Research reportLong-term effects of selective immunolesions of cholinergic neurons of the nucleus basalis magnocellularis on the ascending cholinergic pathways in the rat: A model for Alzheimer's disease
Highlights
► 192 IgG-saporin was injected to the nBM to elicit cholinergic hypofunction in the cortex. ► The immunotoxin differentially affected ChAT- and AChE-positive neurons in the nBM. ► It reduced the number of AChE-positive fibers of the ascending cholinergic pathway. ► Fibers to the primary motor cortex and the somatosensory cortex were similarly affected.
Introduction
The cholinergic basal forebrain, one of the diffusely projecting systems of the brain, comprising of the cholinergic neurons in the medial septum, the vertical and horizontal limbs of the diagonal band of Broca and the nucleus basalis magnocellularis (nBM), provides widespread innervation to the neocortex (Baxter and Chiba, 1999, Fitz et al., 2008, Kasa, 1986, Kasa et al., 1997, Kilgard and Merzenich, 1998, Wrenn and Wiley, 1998). The nBM receives inputs from limbic and paralimbic structures and sends projections to the entire cortex (Mesulam et al., 1983). Degeneration of the nBM gives rise to a number of profound morphological, biochemical and functional effects directly related to the development of Alzheimer's disease (AD). The involvement of the cholinergic system in AD has been documented extensively (Cuello et al., 2010, Kasa et al., 1997, Mufson et al., 2008). The observation of the loss of cholinergic neurons in the nBM, associated with a decreased level of cortical cholinergic innervation and leading to the symptoms characteristic of AD, has stimulated the development of animal models involving a variety of techniques for lesioning of the nBM (Berger-Sweeney et al., 1994, Berger-Sweeney et al., 2001, Mallet et al., 1995, Mohapel et al., 2005, Wenk et al., 1994).
The introduction of the use of 192 IgG-saporin, a highly specific cholinergic immunotoxin, improved the specificity of these studies (Book et al., 1994, Fitz et al., 2008, Wiley et al., 1991). This immunotoxin consists of the monoclonal antibody 192 IgG, disulfide-coupled to saporin, a member of the ribosome-inactivating protein family derived from the plant Saponaria officinalis. The antibody component is directed against rat p75, a low-affinity neurotrophin receptor protein, which ensures the specificity of the toxin since only the cholinergic neurons express p75 in the nBM (Cuello et al., 1990). Following receptor binding and internalization, saporin enzymatically inactivates the large ribosomal subunit, thereby blocking protein synthesis and ultimately resulting in cell death; the neurodegenerative process can be considered complete in about 2 weeks (Wrenn and Wiley, 1998). Its specificity makes 192 IgG-saporin a useful agent with which to establish specific cholinergic lesions modeling the AD-associated cholinergic hypofunction. Although deficits in cholinergic function following several types of lesions to the nBM are well documented (Harati et al., 2008, Nag et al., 2009, Pizzo et al., 1999), there have been only a few reports regarding the time course of the development of this cholinergic hypofunction or its potential recovery (Abdulla et al., 1997, Höhmann and Coyle, 1988, Perry et al., 2001, Rossner et al., 1995a, Rossner et al., 1995b). Our present goal was therefore to demonstrate the persistence of a 192 IgG-saporin-induced lesion in the nBM and the long-term cortical response to this selective cholinergic neuron loss in architectonically well-defined frontal and parietal cortical projection areas. Choline acetyltransferase (ChAT, EC 2.3.1.6) immunohistochemistry and acetylcholinesterase (AChE, EC 3.1.1.7) enzyme histochemistry were used to assess the losses in the number of cholinergic and cholinoceptive neurons, respectively, and the degree of cortical cholinergic innervation. ChAT-positive neurons were considered cholinergic, while AChE-positive neurons were considered cholinoceptive only as AChE activity is also a common, but not obligatory, feature of most cholinergic neurons (Butcher, 1995, Butcher and Woolf, 1984).
Section snippets
Animals
The experimental procedures were carried out in strict compliance with the European Communities Directive 86/609/EEC, and followed the Hungarian legislation requirements (XXVIII/1998 and 243/1998) and the University guidelines regarding the care and use of laboratory animals. The experimental protocols involving the participation of animals were approved by the Institutional Animal Welfare Committee of the University of Szeged (I-74-II/2009/MÁB). Adult (150–180 g) male Sprague-Dawley rats were
Results
The effects of intraparenchymal 192 IgG-saporin injection into the caudal part of the nBM were demonstrated by using the cholinergic and cholinoceptive markers ChAT and AChE, respectively (Fig. 3). The immunotoxin differentially affected ChAT- and AChE-positive neurons in the nBM. The numbers of ChAT-expressing neurons and AChE-positive cell somata in the nBM of the saline-injected animals (Fig. 3A and C, respectively) were markedly reduced 2 weeks after the lesion elicited by 192 IgG-saporin
Discussion
Small animal models are essential in understanding the pathomechanism underlying neurodegenerative disorders (Brooks et al., 2012, Hall and Roberson, 2012). Several animal studies have established that selective lesioning of the cholinergic system produced alterations similar to that seen in AD (Abdulla et al., 1997, Berger-Sweeney et al., 2001, Fitz et al., 2008, Leanza et al., 1995). The possibility of achieving selective induction of the degeneration of cholinergic neurons in certain basal
Conflict of interest
Neither the authors nor the institutions with which they are affiliated have any competing interest in the subject or findings of this study.
Acknowledgements
This work was supported by program project grants to the University of Szeged from the Ministry of National Resources (TÁMOP 4.2.1.B-09/1/KONV-2010-0005, TÁMOP 4.2.2.A-11/1/KONV-2012-0052) through the European Union Cohesion Fund. The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript. The authors thank Mrs. Susan Ambrus and Mrs. Olga Daranyi for excellent technical help.
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