Choline acetyltransferase activity at different ... - Wiley Online Library

Journal of Neurochemistry, 2006, 97, 515–526

doi:10.1111/j.1471-4159.2006.03769.x

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down’s syndrome and related neurodegenerative diseases Andrea Contestabile,* Tatiana Fila,* Renata Bartesaghi,* Antonio Contestabile and Elisabetta Ciani* *Department of Human and General Physiology, University of Bologna, Bologna, Italy Department of Biology, University of Bologna, Bologna, Italy

Abstract Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down’s syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex,

but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer’s disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months). Keywords: Alzheimer’s disease, basal forebrain cholinergic system, choline acetyltransferase, Down’s syndrome, Ts65Dn mice. J. Neurochem. (2006) 97, 515–526.

Down’s syndrome (DS) or trisomy 21, with a prevalence of 1 : 800 live births (Hayes and Batshaw 1993), is the most frequent genetic form of mental retardation. In addition to developmental somatic alterations and mental retardation, individuals with DS develop, by the fourth decade of life, neuropathological alterations resembling those found in Alzheimer’s disease (AD) patients and they often become demented. Main neuropathological alterations include degeneration of basal forebrain cholinergic neurons (BFCN) and occurrence of plaques and neurofibrillary tangles in some brain regions (Casanova et al. 1985; Wisniewski et al. 1985; Mann and Esiri 1989; Mufson et al. 1993). The imbalance of gene dosage consequent to the trisomic condition has been proposed to be responsible for the phenotypic alterations (Korenberg et al. 1994). Some genetic strategies have been developed in order to replicate in animal models of DS the occurrence of the neuropathological and cognitive dysfunction found in human

patients. Among these models, Ts65Dn mice with segmental trisomy of chromosome 16, which is homologous to a region of human chromosome 21 critical for DS (Davisson et al. 1990), have been extensively tested during the last decade. These mice are impaired in several sensorimotor

Received August 31, 2005; revised manuscript received January 4, 2006; accepted January 4, 2006. Address correspondence and reprint requests to Dr Elisabetta Ciani, Department of Human and General Physiology, University of Bologna, Piazza di Porta San Donato 2, 40126 Bologna, Italy. E-mail: [email protected] Abbreviations used: AChE, acetylcholinesterase; AD, Alzheimer’s disease; APLP2, amyloid precursor-like protein 2; APP, amyloid precursor protein; BFCN, basal forebrain cholinergic neurons; ChAT, choline acetyltransferase; DS, Down’s syndrome; FISH, fluorescent in situ hybridization; GAD, glutamate decarboxylase; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; MCI, mild cognitive impairment; NGF, nerve growth factor.

2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 515–526

515

516 A. Contestabile et al.

performances, reproductive behavior, learning and memory (Escorihuela et al. 1995; Reeves et al. 1995; Coussons-Read and Crnic 1996; Klein et al. 1996; Demas et al. 1998; Hyde and Crnic 2001; Driscoll et al. 2004; Hampton et al. 2004; Kleschevnikov et al. 2004). When some of these deficits were tentatively correlated with neuropathological alterations of BFCN, however, somewhat contradictory results arose. In a first report, a decrease in the number of BFCN in Ts65Dn mice was reported to become significant only at advanced age (20 months) and to be accompanied by cellular atrophy (Holtzman et al. 1996). In a subsequent paper (Cooper et al. 2001), a significant decrease in number of medial septum cholinergic neurons and in their cross-sectional area was reported in Ts65Dn mice aged 12 and 18 months. The decrease in number of BFCN at 12 months of age was recently confirmed, together with an increase of the catalytic activity of choline acetyltransferase (ChAT) in the hippocampus of Ts65Dn mice (Seo and Isacson 2005). Other authors, instead, have reported a decrease in BFCN of Ts65Dn mice at 6–10 months of age (Granholm et al. 2000; Hunter et al. 2004). In this animal model, other neurodegenerative hallmarks, such as astrocytosis and microgliosis, have been noticed to develop with age to a higher extent than in normogenic mice (Holtzman et al. 1996; Hunter et al. 2004). No development of amyloid plaques or neurofibrillary tangles has been described in Ts65Dn mice, even though the expression of amyloid precursor protein (APP) was found to be increased in some brain regions (Holtzman et al. 1996; Hunter et al. 2003a). On the basis of the above-summarized results, Ts65Dn mice are currently considered a valuable tool to study not only DS but also AD, due to the similarity in cognitive deficits and impairment of BFCN characterizing the two diseases. Surprisingly, however, only a very recent study has addressed, limited to the hippocampal region, the direct evaluation of cholinergic activity in the brain of Ts65Dn mice (Seo and Isacson 2005). This kind of information appears crucial in the light of some recent data demonstrating that the specific cholinergic marker, ChAT, which is severely affected at end stages of AD, is not compromised at early stages of the disease (Davis et al. 1999) and may be, instead, up-regulated in a form of cognitive impairment considered prodromal to AD, mild cognitive impairment (MCI) (DeKosky et al. 2002). BFCN, characterized through immunostaining for ChAT and the vesicular acetylcholine transporter were preserved in individuals with diagnosis of MCI or early AD (Gilmor et al. 1999), even though the same neurons appeared decreased at equivalent disease stages when immunostained for the specific proteins acting as receptors for nerve growth factor (NGF) (Mufson et al. 2000, 2002). In another study, measurement of ChAT activity carried out in nine cortical regions revealed significant decreases only in subjects with severe dementia, but not in subjects clinically scored as mild or moderate demented

individuals (Davis et al. 1999). Furthermore, ChAT activity was not only found decreased in brain regions of individuals with severe dementia and preserved in those showing mild forms of AD but, surprisingly, significantly increased in the superior frontal cortex and hippocampus of subjects scored as affected by MCI (DeKosky et al. 2002). The interpretation of these data on BFCN stability and preserved, or even increased, ChAT activity in MCI and mild AD, should be, however, confronted with results demonstrating hypersensitivity of AD patients to cholinergic blockade with scopolamine (Sunderland et al. 1995). With the present report, we demonstrate that middle-aged (10 month old) Ts65Dn mice show up-regulated ChAT activity in the hippocampus, olfactory bulb and some cortical areas and that this up-regulation is not present any more in older (19 month old) animals. At this rather advanced age, none of the tested areas revealed any impairment of biochemically determined cholinergic activity. Compared to the progression of the disease in humans, Ts65Dn mice seem to recapitulate the situation present at the MCI stage when middle-aged and that of mild AD when getting older.

Experimental procedures Animals Female Ts65Dn mice carrying a partial trisomy of chromosome 16 (Davisson et al. 1993) were obtained from Jackson Laboratories, Bar Harbor, ME, USA and maintained on the original genetic background by mating them to C57BL/6JEi · C3SnHeSnJ (B6EiC3) F1 males. Animals where karyotyped by fluorescent in situ hybridization (FISH) on cultures of tail fibroblasts prepared at one month of age as previously described (Strovel et al. 1999). For karyotyping, the BAC clone 189N10 (a kind gift from Dr W. Song, Hanwha R & D Institute, South Korea) carrying the mouse Dyrk1a genomic region (mapping to the distal part of mouse chromosome 16) was used as probe. Biotinylated DNA probe was synthesized with Biotin-Nick Translation Mix (Roche Applied Science, Mannheim, Germany). FISH assay was carried out as previously described (Saccone et al. 1992). In total, 30 2N and 25 Ts65Dn mice, aged 10 or 19 months, were used for the present experiments. Experimental groups were composed of an approximately equal number of male and female mice, in order to balance for possible sex-related differences. Experiments were performed in accordance with the Italian and European Community law for the use of experimental animals and were approved by a local bioethical committee. Neurochemical assays and western blotting At 10 or 19 months of age, 2N and Ts65Dn mice were killed by decapitation, the brain was immediately dissected from the skull and the cerebellum and olfactory bulbs were collected. The rest of the brain was cut in transverse slices (approximately 400-lm thick) by means of a Sorvall tissue chopper and the various brain regions were microdissected under the stereomiscrope using a mouse brain atlas (Franklin and Paxinos 1997) as a topographical guide. Samples were immediately frozen in dry ice and stored in the deep freezer until used.


ChAT in a Down’s syndrome mice model 517

For neurochemical assays, tissue was homogenized in ice-cold 50 mM Tris-HCl buffer at pH 7.4 and added with Triton X-100 to a final 0.5% concentration. Aliquots were used to measure protein content (Lowry et al. 1951) and to assay the activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), glutamine synthetase (GS) and glutamate decarboxylase (GAD). For ChAT activity a radiochemical method using [14C]acetyl coenzyme A as substrate was used (Fonnum 1975). GAD activity was assayed through a radiochemical method based on the use of [1-14C]glutamate as substrate (Fonnum et al. 1977). AChE and GS were assayed by colorimetric methods (Ellman et al. 1961; Patel et al. 1983). Catalytic activity of the different enzymes was expressed per unit of protein content of the homogenates. For western blotting, crude homogenates were added with 2% sodium dodecyl sulfate, 10 mM dithiothreitol, 1% protease and phosphatase inhibitors cocktails (Sigma, St Louis, MO, USA), boiled for 10 min and cleared by centrifugation at 13 000 g for 5 min. Equivalent (30 lg) amounts of proteins per sample were subjected to electrophoresis on a 10% or, in some cases, 18% sodium dodecyl sulfate–polyacrylamide gel. The gel was then blotted onto a nitrocellulose membrane and equal loading of protein in each lane was assessed by brief staining of the blot with 0.1% Ponceau S. Blotted membranes were blocked for 1 h in 5% defatted milk in Tris-buffered saline (Tris-HCl 10 mM, NaCl 150 mM, pH 8.0) Triton X-100 (0.1%) and incubated overnight at 4C with primary antibodies. The following primary antibodies were used: anti-b-actin (rabbit polyclonal 1 : 2000, Sigma); anti-p75NTR (rabbit polyclonal 1 : 1000, Promega, Madison, WI, USA); affinity purified anti-ChAT (goat polyclonal 1 : 1000, Chemicon Int., Temecula, CA, USA); anti-glial fibrillary acidic protein (GFAP) clone GA5 (mouse monoclonal 1 : 2000, Sigma); anti-APP/amyloid precursor-like protein 2 (APLP2) clone 22C11 raised against Nterminus amino acids 66–81 of amyloid precursor protein (mouse monoclonal 1 : 1,500, Chemicon) (Slunt et al. 1994; Hunter et al. 2003a; Esh et al. 2005; Seo and Isacson 2005). Membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit, anti-mouse (Amersham, Piscataway, NJ, USA) or anti-goat (Invitrogen, Carlsbad, CA, USA) secondary antibodies (diluted 1 : 1000). Specific reactions were revealed by the ECL western blotting detection system (Amersham). Immunocytochemistry Ten- and 19-month-old 2N and Ts65Dn mice were deeply anesthetized and perfused through the heart with saline, followed by 4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4. The brains were dissected from the skull and additionally fixed overnight in the same fixative, washed and immersed overnight in 18% sucrose in phosphate buffer. Brains were cut in 40-lm thick transverse slices with a freezing microtome and serially collected in phosphate-buffered saline. The following primary antibodies were used: anti-p75NTR (rabbit polyclonal 1 : 500, Promega); anti-GFAP (mouse monoclonal 1 : 400, Sigma), affinity purified anti-ChAT (goat polyclonal 1 : 100, Chemicon). After inactivation of endogenous peroxidases, slices were blocked for 1 h in phosphatebuffered saline containing 1.5% normal serum and 0.1% Triton X-100 and incubated overnight at 4C in the same mixture containing the primary antibody. Slices were then incubated with HRP-conjugated anti-rabbit, anti-mouse or anti-goat (dilution

1 : 200, Amersham) secondary antibody for 1.5 h at room temperature. After all incubations, specimens were extensively washed with phosphate-buffered saline containing 0.1% Triton X-100. Detection was performed with DAB kit (Vector Laboratories, Burlingame, CA, USA). Bright field images where taken on a Leitz Diaplan microscope equipped with a motorized stage and a Coolsnap-Pro Color digital camera (Media Cybernetics, Silver Spring, MD, USA). p75NTR and ChAT positive cells in the medial septum (MS) and in the vertical limb of the diagonal band (VLDB) were separately counted in a series of 40 lm-thick sections covering the complete area from Bregma 1.54 mm to Bregma 0.14 mm (referring to Franklin and Paxinos 1997). For double immunofluorescence histochemistry, the anti-ChAT and anti-p75NTR primary antibodies were used, together with anti-rabbit FITC-conjugated (dilution 1 : 200, Vector) or anti-goat TRITC-conjugated (dilution 1 : 200, Santa Cruz) secondary antibodies. Fluorescent confocal images were taken on a Leica TCS confocal microscope (Leica Microsystems, Wetzlar, Germany). Statistics The results are expressed as mean ± SE of the number of experiments indicated in the figure and table legends. The data were analyzed by ANOVA followed by post hoc comparison through Bonferroni’s test. The differences between means were considered statistically significant when p < 0.05.

Results

Choline acetyltransferase activity in brain of Ts65Dn mice at different ages Assay of ChAT activity in forebrain areas receiving extensive cholinergic inputs from the medial septal area (Mesulam et al. 1983; Woolf 1991), revealed a significantly higher enzyme catalytic activity in Ts65Dn mice at 10 months of age compared to 2N animals, a difference that was not present any more at 19 months of age (Fig. 1). This was true concerning the hippocampus (Fig. 1a), the pre-frontal cortex (Fig. 1b), the olfactory cortex (Fig. 1c) and the olfactory bulb (Fig. 1d). Other cortical areas, such as the frontoparietal and the occipital cortices, receiving their main cholinergic input from more posterior areas of the basal forebrain cholinergic complex, the horizontal diagonal band and the nucleus basalis magnocellularis (Mesulam et al. 1983; Woolf 1991), did not show any relevant difference in ChAT activity between 2N and Ts65Dn mice at any age (Figs 1e and f). In the cerebellum, ChAT activity was significantly higher in Ts65Dn mice than in 2N animals at both 10 and 19 months of age (Fig. 2a). In the striatum, a significant increase of ChAT activity was only measured in Ts65Dn mice aged 19 months (Fig. 2b). Possible differences in the kinetic parameters of ChAT between Ts65Dn and 2N mice might, theoretically, affect the measure of the enzyme catalytic activity. We therefore determined in cortical samples obtained from the two type of mice the affinity of the enzyme



Fig. 1 Choline acetyltransferase activity in forebrain regions of 2N and Ts65Dn mice at 10 or 19 months of age. Bars are the mean ± SE of seven to 10 animals. *p < 0.05 compared to 2N animals of the same age; Bonferroni’s test after ANOVA.

for the rate-limiting substrate acetyl-CoA. By increasing substrate concentration in the assay medium from 2 to 200 lM, we measured a similar value of Km (around 40 lM) in both these samples. Quantitative evaluation by western blots of p75NTR and ChAT protein content in whole homogenates of some of these regions did not reveal significant differences between Ts65Dn and 2N mice at both 10 and 19 months of age, even if a tendency towards increased levels in the hippocampus of 10-month-old Ts65Dn mice was noticed (Fig. 3). The absence of significant differences in ChAT content evaluated through western blotting indicates that a more sensitive method, such as the radioenzymatic assay, is needed to detect differences related to cholinergic innervation in this model.

Basal forebrain cholinergic neurons in Ts65Dn mice at different ages Somewhat contradictory results have been recently published concerning alterations in the number of BFCN at various ages in Ts65Dn mice, compared to 2N littermates (Granholm et al. 2000; Cooper et al. 2001; Hunter et al. 2004). As the presence of the low affinity NGF receptor protein, p75NTR, specifically marks BFCN (Koh and Loy 1989; Cooper et al. 2001), we counted in p75NTR immunostained sections the cholinergic neurons present in the medial septum of Ts65Dn and 2N mice at the same ages used for neurochemical and western blot experiments. Our results, similar to those recently reported by others (Holtzman et al. 1996; Cooper et al. 2001), revealed no significant differences in total



this, we counted ChAT immunoreactive neurons in series of sections taken from 19-month-old 2N and Ts65Dn mice and we exactly replicated the result obtained for p75NTR, with ChAT-labeled neurons in the medial septum closely matching those labeled for p75NTR (compare Figs 4a and 4i). In agreement with this result, ChAT protein content, assayed through western blot at 19 months of age, was higher in medial septum samples from 2N, compared to Ts65Dn mice (Fig. 4i). Acetylcholinesterase activity in the brain of Ts65Dn mice at different ages In the same samples of Ts65Dn and 2N mice used for the determination of ChAT activity, we also measured the activity of the acetylcholine-degrading enzyme, AChE (Fig. 5). A significant difference was detected in the olfactory cortex, where AChE activity was higher in Ts65Dn mice at 10 months of age, and in the striatum, where the same situation was present at 19 months of age (Fig. 5). Furthermore, in the pre-frontal, fronto-parietal and olfactory cortex, AChE activity showed a trend towards decrease between 10 and 19 months of age in 2N mice and, in the last two regions, also in Ts65Dn mice (Fig. 5). To assess possible differences related to another widespread neurotransmitter system, namely the GABAergic one, we also measured in some brain areas (cortex, hippocampus, striatum, cerebellum) of both mice types the activity of the GABA synthetic enzyme, GAD. No significant differences were detected between Ts65Dn and 2N mice at both 10 and 19 months of age (data not shown).

Fig. 2 Choline acetyltransferase activity in the cerebellum and striatum of 2N and Ts65Dn mice at 10 and 19 months of age. Bars are the mean ± SE of seven to 10 animals. *p < 0.05, **p < 0.01 compared to 2N mice of the same age; Bonferroni’s test after ANOVA.

number of labeled neurons in the medial septal nucleus at 10 months of age and a significant difference in older animals, due to an increased number of p75NTR positive neurons in 2N mice (Fig. 4a). No significant differences were detected at both the examined ages in the number of positive p75NTR neurons in the more ventrally located vertical limb of the diagonal band of Broca (VLDB) (Fig. 4b), in line with similar evidence recently reported for ChAT-immunoreactive neurons (Hunter et al. 2004). Confocal microscopy observations showed that, with no apparent exception, every neuron labeled for p75NTR in these areas was also ChAT positive in both 2N and Ts65Dn mice (Figs 4c–h). This clearly suggested that the well-documented identity between p75NTR and ChAT containing neurons in the medial septum/diagonal band region (Koh and Loy 1989), also held true concerning the mice used in the present study. To quantitatively confirm

Amyloid precursor protein/amyloid precursor-like protein 2 expression The gene coding for APP is among those present in trisomic dosage in Ts65Dn mice and its expression at the mRNA level has been found significantly increased in the brain of Ts65Dn mice (Holtzman et al. 1996). It may therefore be expected that the APP protein content is higher in brain regions of these mice compared to 2N animals. By using an antibody against APP (mAb 22C11), we observed an increased expression of the protein in the hippocampus of Ts65Dn mice at both 10 and 19 months of age, whereas in the frontoparietal cortex the difference with respect to 2N mice only attained statistical significance in animals aged 19 months (Fig. 6). It should be noted, however, that the used antibody also recognizes the APP-like protein 2, APLP2 (Slunt et al. 1994). Thus, we cannot rule out that densitometric quantification includes both APP and APLP2. Astrocytic markers Several neurodegenerative diseases, including DS and AD, are characterized by the occurrence of gliosis in some brain areas. By western blotting, we observed a significantly increased expression of the glial marker, GFAP, in the



Fig. 3 Quantification through western blot analysis of p75 or ChAT protein content in the hippocampus and the fronto-parietal cortex of 2N and Ts65Dn mice at 10 or 19 months of age. Bars are the mean ± SE of five animals.

hippocampus and cortex of Ts65Dn mice at 19 months of age (Fig. 7a). This was confirmed through immunocytochemistry, which revealed an increased staining (Figs 7b and c) and a clear hypertrophy of GFAP-positive cells (Figs 7d and e) in the hippocampus of Ts65Dn mice. To further evaluate the gliotic process, we measured the catalytic activity of GS, a marker for astrocytic metabolic activation and hypertrophy (Fonnum 1985). In both the fronto-parietal cortex and the hippocampus, GS activity was significantly higher at 19 months of age, but not at 10 months of age, in Ts65Dn mice, compared to 2N animals (Figs 7f and g). No significant differences were, instead, found concerning GS activity in other regions, such as the cerebellum, the prefrontal cortex and the olfactory cortex (data not shown). Discussion

We report here new data on the cholinergic systems in several brain regions of Ts65Dn mice, a proposed animal model for DS and related neurodegenerative diseases, such as AD. The essential novel information provided by the present report is that the specific cholinergic marker, ChAT, is up-regulated in some regions, in middle-aged (10-monthold) Ts65Dn mice, in agreement with what was previously reported for the hippocampus of 12-month-old mice (Seo and Isacson 2005), and that no significant decrease of activity occurs in target regions of BFCN in older (19-month-old) animals. These results suggest that the alterations of the forebrain cholinergic system occurring in Ts65Dn mice, up to ages close to their maximum lifespan,

are not representative of the profound cholinergic deficit characterizing advanced forms of DS and AD. Rather, the evolution of the cholinergic parameters in Ts65Dn mice described in the present report is reminiscent of similar alterations reported in cases of MCI and mild AD (Davis et al. 1999; DeKosky et al. 2002; Mufson et al. 2003). In 10-month-old Ts65Dn mice, indeed, ChAT activity increased in some target areas of BFCN, as described in human patients with MCI diagnosis (DeKosky et al. 2002). On the other hand, the situation found in mice aged 19 months, in which no significant differences of ChAT catalytic activity were found in the hippocampus and in the cortical target areas, is similar to the assessment of patients with mild or moderate clinical dementia rating (CDR) scores of AD (Davis et al. 1999; DeKosky et al. 2002; Mufson et al. 2003). In humans, the finding that ChAT activity is up-regulated in some brain areas in MCI cases, has been interpreted as a plastic response through which cholinergic sprouting tends to restore innervations of synaptic sites vacated by the loss of other synaptic inputs (DeKosky et al. 2002). This may also be the case for Ts65Dn mice, on the basis of the present results as well as of those recently demonstrating increased ChAT activity in the hippocampus of 12-month-old Ts65Dn mice (Seo and Isacson 2005). Such a spontaneous plastic response may be relevant for perspective medical treatments, as the transition from MCI to AD offers a temporal window that may be used to target degenerative alterations and to slow down their course (DeKosky et al. 2002). Our finding that Ts65Dn mice recapitulate some relevant aspects of this



(a)

Fig. 4 Total number of p75-positive cells in the medial septum (a) and vertical limb of the diagonal band of Broca (b) in 2N and Ts65Dn mice at 10 or 19 months of age. Bars are the mean ± SE of five animals; *p < 0.05 compared to 2N mice of 19 months, #p < 0.05 compared to 2N animals of 10 months, Bonferroni’s test after ANOVA. (c–h) Confocal microscopic demonstration through double immunocytochemistry of the coincidence of medial septal neurons expressing p75 (green) and choline acetyltransferase (ChAT, red) in 2N (c–e) and Ts65Dn (f–h) mice; (e) and (h) are the merge of (c), (d) and (f), (g), respectively. Calibration bar 150 lm. (i) Left panel: total number of ChAT-positive cells in the medial septum of 2N and Ts65Dn mice at 19 months of age; bars are the mean ± SE of four animals, *p < 0.05 compared to 2N animals, Bonferroni’s test after ANOVA. Right panel: western blot analysis of ChAT protein expression, relative to b-actin content in homogenate of the medial septal region from a 2N or a Ts65Dn animal.

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

transition makes this animal model potentially useful to test this opportunity at the pre-clinical level. The present report provides for the first time evidence that in the brain of Ts65Dn mice there are also alterations in the regional activity of the acetylcholine degrading enzyme, AChE. In some regions (for instance the olfactory cortex and the striatum) the observed alterations of AChE activity were in the same direction of changes measured for ChAT, whereas in other regions (for instance the hippocampus) positive correlation was lacking. This apparent discrepancy is easily explained by the fact that ChAT is a specific cholinergic marker, whereas AChE is also present in cholinoceptive neurons and may therefore mask alterations occurring in cholinergic neurons and terminals. Behavioural studies are needed to ascertain whether some specific alterations in learning and memory performances in

Ts65Dn mice are associated with the age-related alterations in cholinergic markers described here. In 2N mice, neurons of the basal forebrain phenotypically characterized by p75NTR immunoreactivity increase in number between 10 and 19 months of age, an increase not matched by Ts65Dn mice. However, in Ts65Dn mice this phenotypical increase can be restored by intraventricular injection of NGF, a response clearly suggesting the cholinergic nature of these neurons (Cooper et al. 2001). Our parallel cell counting in sections stained for p75NTR or ChAT antibodies, together with observations on co-localization at the confocal microscope, makes us confident that all p75NTR positive neurons in the basal forebrain are bona fide, cholinergic neurons. Interestingly, a differential phenotypic expression of various markers for basal forebrain cholinergic neurons (p75NTR, TrkA, ChAT content, vesicular acetylcholine transporter) has



Fig. 5 Acetylcholinesterase activity in various brain regions of 10- and 19-month-old 2N and Ts65Dn mice. Data are the mean ± SE of 7–10 animals. #p < 0.05, ##p < 0.01 with respect to 2N mice of the same age; *p < 0.05, **p < 0.01 with respect to 2N mice of 10 months of age; oop < 0.01 with respect to Ts65Dn mice of 10 months of age; Bonferroni’s test after ANOVA.

been described in human patients at prodromal and early stages of AD (Gilmor et al. 1999; Mufson et al. 2000, 2002, 2003). The present study not only confirms that the BFCN of Ts65Dn mice are preserved up to 10 months of age and only decrease at later stages, as previously shown (Holtzman et al. 1996; Cooper et al. 2001), but additionally demonstrates that, similar to what has been described in MCI and mild AD cases (Davis et al. 1999; DeKosky et al. 2002), ChAT activity is also preserved, and transitorily up-regulated, in some brain regions of Ts65Dn mice. Interestingly, the cognitive impairment is evident in these animals by the sixth month of age (Holtzman et al. 1996; Moran et al. 2002; Hunter et al. 2003b; Seo and Isacson 2005). Therefore, in Ts65Dn mice a clear temporal gap exists between the early appearance of cognitive deficit and the later impairment of BFCN, similar to what has been described in human patients (Davis et al. 1999; DeKosky et al. 2002; Ikonomovic et al. 2003; Mufson et al. 2003). A functional interpretation of these findings in terms of the actual involvement of cholinergic deficits in the cognitive decline characterizing both human patients and segmentally trisomic mice, must take into account the fact that single photon emission computed tomography has revealed hypersensitivity of muscarinic receptors to scopolamine blockade in mild AD patients (Sunderland et al. 1995). In this respect, it could be important to verify whether such hypersensitivity is also present in MCI cases, as well as to find other neural systems whose derangement could be related to the cognitive deficit. In humans, the early degeneration of specific neurons of the entorhinal cortex in cases of MCI and mild AD, with the consequent alteration of the projection to the hippocampus, appears a good candidate for the initial phases of cognitive impairment (Gomez-Isla et al. 1996; Kordower et al. 2001; DeKosky et al. 2002; Ikonomovic et al. 2003). Whether an equivalent degenerative process also contributes to the early cognitive impairment of Ts65Dn mice, should be the matter of future investigation. The recently described impairment of long-term potentiation in the dentate gyrus of Ts65Dn mice (Kleschevnikov et al. 2004), a region receiving glutamatergic input from the entorhinal cortex, suggests that this connection may be actually altered in this model. A further element that may, theoretically, be involved in cognitive deficits observed in relatively young Ts65Dn mice is the altered number of neurons and the altered synaptic density detected in some hippocampal regions (Insausti et al. 1998; Kurt et al. 2004). A detailed morphometric study, recently performed in cortical and hippocampal regions, has revealed several abnormalities involving both pre- and postsynaptic structures in Ts65Dn, compared to 2N mice (Belichenko et al. 2004). Interestingly some of these structural alterations, in particular those involving an increased size of presynaptic terminals in some synaptic fields, are more evident at middle age than in older animals (Belichenko



(a)

(b)

Fig. 6 Quantification (left panels) and examples of western blots (right panels) for amyloid precursor protein/amyloid precursor-like protein 2 (APP/APLP2), from the hippocampus (a) and the fronto-parietal cortex (b) of 2N and Ts65Dn mice at 10 or 19 months of age. Bars are the mean ± SE of 3 (10 months) or 4 (19 months) animals; *p < 0.05, **p < 0.01 compared to 2N mice of the same age; Bonferroni’s test after ANOVA.

et al. 2004). Thus, the transitory increase of ChAT activity found in the present study in some brain regions of Ts65Dn mice may be related to a transitory hypertrophy of cholinergic terminals in these regions. At least concerning the hippocampus, an increased cholinergic innervation in Ts65Dn mice seems to be present both before (6 month of age, Cooper et al. 2001 and 10 months of age, present results) and shortly after (12 months of age, Seo and Isacson 2005), the beginning of the decrease in number of BFCN. The present report provides a further novel observation concerning the increased ChAT activity in the cerebellum of Ts65Dn mice. This is very likely due to the fact that the cerebellum of Ts65Dn mice, similar to the cerebellum of Down’s syndrome individuals, is underdeveloped and that this hypotrophic condition is due to a reduced number and density of the most abundant neuronal cerebellar population, the granule cells (Aylward et al. 1997; Baxter et al. 2000). Previous researches in rodents reported that experimental conditions leading to altered cerebellar development and decreased granule cell population resulted in increased cholinergic innervation due to the smaller terminal field served by the exogenously derived cerebellar cholinergic system (Slevin et al. 1982; Tregnago et al. 1998). We have also observed an increased ChAT activity in the striatum of 19-month-old Ts65Dn mice. As the cholinergic activity in

this region depends entirely on the presence of a widespread population of cholinergic interneurons, this suggests the occurrence of age-dependent differences, presently unknown in nature, involving these neurons in Ts65Dn mice. The trisomic condition of the gene for the amyloid precursor protein (APP) is considered to be related to the development of AD-type neuropathology in Down’s syndrome individuals (Neve et al. 2000). Pioneer studies in Ts65Dn mice (Holtzman et al. 1996) demonstrated an approximate doubling of the expression of this gene at the mRNA level in the forebrain of young-adult animals (4 months). At the protein level, an increase in APP expression was seen in the striatum of Ts65Dn mice aged over 6 months and in the hippocampus and cortex of mice aged at least 12 months (Hunter et al. 2003a; Seo and Isacson 2005). The antibody used by these authors, as well as by us in the present study, also recognizes another member of the family to which APP belongs, the APLP2, for which a role in neurogenesis and axonogenesis has been recently postulated (Slunt et al. 1994; Cappai et al. 1999; Caille et al. 2004). On the basis of the increased expression of App mRNA (Holtzman et al. 1996), it seems likely that the increase in the detected protein reflects APP. This assumption could be, however, only hypothetical in view of the topographically and temporally restricted expression of the



(a)

(f)

(b)

(c)

(d)

(e)

(g)

immunoreactive protein detected through western blotting (Hunter et al. 2003a; Seo and Isacson 2005; and present results). Glial abnormalities, have been described in the brain of Down’s syndrome individuals (Griffin et al. 1989; Schubert et al. 2001). Our present observations add some new information concerning astrocytes at an advanced age in this animal model. A well-known marker of astrocyte metabolic hyperactivity, the glutamate converting enzyme GS (Fonnum 1985), was significantly increased in the hippocampus and fronto-parietal cortex of Ts65Dn mice at

Fig. 7 (a) Representative western blots and quantification of glial fibrillary acidic protein (GFAP) content in the fronto-parietal cortex and the hippocampus of 2N and Ts65Dn mice at 19 months of age. Bars are the mean ± SE of four animals; *p < 0.05, Bonferroni’s test after ANOVA. Visualization of gliosis trough GFAP immunohistochemistry in the hippocampus of 2N (b, d) and Ts65Dn (c, e) mice at 19 months of age; calibration bars 500 lm (b, c) or 50 lm (d, e). (f, g) Glutamine synthetase activity in the fronto-parietal cortex (f) and the hippocampus (b) of 2N and Ts65Dn mice at 10 or 19 months of age. Bars are the mean ± SE of 7–10 animals; *p < 0.05, Bonferroni’s test after ANOVA.

19, but not at 10 months of age. This neurochemical result very well correlated with a parallel increase of GFAP immunoreactivity in the same regions, indicating a topographically restricted hypertrophic condition of astrocytes in aged Ts65Dn mice. In conclusion, the present work reports new data on the level of ChAT activity in the brain of Ts65Dn mice at different ages. This novel information is essential in order to more precisely assess the relevance of this animal model in relation to the corresponding human pathologies, particularly in the light of recent findings on alterations of cholinergic



markers characterizing the transition from MCI to mild and advanced AD. Other data reported here, also significantly contribute to a better understanding of the neurochemical alterations occurring in this animal model. Acknowledgements The present work was funded by a grant for basic research from the Italian Ministry for Universities and Research (FIRB grant RBAU01BS5L) to EC. The skilful technical assistance of Miss Monia Bentivogli for neurochemical assays and Miss Lucia Di Pietrangelo for confocal microscopy observations is gratefully acknowledged. The authors are very grateful to Dr W. Song for providing the BAC clone 189N10 probe and to Dr S. Saccone for expert advice and assistance with the FISH technique.

References Aylward E. H., Habbak R., Warren A. C., Pulsifer M. B., Barta P. E., Jerram M. and Pearlson G. D. (1997) Cerebellar volume in adults with Down syndrome. Arch. Neurol. 54, 209–212. Baxter L. L., Moran T. H., Richtmeister J. T., Troncoso J. and Reeves R. H. (2000) Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse. Hum. Mol. Gen. 9, 195–202. Belichenko P. V., Masliah E., Kleschevnikov A. M., Villar A. J., Epstein C. J., Salehi A. and Mobley W. C. (2004) Synaptic structural abnormalities in the Ts65Dn mouse model of Down syndrome. J. Comp. Neurol. 480, 281–298. Caille I., Allinquant B., Dupont E., Bouillot C., Langer A., Muller U. and Prochiantz A. (2004) Solubile form of amyloid precursore protein regulates proliferation of progenitors in the adult subventricular zone. Development 131, 2173–2181. Cappai R., Mok S. S., Galatis D., Tucker D. F., Henry A., Beyreuther K. and Small D. H. and Masters C. L. (1999) Recombinant human amyloid precursor-like protein 2 (APLP2) expressed in the yeast Picchia pastoris can stimulate neurite outgrowth. FEBS Lett. 442, 95–98. Casanova M. F., Walker L. C., Whitehous P. J. and Price D. L. (1985) Abnormalities of the nucleus basalis in Down’s syndrome. Ann. Neurol. 18, 310–313. Cooper J. D., Salehi A., Delcroix J.-D., Howe C. L., Belichenko P. V., Chua-Couzens J., Kilbridge J. F., Carlson E. J., Epstein C. J. and Mobley W. C. (2001) Failed retrograde transport of NGF in a mouse model of Down’s syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion. Proc. Natl Acad. Sci. USA 98, 10 439–10 444. Coussons-Read M. E. and Crnic L. S. (1996) Behavioral assessment of the Ts65Dn mouse, a model for Down syndrome: altered behavior in the elevated plus maze and open field. Behav. Genet. 26, 7–13. Davis K. M., Mohs R. C., Marin D., Purhoit D. P., Perl D. P., Lantz M., Austin G. and Haroutunian V. (1999) Cholinergic markers in elderly patients with early signs of Alzheimer’s disease. JAMA 281, 1401–1406. Davisson M. T., Schmidt C. and Akeson E. C. (1990) Segmental trisomy of murine chromosome 16: a new model for studying Down syndrome. Prog. Clin. Biol. Res. 360, 263–280. Davisson M. T., Schmidt C., Reeves R. H., Irving N. G., Akeson E. C., Harris B. S. and Bronson R. T. (1993) Segmental trisomy as a mouse model for Down syndrome. Prog. Clin. Biol. Res. 384, 117– 133.

DeKosky S. T., Ikonomovic M. D., Styren S. D., Beckett L., Wisniewski S., Bennet D. A., Cochran E. J., Kordower J. H. and Mufson E. J. (2002) Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment. Ann. Neurol. 51, 145–155. Demas G. E., Nelson R. J., Kreuger B. K. and Yarowsky P. J. (1998) Impaired spatial working and reference memory in segmental trisomy (Ts65Dn) mice. Behav. Brain Res. 90, 199–201. Driscoll L. L., Carrol J. C., Moon J., Crnic L. S., Levitsky D. A. and Strupp B. J. (2004) Impaired sustained attention and error-induced stereotipy in the aged Ts65Dn mouse: a mouse model of Down syndrome and Alzheimer’s disease. Behav. Neurosci. 118, 1196– 1205. Ellman G. L., Courtney K. D., Andres V. Jr and Featherstone V. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Escorihuela R. M., Fernandez-Teruel A., Vallina I. F., Baamonde C., Lumberas M. A., Dierssen M., Tobena A. and Florez J. (1995) A behavioral assessment of Ts65Dn mice: a putative Down syndrome model. Neurosci. Lett. 247, 171–174. Esh C., Patton L., Kalback W. et al. (2005) Altered APP processing in PDAPP (Val717 fi Phe) transgenic mice yields extended-length Abeta peptides. Biochemistry 44, 13 807–13 819. Fonnum F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 25, 407–409. Fonnum F. (1985) Determination of transmitter amino acid turnover, in Neuromethods, Vol. 3, Amino Acids (Boulton, A. A., Backer, G. B. and Wood, J. D., eds.), pp. 201–237. Humana Press, Clifton, NJ. Fonnum F., Walaas I. and Iversen E. (1977) Localization of GABAergic, cholinergic and aminergic structures in the mesolimbic system. J. Neurochem. 29, 221–230. Franklin K. B. J. and Paxinos G. (1997) The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York. Gilmor M. L., Erickson J. D., Varoqui H., Hersh L. B., Bennet D. A., Cochran E. J., Mufson E. J. and Levey A. I. (1999) Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer’s disease. J. Comp. Neurol. 411, 693–704. Gomez-Isla T., Price J. L., McKeel D. W. Jr, Morris J. C., Growdon J. H. and Hyman B. T. (1996) Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer’s disease. J. Neurosci. 16, 4491–4500. Granholm A.-C., Sanders L. A. and Crnic L. S. (2000) Loss of cholinergic phenotype in basal forebrain coincides with cognitive decline in a mouse model of Down’s syndrome. Exp. Neurol. 161, 647–663. Griffin W. S., Stanley L. C., Ling C., White L., MacLeod V., Perrot L. J., Withe C. L. and Araoaz C. (1989) Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl Acad. Sci. USA 86, 7611–7615. Hampton T. G., Stasko M. R., Kale A., Amende J. and Costa A. C. (2004) Gait dynamics in trisomic mice: quantitative neurological traits of Down syndrome. Physiol. Behav. 82, 381–389. Hayes A. and Batshaw M. L. (1993) Down syndrome. Pediatr. Clin. North. Am. 40, 523–529. Holtzman D. M., Santucci D., Kilbridge J. et al. (1996) Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome. Proc. Natl Acad. Sci. USA 93, 13 333–13 338. Hunter C. L., Isacson O., Nelson M., Bimonte-Nelson H., Seo H., Lin L., Ford K., Kindy M. S. and Granholm A.-C. (2003a) Regional alterations in amyloid precursor protein and nerve growth factor across age in a mouse modl of Down’s syndrome. Neurosci. Res. 45, 437–445.



Hunter C. L., Bimonte H. A. and Granholm A.-C. (2003b) Behavioral comparison of 4 and 6 month-old Ts65Dn mice: age-related impairments in working and reference memory. Behav. Brain Res. 138, 121–131. Hunter C. L., Bachman D. and Granholm A.-C. (2004) Minocycline prevents cholinergic loss in a mouse model of Down’s syndrome. Ann. Neurol. 56, 675–688. Hyde L. A. and Crnic L. S. (2001) Age-related deficits in context discrimination learning in Ts65Dn mice that model Down syndrome and Alzheimer’s disease. Behav. Neurosci. 115, 1239–1246. Ikonomovic M. D., Mufson E. J., Wuu J., Cochran E. J., Bennet D. A. and DeKoski S. T. (2003) Cholinergic plasticity in hippocampus of individuals with mild cognitive impairment: correlation with Alzheimer’s neuropathology. J. Alzheimer Dis. 5, 39–48. Insausti A. M., Megias M., Crespo D., Cruz-Orive L. M., Vallina T. F., Insausti R. and Florez J. (1998) Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome. Neurosci. Lett. 253, 175–178. Klein S. L., Kriegsfield L. J., Hairston J. E., Rau V., Nelson R. J. and Yarowsky P. J. (1996) Characterization of sensorimotor performance, reproductive and aggressive behaviors in segmental trisomic 16 mice. Physiol. Behav. 60, 1159–1164. Kleschevnikov A. M., Belichenko P. V., Villar A. J., Epstein C. J., Malenka R. C. and Mobley W. C. (2004) Hippocampal long-term potentiation suppressed by increased inhibition in the Ts65Dn mouse, a genetic model of Down syndrome. J. Neurosci. 24, 8153– 8160. Koh S. and Loy R. (1989) Localization and development of nerve growth factor-sensitive rat basal forebrain neurons and their afferent projections to hippocampus and neocortex. J. Neurosci. 9, 2999–3018. Kordower J. H., Chu Y., Stebbins J. T., DeKosky S. T., Cochran E. J., Bennet D. A. and Mufson E. J. (2001) Loss and atrophy of layer II entorhinal cortex neurons in elderly people with mild cognitive impairmant. Ann. Neurol. 49, 202–213. Korenberg J. R., Chen X. N., Schipper R., Sun Z., Gonsky R., Gerwehr S., Carpenter N., Daumer C., Dignan P. and Disteche C. (1994) Down syndrome phenotypes: the consequences of chromosomal imbalance. Proc. Natl Acad. Sci. USA 91, 4997–4501. Kurt M. A., Kafa M. I., Dierssen M. and Davies D. C. (2004) Deficits of neuronal density in CA1 and synaptic density in the dentate gyrus, CA3 and CA1, in a mouse model of Down syndrome. Brain Res. 1022, 101–109. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mann D. M. and Esiri M. M. (1989) The pattern of acquisition of plaques and tangles in the brain of patients under 50 years of agewith Down’s syndrome. J. Neurol. Sci. 89, 169–179. Mesulam M. M., Mufson E. J., Wainer B. H. and Levey A. I. (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature. Neuroscience 10, 1185–1201. Moran T. H., Capone G. T., Knipp S., Davisson M. T., Reeves M. H. and Gearhart J. D. (2002) The effects of piracetam on cognitive performance in a mouse model of Down’s syndrome. Physiol. Behav. 77, 103–109. Mufson E. J., Cochran E., Benzing W. and Kordower J. H. (1993) Galaninergic innervation of the cholinergic vertical limb of the diagonal band (Ch2) and bed nucleus of the stria terminalis in

aging, Alzheimer’s disease and Down’s syndrome. Dementia 4, 237–250. Mufson E. J., Ma S. Y., Cochran E. J., Bennet D. A., Beckett L. A., Jaffar S., Saragovi H. U. and Kordower J. H. (2000) Loss of nucleus basalis neurons containing trkA immunoreactivity in individuals with mild cognitive imapirment and early Alzheimer’s disease. J. Comp. Neurol. 427, 19–30. Mufson E. J., Ma S. Y., Dills J. et al. (2002) Loss of basal forebrain P75 (NTR) immunoreactivity in subjects with mild cognitive impairment and Alzheimer’s disease. J. Comp. Neurol. 443, 136–153. Mufson E. J., Ginsberg S. D., Ikonomovic M. D. and DeKosky S. T. (2003) Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction. J. Chem. Neuroanat. 26, 233–242. Neve R. L., McPhie D. L. and Chen Y. (2000) Alzheimer’s disease: a dysfunction of the amyloid precursor protein. Brain Res. 886, 54– 66. Patel A. J., Hunt A. and Tahourdin C. S. M. (1983) Regional development of glutamine synthetase activity in rat brain and its association with the differentiation of astrocytes. Dev. Brain Res. 8, 31– 37. Reeves R. H., Irving N. J., Moran T. H., Wohn A., Kitt C., Sisodia S. S., Schmidt C., Bronson R. T. and Davisson M. T. (1995) A mouse model for Down syndrome exhibits learning and behavior deficits. Nat. Gen. 11, 177–184. Saccone S., De Sario A., Della Valle G. and Bernardi G. (1992) The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc. Natl Acad. Sci. USA 89, 4913–4917. Schubert P., Ogata T., Marchini C. and Ferroni S. (2001) Glia-related mechanisms in AD: a therapeutic target? Mech. Ageing Dev. 123, 47–57. Seo H. and Isacson O. (2005) Abnormal APP, cholinergic and cognitive function in Ts65Dn Down’s model mice. Exp. Neurol. 193, 469– 480. Slevin J. T., Johnston M. V., Biziere K. and Coyle J. T. (1982) Methylazoxymethanol acetate ablation of mouse cerebellar granule cells: effects on synaptic neurochemistry. Dev. Neurosci. 5, 3–12. Slunt H. H., Thinakaran G., Von Koch C., Lo A. C., Tanzi R. E. and Sisodia S. S. (1994) Expression of a ubiquitous, cross-reactivity homologue of the mouse beta-amyloid precursor protein (APP). J. Biol. Chem. 269, 2637–2644. Strovel J., Stamberg J. and Yarowsky P. J. (1999) Interphase FISH for rapid identification of a Down syndrome animal model. Cytogenet. Cell Genet. 86, 285–287. Sunderland T., Esposito G., Molchan S. E., Coppola R., Jones D. W., Gorey J., Little J. T., Bahro M. and Weinberger D. R. (1995) Differential cholinergic regulation in Alzheimer’s patients compared to controls following chronic blockade with scopolamine: a SPECT study. Psychopharmacology 121, 231–241. Tregnago M., Virgili M., Monti B., Guarnieri T. and Contestabile A. (1998) Alteration of neuronal nitric oxide synthase activity and expression in the cerebellum and the forebrain of microencephalic rats. Brain Res. 793, 54–60. Wisniewski K. E., Wisniewski H. M. and Wen G. Y. (1985) Occurrence of neuropathological changes and dementia of Alzheimer’s disease in Down’s syndrome. Ann. Neurol. 17, 278–282. Woolf N. J. (1991) Cholinergic systems in mammalian brain and spinal cord. Prog. Neurobiol. 37, 475–524.
