Elsevier

Brain Research Bulletin

Volume 94, May 2013, Pages 9-16
Brain Research Bulletin

Research report
Long-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

https://doi.org/10.1016/j.brainresbull.2013.01.007Get rights and content

Abstract

Alzheimer's disease is associated with a significant decrease in the cholinergic input to the neocortex. In a rat model of this depletion, we analyzed the subsequent long-term changes in cholinergic fiber density in two well-defined areas of the frontal and parietal cortices: Fr1, the primary motor cortex, and HL, the hindlimb area of the somatosensory (parietal) cortex, two cortical cholinergic fields that receive inputs from the nucleus basalis magnocellularis (nBM). A specific cholinergic lesion was induced by the intraparenchymal injection of 192 IgG-saporin into the nBM. Choline acetyltransferase (ChAT) immunohistochemistry was applied to identify the loss of cholinergic neurons in the nBM, while acetylcholinesterase (AChE) enzyme histochemistry was used to analyze the decreases in the number of cholinoceptive neurons in the nBM and the cholinergic fiber density in the Fr1 and HL cortical areas in response to the nBM lesion. The immunotoxin differentially affected the number of ChAT- and AChE-positive neurons in the nBM. 192 IgG-saporin induced a massive, irreversible depletion of the ChAT-positive (cholinergic) neurons (to 11.7% of the control level), accompanied by a less dramatic, but similarly persistent loss of the AChE-positive (cholinoceptive) neurons (to 59.2% of the control value) in the nBM within 2 weeks after the lesion. The difference seen in the depletion of ChAT- and AChE-positive neurons is due to the specificity of the immunotoxin to cholinergic neurons. The cholinergic fiber densities in cortical areas Fr1 and HL remained similarly decreased (to 62% and 68% of the control values, respectively) up to 20 weeks. No significant rebound in AChE activity occurred either in the nBM or in the cortices during the period investigated. This study therefore demonstrated that, similarly to the very extensive reduction in the number of ChAT-positive neurons in the nBM, cortical areas Fr1 and HL underwent long-lasting reductions in the number of AChE-positive fibers in response to specific cholinergic lesioning of the nBM.

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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