.5 mm.



21 May 2016

the neurons of 3xTg-AD mice (Oddo et al., 2003), and

there is also a deficit in long-term retention that correlates

with the accumulation of intraneuronal bA in hippocampus

and amygdala (Billings et al., 2005). Moreover, there is

little evidence of a decrease in the number of neurons in

3xTg-AD mice at 4 months, but a reduction in cells

has been observed at the medial septum/vertical limb of

the diagonal band of Broca in aged 3xTg-AD mice

(18–20 months) (Perez et al., 2011).

384

385

386

387

388

389

390

391

392

393

394

395

Activity mediated by muscarinic receptors

The cholinergic alterations in 3xTg-AD mice include

degeneration of the cholinergic septo-hippocampal

pathway, reflected by a decrease in ChAT-positive

fibers in the hippocampus and a decrease in positive

neurons in the medial septum at 4 months of age (Girao

da Cruz et al., 2012). Regarding the observed decrease

of the MR response in amygdala, it is worth noting that

this nucleus has an important role in the processing of

anxiety and emotions. In 3xTg-AD mice, an increase of



anxiety and fear-related behavior has been observed at

a similar age (Espana et al., 2010). The cholinergic con-

trol of cortical GABA activity involves M2 muscarinic

receptors on GABAergic terminals (Kawaguchi and

Kubota, 1997; Steriade and Descarries, 2006). The loss

of M2 receptors on GABA terminals in the amygdala,

could also account for the detected impairment of the

muscarinic receptor signaling.

The up-regulation of MR activity in the motor cortex

may be a response to the described increase in positive

ChAT-ir dystrophic neurites in the cortex in aged

animals (Perez et al., 2011), accompanied by the intra-

neuronal bA in the primary motor cortex (Mastrangelo

and Bowers, 2008). In other murine models of AD,

expressing the APP Swedish and the PS1DE9 mutations,

the ChAT activity was diminished at advanced ages

(Perez et al., 2007). The cholinergic regulation or alter-

ation in these transgenic models is only detected in aged

animals, as is also the case for the regulation of the MR

activity. Nevertheless, the TgCRND8 mice, which develop

demyelination and many Ab plaques, also show choliner-



396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

Table 1. [35S]GTPcS binding in different areas of 4-month-old 3xTg-AD and NTg mice induced by carbachol (100 lM) and galanin (1 lM) expressed as

the percentage of stimulation over the basal

Brain region Carbachol stimulation (%) Galanin stimulation (%)

NTg 3xTg-AD NTg 3xTg-AD

Amygdala

Anterior 101.5 ± 29.2 128.9 ± 25.9 19.6 ± 5.4 16.6 ± 15.1

Posterior 59.8 ± 14.1 25.4 ± 7.9* 39.9 ± 10.3 44.6 ± 5.5

Thalamic nuclei

Anteroventral 165.1 ± 21.8 208.7 ± 51.7 40.7 ± 30.3 70.8 ± 21.3

Thalamus 134.1 ± 21.3 146.5 ± 5.8 �3.1 ± 5.8 12.7 ± 12.5

Striatum 210.8 ± 24.2 193.0 ± 22.4 10.8 ± 7.9 5.7 ± 4.6

Hypothalamus 70.9 ± 11.1 74.5 ± 27.9 14.4 ± 15.1 46.6 ± 12.4*

Cortex

Cingular 118.5 ± 30.9 202.6 ± 99.5 22.4 ± 6.3 43.9 ± 30.1

Motor 111.7 ± 12.8 98.2 ± 32.6 �4.3 ± 4.2 �15.7 ± 5.5

Hippocampus

CA1 94.6 ± 14.8 92.7 ± 32.3 �5.4 ± 5.8 3.2 ± 7.9

Dentate gyrus 33.2 ± 16.2 66.9 ± 27.5 �17.6 ± 11.4 9.9 ± 12.2

Basal nucleus 118.0 ± 25.4 75.9 ± 16.6 35.0 ± 13.2 23.7 ± 8.7

Mean ± SEM of six animals by genotype.

Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL (ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05 (two tailed Student’s ‘‘t” test).

Table 2. [35S]GTPcS binding in different areas of 4-month-old 3xTg-AD

and NTg mice induced by WIN55,212-2 (100 lM) expressed as the

percentage of stimulation over the basal

Brain region WIN 55212-2 stimulation (%)

NTg 3xTg-AD

Amygdala

Anterior 124.8 ± 50.1 55.2 ± 17.0

Posterior 147.4 ± 14.3 187.5 ± 24.3

Thalamic nuclei

Anteroventral 64.8 ± 21.2 136.3 ± 15.5*

Thalamus 73.4 ± 24.0 115.9 ± 30.9

Striatum 254.2 ± 39.4 247.9 ± 18.9

Cortex

Cingular 364.9 ± 87.7 456.5 ± 58.2

Motor 381.4 ± 39.5 360.1 ± 25.5

Hippocampus

CA1 309.2 ± 43.4 304.9 ± 34.0

Dentate gyrus 276.2 ± 62.4 369.7 ± 64.6

Basal nucleus 203.5 ± 21.5 359.8 ± 70.2

Hypothalamus 93.3 ± 6.1 101.0 ± 18.7

Globus pallidus 507.8 ± 123.0 594.5 ± 88.8

Sustantia nigra 549.6 ± 224.7 737.6 ± 152.6


Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL

(ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05 (two tailed Student’s ‘‘t” test).



21 May 2016

gic dysfunction with a reduction in ChAT in the nbM and

M2 reduction in the cortex (Bellucci et al., 2006). The

cholinergic activity seems to depend on the different

mutations expressed in each transgenic mouse model

of familial AD.



Activity mediated by galaninergic receptors

The GalR1-mediated activity was measured using rat

galanin. The GalR1 subtype is the main galanin receptor

present in the CNS and is coupled to Gi/o family of G

proteins. Therefore, the recorded galanin stimulations of

[35S]GTPcS binding should be attributable to GalR1-

mediated activity receptors. Studies assessing different

behavior parameters of 4-month-old mice using the

open field test have described an anxious-like state in

these animals. The freezing latency was more

pronounced in 3xTg-AD mice than in NTg, and they also

showed a decrease in locomotion during the first

minute. Other fear-associated parameters such as

increased defecations and a delay in the onset of

grooming could also be an indication of the fact that the

anxious responses are exacerbated in these mice

(Gimenez-Llort et al., 2007). The increase in the activity

of GalR1 in limbic brain areas involved in the control of

anxiety, such as the hypothalamus, could be related to

the reported increase in anxious-like behavior.

Galanin seems to exert an important role in the

hypothalamus by regulating glutamate release in that

area (Kinney et al., 1998). Furthermore, under stress con-

ditions, an increase in the production of galanin in the

hypothalamus does exist (Palkovits, 2000), but both

exogenous galanin administration and agonists are able

to induce anxiolytic-like effects in rats (Bing et al., 1993;

Moller et al., 1999; Rajarao et al., 2007). A possible up-

regulation of GalR1 activity in 4-month-old 3xTg-AD mice

could be a physiological response to compensate for the

increased anxiety during their development that is not

necessary in more aged mice. In other mice models of

AD in which there is an overexpression of Ab, such as

PDAPP, APPSwe/PS1DE9 double transgenic or APP23,

an increase in galanin has also been described, but



436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

Fig. 3. Autoradiographic images of sagittal tissue sections showing basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note

that basal binding is higher in the anterior amygdala of 3xTg-AD mice in comparison with NTg mice. Scale bar = 2.5 mm.

Table 3. Basal [35S]GTPcS binding in different areas of 15-month-old

3xTg-AD and NTg mice

Brain region Basal binding (nCi/g t.e.)

NTg 3xTg-AD

Amygdala

Anterior 639 ± 18 817 ± 31**

Posterior 506 ± 19 711 ± 39

Thalamic nuclei

Anteroventral 578 ± 32 621 ± 18**

Thalamus 471 ± 29 562 ± 15*

Striatum 665 ± 24 752 ± 16*

Cortex

Cingular 677 ± 32 761 ± 22

Motor 542 ± 19 601 ± 38

Hippocampus

CA1 665 ± 38 714 ± 36

Dentate gyrus 436 ± 20 474 ± 24

Basal nucleus 607 ± 19 752 ± 14**

Locus coeruleus 342 ± 29 314 ± 22

Globus pallidus 672 ± 18 741 ± 20*

Sustantia nigra 635 ± 31 670 ± 31


Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL

(ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05.

** p< 0.01 (two tailed Student’s ‘‘t” test).



21 May 2016

mainly in hippocampus and cortex (Diez et al., 2000;

Mufson et al., 2005).

477

478

479

480

481

482

483

484

485

486

487

488

Activity mediated by cannabinoid receptors

The endocannabinoid system is implicated in

inflammatory responses and neurogenesis, but also in

memory deficits. Memory impairments induced by bApeptide administration are reverted by the CB1

antagonist, SR141716A or rimonabant (Mazzola et al.,

2003). Nevertheless, cannabinoid agonist administration

(HU-210) did not have any effects either in memory tests

or in amyloid progression in APP23/PS45 double trans-

genic mice (Chen et al., 2010). However, other authors



using both CB1 and CB2 agonists described some protec-

tive effects in Tg APP mice (Martın-Moreno et al., 2012).

Moreover, the administration of the CB2 specific agonist,

JWH-133, could improve some inflammatory responses

present in AbetaPP/PS1 transgenic mice, but not the bAproduction or deposition in cortex and hippocampus

(Aso et al., 2013). Cannabinoids seem to act in excitotoxic

and inflammatory processes by increasing cannabinoid

receptor activity (Fowler et al., 2010). Thus, 4-month-old

3xTg-AD mice that only present intraneuronal bA, onlyhave an up-regulation of CB1-mediated activity in thala-

mus, while this is decreased in nbM when CNS damage

is evident at 15 months of age. The CB1-mediated activity

is increased in AD patients from the early stages of the

disease in hippocampal and cortical areas, suggesting

the existence of a possible neuroprotective response that

is not clearly observed in 3xTg-AD mice (Manuel et al.,

2014). Other early damaging factors or causes inherent

to the sporadic forms of AD, not present in the hereditary

forms of AD represented by 3xTg-AD mice, could be

responsible for initiating the early response of the endo-

cannabinoid system.

CONCLUSIONS

The 3xTg-AD mice represent an animal model of three

human transgenes and allow us to reproduce the

classical histopathological markers of AD, neuritic

plaques and neurofibrillary tangles. The model develops

the most representative biochemical modifications

associated with AD, i.e. the hyperphosphorylation of tau

and bA accumulation, which have been widely analyzed

in this mouse model at different ages. The behavior of

these mice has also been an important subject of study,

but only a few neurotransmission systems more

associated with memory impairment, such as the

cholinergic system, have been analyzed.

The activity of cholinergic muscarinic receptors is

analyzed in the present study together with the activity

mediated by a system of neuropeptides, the

galaninergic, and a system of neurolipids, the

endocannabinoid. Both systems modulate cholinergic

inputs from the basal forebrain to cortical and



489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

Fig. 5. Autoradiographic images of sagittal tissue sections of basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note that

[35S]GTPcS binding stimulated by WIN55,212-2 is not modified in 3xTg-AD mice in comparison with NTg mice. Scale bar = 2.5 mm.

Fig. 4. Autoradiographic images of sagittal tissue sections of basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note that

[35S]GTPcS binding stimulated by carbachol is increased in the motor cortex and dentate gyrus of the hippocampus of 3xTg-AD mice in comparison

with NTg mice. Scale bar = 2.5 mm.



21 May 2016

subcortical areas involved in the control of learning and

memory processes, and both systems are up-regulated

in AD patients.

The present results are similar to those reported in

postmortem brain samples of AD patients in several

aspects in relation to the regulation of receptor

activities, but are not found in the same brain areas. MR

activity is decreased in 3xTg-AD mice at the age of

4 months, which mimics the early stages of AD, but only

in some limbic areas such as the amygdala, whereas a

general reduction of M2 MR densities has been found in

hippocampus and cortex of AD patients. Furthermore,

MR activity is increased in motor cortex in 15-month-old

transgenic mice. On the other hand, it is not

counteracted by a regulation of GalR1 and CB1 activity,

as seems to be the case in AD patients, in whom

hyperactivity of galanin and CB1 receptor has been

described mainly in hippocampal areas. The up-

regulation of both neuromodulators of the basal

cholinergic signaling has been interpreted as a

neuroprotective response.

In summary, the 3xTg-AD model has a mild

impairment of the cholinergic muscarinic activity and the



regulation of neuromodulatory systems by

neuropeptides, such as galanin, and endocannabinoids

have a role that is likely to be related to the anxious

phenotype, which is characteristic of the prodromal

stages of AD, prior to the onset of clear clinical cognitive

symptoms of the disease.

UNCITED REFERENCE

Blazquez et al. (2014).

Acknowledgments—Supported by grants from the Basque

Government IT584-13 and Spanish Government, Ministry for

Health, I. S. C. III PI 10/01202- co-funded by European Research

Development. Thanks to the Animal facilities of the UPV/EHU at

the Biscay Campus, SGiker. We thank Adele Hopley and M. Ter-

esa Giralt for critical and careful reading of the manuscript. There

are no potential conflicts of interest.

REFERENCES

Aaltonen N, Palomaki VA, Lecklin A, Laitinen JT (2008)

Neuroanatomical mapping of juvenile rat brain regions with



530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669



21 May 2016

prominent basal signal in [(35)S]GTPgammaS autoradiography. J

Chem Neuroanat 35(2):233–241.

Aaltonen N, Lehtonen M, Varonen K, Goterris GA, Laitinen JT (2012)

Lipid phosphate phosphatase inhibitors locally amplify

lysophosphatidic acid LPA1 receptor signalling in rat brain

cryosections without affecting global LPA degradation. BMC

Pharmacol 12:7.

Aso E, Juves S, Maldonado R, Ferrer I (2013) CB2 cannabinoid

receptor agonist ameliorates Alzheimer-like phenotype in AbPP/PS1 mice. J Alzheimers Dis 35(4):847–858.

Bailey P (2007) Biological markers of Alzheimer’s disease. Can J

Neurol Sci 34:72–76.

Bellucci A, Luccarini I, Scali C, Prosperi C, Giovannini MG, Pepeu G,

Casamenti F (2006) Cholinergic dysfunction, neuronal damage

and axonal loss in TgCRND8 mice. Neurobiol Dis 23(2):260–272.

Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla FM (2005)

Intraneuronal Abeta causes the onset of early Alzheimer’s

disease-related cognitive deficits in transgenic mice. Neuron 45

(5):675–688.

Bing O, Moller C, Engel JA, Soderpalm B, Heilig M (1993) Anxiolytic-

like action of centrally administered galanin. Neurosci Lett 164(1–

2):17–20.

Blazquez G, Canete T, Tobena A, Gimenez-Llort L, Fernandez-

Teruel A (2014) Cognitive and emotional profiles of aged

Alzheimer’s disease (3xTg-AD) mice: effects of environmental

enrichment and sexual dimorphism. Behav Brain Res

268:185–201.

Canete T, Blazquez G, Tobena A, Gimenez-Llort L, Fernandez-

Teruel A (2015) Cognitive and emotional alterations in young

Alzheimer’s disease (3xTg-AD) mice: effects of neonatal handling

stimulation and sexual dimorphism. Behav Brain Res

281:156–171.

Chan-Palay V (1988) Galanin hyperinnervates surviving neurons of

the human basal nucleus of Meynert in dementias of Alzheimer’s

and Parkinson’s disease: a hypothesis for the role of galanin in

accentuating cholinergic dysfunction in dementia. J Comp Neurol

273(4):543–557.

Chen B, Bromley-Brits K, He G, Cai F, Zhang X, Song W (2010)

Effect of synthetic cannabinoid HU210 on memory deficits and

neuropathology in Alzheimer’s disease mouse model. Curr

Alzheimer Res 7(3):255–261.

Diez M, Koistinaho J, Kahn K, Games D, Hokfelt T (2000)

Neuropeptides in hippocampus and cortex in transgenic mice

overexpressing V717F beta-amyloid precursor protein–initial

observations. Neuroscience 100(2):259–286.

Ehrhart J, Obregon D, Mori T, Hou H, Sun N, Bai Y, Klein T,

Fernandez F, Tan J, Shytle RD (2005) Stimulation of cannabinoid

receptor 2 (CB2) suppresses microglial activation. J

Neuroinflammation 2:29.

Espana J, Gimenez-Llort L, Valero J, Minano A, Rabano A,

Rodriguez-Alvarez J, LaFerla FM, Saura CA (2010)

Intraneuronal beta-amyloid accumulation in the amygdala

enhances fear and anxiety in Alzheimer’s disease transgenic

mice. Biol Psychiatry 67(6):513–521.

Fowler CJ, Rojo ML, Rodriguez-Gaztelumendi A (2010) Modulation of

the endocannabinoid system: neuroprotection or neurotoxicity?

Exp Neurol 224(1):37–47.

Gimenez-Llort L, Blazquez G, Canete T, Johansson B, Oddo S,

Tobena A, LaFerla FM, Fernandez-Teruel A (2007) Modeling

behavioral and neuronal symptoms of Alzheimer’s disease in

mice: a role for intraneuronal amyloid. Neurosci Biobehav Rev 31

(1):125–147.

Girao da Cruz MT1, Jordao J, Dasilva KA, Ayala-Grosso CA,

Ypsilanti A, Weng YQ, LaFerla FM, McLaurin J, Aubert I (2012)

Early increases in soluble amyloid-b levels coincide with

cholinergic degeneration in 3xTg-AD mice. J Alzheimers Dis 32

(2):267–272.

Gonzalez de San Roman E, Manuel I, Giralt MT, Chun J, Estivill-

Torrus G, Rodrıguez de Fonseca F, Santın LJ, Ferrer I,

Rodrıguez-Puertas R (2015) Anatomical location of LPA1



activation and LPA phospholipid precursors in rodent and

human brain. J Neurochem 134(3):471–485.

Gonzalez-Maeso J, Torre I, Rodrıguez-Puertas R, Garcıa-Sevilla JA,

Guimon J, Meana JJ (2002) Effects of age, postmortem delay and

storage time on receptor-mediated activation of G-proteins in

human brain. Neuropsychopharmacology 26(4):468–478.

Harrison C, Traynor JR (2003) The [35S]GTPgammaS binding assay:

approaches and applications in pharmacology. Life Sci 74(4).

489-08.

Kawaguchi Y, Kubota Y (1997) GABAergic cell subtypes and their

synaptic connections in rat frontal cortex. Cereb Cortex 7

(6):476–486.

Kinney GA, Emmerson PJ, Miller RJ (1998) Galanin receptor-

mediated inhibition of glutamate release in the arcuate nucleus

of the hypothalamus. J Neurosci 18(10):3489–3500.

Llorente A, Gonzalez de San Roman E, Moreno M, Manuel I, Giralt

MT, Rodrıguez-Puertas R (2014) Activity mediated by neurolipid

(CB1 and LPA1) and neuropeptide (GAL1) receptors in a rat

model with cholinergic basal forebrain lesion. American Society

for Neuroscience Meeting. Washington 2014. USA. 307.25/D71.

Manuel I, Gonzalez de San Roman E, Giralt MT, Ferrer I, Rodrıguez-

Puertas R (2014) Type-1 cannabinoid receptor activity during

Alzheimer’s disease progression. J Alzheimers Dis 42

(3):761–766.

Marcyniuk B, Mann DM, Yates PO (1986) The topography of cell loss

from locus caeruleus in Alzheimer’s disease. J Neurol Sci 76(2–

3):335–345.

Marsicano G, Goodenough S, Monory K, Hermann H, Eder M,

Cannich A, Azad SC, Cascio MG, Ortega-Gutierrez S, Van der

Stelt M, Lopez-Rodrıguez ML, Casanova E, Schutz G,

Zieglgansberger W, Di Marzo V, Behl C, Lutz B (2003) CB1

Cannabinoid receptors and on-demand defense against

excitotoxicity. Science 302:84–88.

Martın-Moreno AM, Brera B, Spuch C, Carro E, Garcıa-Garcıa L,

Delgado M, Pozo MA, Innamorato NG, Charade A, de Ceballos

ML (2012) Prolonged oral cannabinoid administration prevents

neuroinflammation, lowers b-amyloid levels and improves

cognitive performance in Tg APP 2576 mice. J

Neuroinflammation 9:8.

Mastrangelo MA, Bowers WJ (2008) Detailed immunohistochemical

characterization of temporal and spatial progression of

Alzheimer’s disease-related pathologies in male triple-transgenic

mice. BMC Neurosci 2(9):81.

Mazzola C, Micale V, Drago F (2003) Amnesia induced by beta-

amyloid fragments is counteracted by cannabinoid CB1 receptor

blockade. Eur J Pharmacol 477(3):219–225.

Moller C, Sommer W, Thorsell A, Heilig M (1999) Anxiogenic-like

action of galanin after intra-amygdala administration in the rat.

Neuropsychopharmacology 21(4):507–512.

Mufson EJ, Deecher DC, Basile M, Izenwasse S, Mash DC (2000)

Galanin receptor plasticity within the nucleus basalis in early and

late Alzheimer’s disease: an in vitro autoradiographic analysis.

Neuropharmacology 39(8):1404–1412.

Mufson EJ, Counts SE, Perez SE, Binder L (2005) Galanin plasticity

in the cholinergic basal forebrain in Alzheimer’s disease and

transgenic mice. Neuropeptides 39(3):233–237.

Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R,

Metherate R, Mattson MP, Akbari Y, LaFerla FM (2003) Triple-

transgenic model of Alzheimer’s disease with plaques and

tangles: intracellular Abeta and synaptic dysfunction. Neuron 39

(3):409–421.

Palkovits M (2000) Stress-induced expression of co-localized

neuropeptides in hypothalamic and amygdaloid neurons. Eur J

Pharmacol 405(1–3):161–166.

Palmer AM, Francis PT, Benton JS, Sims NR, Mann DM, Neary D,

Snowden JS, Bowen DM (1987) Presynaptic serotonergic

dysfunction in patients with Alzheimer’s disease. J Neurochem

48(1):8–15.

Perez S, Basile M, Mash DC, Mufson EJ (2002) Galanin receptor

over-expression within the amygdala in early Alzheimer’s disease:



670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

707

708

709



21 May 2016

an in vitro autoradiographic analysis. J Chem Neuroanat 24

(2):109–116.

Perez SE, Dar S, Ikonomovic MD, DeKosky ST, Mufson EJ (2007)

Cholinergic forebrain degeneration in the APPswe/PS1DeltaE9

transgenic mouse. Neurobiol Dis 28(1):3–15.

Perez SE, He B, Muhammad N, Oh KJ, Fahnestock M, Ikonomovic

MD, Mufson EJ (2011) Cholinotrophic basal forebrain system

alterations in 3xTg-AD transgenic mice. Neurobiol Dis 41

(2):338–352.

Rajarao SJ, Platt B, Sukoff SJ, Lin Q, Bender CN, Nieuwenhuijsen

BW, Ring RH, Schechter LE, Rosenzweig-Lipson S, Beyer CE

(2007) Anxiolytic-like activity of the non-selective galanin receptor

agonist, galnon. Neuropeptides 41(5):307–320.

Rodrıguez-Puertas R, Pascual J, Vilaro T, Pazos A (1997a)

Autoradiographic distribution of M1, M2, M3, and M4 muscarinic

receptor subtypes in Alzheimer’s disease. Synapse 26:341–350.

Rodrıguez-Puertas R, Nilsson S, Pascual J, Pazos A, Hokfelt T

(1997b) 125I-galanin binding sites in Alzheimer’s disease:

706



increases in hippocampal subfields and a decrease in the

caudate nucleus. J Neurochem 68(3):1106–1113.

Sperling RA, Karlawish J, Johnson KA (2013) Preclinical Alzheimer’s

disease – the challenges ahead. Nat Rev Neurol 9:54–58.

Steriade M, Descarries L (2006) Cholinergic modulation of cortical

activity. In: Giacobini E, Pepeu G, editors. The brain cholinergic

system in health and disease. Abingdon, UK: Informa Healthcare.

p. 191–207.

Welsh-Bohmer KA (2008) Defining ‘‘prodromal” Alzheimer’s disease,

frontotemporal dementia, and Lewy body dementia: are we there

yet? Neuropsychol Rev 18(1):70–72.

Westlake TM, Howlett AC, Bonner TI, Matsuda LA, Herkenham M

(1994) Cannabinoid receptor binding and messenger RNA

expression in human brain: an in vitro receptor autoradiography

and in situ hybridization histochemistry study of normal aged and

Alzheimer’s brains. Neuroscience 63(3):637–652.

Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delon MR

(1982) Alzheimer’s disease and senile dementia: loss of neurons

in the basal forebrain. Science 215(4537):1237–1239.

(Accepted 10 May 2016)(Available online xxxx)



Documents

ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID … · 1 2 ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID RECEPTORS 3 IN THE PRODROMAL AND ADVANCED STAGES IN THE TRIPLE 4 TRANSGENIC