Review
Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction

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Abstract

The human cholinergic basal forebrain (CBF) is comprised of magnocellular hyperchromic neurons within the septal/diagonal band complex and nucleus basalis (NB) of Meynert. CBF neurons provide the major cholinergic innervation to the hippocampus, amygdala and neocortex. They play a role in cognition and attentional behaviors, and are dysfunctional in Alzheimer's disease (AD). The human CBF displays a continuum of large cells that contain various cholinergic markers, nerve growth factor (NGF) and its cognate receptors, calbindin, glutamate receptors, and the estrogen receptors, ERα and ERβ. Admixed with these cholinergic neuronal phenotypes are smaller interneurons containing the m2 muscarinic acetylcholine receptor (mAChRs), NADPH-diaphorase, GABA, calcium binding proteins and several inhibitory neuropeptides including galanin (GAL), which is over expressed in AD. Studies using human autopsy material indicate an age-related dissociation of calbindin and the glutamate receptor GluR2 within CBF neurons, suggesting that these molecules act synergistically to induce excitotoxic cell death during aging, and possibly during AD. Choline acetyltrasnferease (ChAT) activity and CBF neuron number is preserved in the cholinergic basocortical system and up regulated in the septohippocampal system during prodromal as compared with end stage AD. In contrast, the number of CBF neurons containing NGF receptors is reduced early in the disease process suggesting a phenotypic silence and not a frank loss of neurons. In end stage AD, there is a selective reduction in trkA mRNA but not p75NTR in single CBF cells suggesting a neurotrophic defect throughout the progression of AD. These observations indicate the complexity of the chemoanatomy of the human CBF and suggest that multiple factors play different roles in its dysfunction in aging and AD.

Introduction

A diverse group of telencephalic structures situated on the medial and ventral aspects of the cerebral hemispheres together compose the basal forebrain in the primate brain. This complex region contains structures including the septal area, the vertical and the horizontal limbs of the diagonal band of Broca as well as the region termed the substantia innominata (for review see Mufson and Kordower, 2000). Historically, this later region has been termed the nucleus of the ansa lenticularis (Meynert, 1872) and later renamed the nucleus basalis (NB; Kolliker, 1896). Part of the difficulty in understanding this region is the complexity of its chemoanatomical organization. These regions also have a diversity of cells displaying different neurotransmitters, morphologies and projection patterns (see de Lacalle et al., 1994, Mufson and Kordower, 2000 for reviews). These cholinergic corticopetal projection neurons have received extensive attention due to their reduction in Alzheimer's disease (AD) and their close association with the neurotrophic substance nerve growth factor (NGF) and its high (trkA) and low (p75NTR) affinity receptors which are involved in basal forebrain cell survival (see Mufson and Kordower, 1999, Mufson et al., 1997 for reviews). This article will discuss the central cholinergic projection neurons of the human basal forebrain and their dysfunction in normal aging and in AD.

Section snippets

Subjects

The human brain material discussed in this compilation report was harvested from several brain banks including Rush Presbyterian St. Luke's Medical Center, Harvard Medical School Alzheimer's Disease Center, University of Pittsburgh Alzheimer's Disease Center and Sun City Research Institute. In general, brains are removed and soaked in 4% paraformaldehyde and cryoprotected (Mufson et al., 1989c, Mufson et al., 1998) overnight and cut at 40 μm thickness on a freezing sliding microtome. Material

Immunocytochemistry

Immunohistochemistry was performed for each antibody according to previously reported procedures (for choline acetyltrasnferease (ChAT) (see Mufson et al., 1989c); for trkA (see Mufson et al., 1997); for p75NTR (see Mufson et al., 2002b); for galanin (GAL) (see Mufson et al., 1993); for m2 receptor (see Mufson et al., 1998); for calbindin (see Geula et al., 2003); for glutamate receptors (see Ikonomovic et al., 2000)). ERβ (Zymed Labs, San Francisco, CA) staining was performed at a dilution of

Choline acetyltransferase activity

ChAT activity was measured from snap frozen tissue samples obtained from the anterior cingulate, superior frontal, superior temporal cortex, and hippocampus using radioactive carbon-14 labeled acetyl Co-A (New England Nuclear, Boston, MA) (see Fonnum, 1975). Protein content was determined by a BCA protein assay kit (Pierce, Rockford, IL). Calculations were based on net counts per minute (CPM) of samples multiplied by specific activity of labeled substrate and divided by protein concentration to

Single cell RNA amplification

Procedures for single cell micro aspiration of NB neurons, RNA amplification and analysis used fixed tissue as described previously (Ginsberg and Che, 2002, Ginsberg et al., 1999, Hemby et al., 2003, Mufson et al., 2002a). Briefly, terminal continuations (TC) amplified RNA probes generated from individual NB cholinergic basal forebrain (CBF) neurons were hybridized to custom-designed cDNA arrays, which consisted of nylon membranes (Hybond XL, Amersham Biosciences, Piscataway, NJ) adhered with

Quantitative stereology

Total neuron counts were derived using an unbiased, stereologic cell counting method (see Kordower et al., 2001, Mufson et al., 2000b, West et al., 1996). The total number of neurons was calculated by using the formula N=NV×VSN, where NV is the numerical density and VSN is the volume the NB (West et al., 1996). Variability within groups was assessed by mean of the coefficient of error (CE: West et al., 1996).

Anatomy of the human cholinergic basal forebrain subgroups

Nissl stained preparations reveal that the main cell groups of the CBF exhibit a distinct magnocellular appearance (Fig. 1A). Studies using the non-specific cholinergic marker acetylcholinesterase (AChE), the synthesizing enzyme for acetycholine, ChAT, the vesicular acetylcholine transporter, and the high-affinity choline transporter have delimited a continuum of cholinergic neurons within the CBF (Fig. 1B–D and G; see Gilmor et al., 1999, Kus et al., 2003, Mesulam et al., 1983, Mesulam et al.,

NGF, estrogen receptors, gabaergic and glutaminergic markers in CBF neurons nerve growth factor receptors

Immunocytochemical and in situ hybridization studies have shown that the high (trkA) and low (p75NTR) affinity NGF receptors as well as retrogradely transported NGF are localized within human CBF neurons (Fig. 1B–E; Schatteman et al., 1988, Strada et al., 1992, Boissiere et al., 1997, Chu et al., 2001, Hefti, 1983, Hefti, 1986, Hefti and Mash, 1989, Kordower et al., 1988, Kordower et al., 1989a, Kordower et al., 1989b, Mufson et al., 1989b, Mufson et al., 1999b, Mufson et al., 1997, Mufson et

Calcium binding proteins

Calbindin and ChAT colocalize within human CBF neurons of the NB (Ch4) (Wu et al., 1997, de Lacalle and Saper, 1997, Geula et al., 2003, Geula et al., 1993). Geula et al., 2003, Geula et al., 1993 have shown an age-related loss of calbindin stained human NB neurons (compare Fig. 1H and I) suggesting a loss of the phenotypic expression of calbindin in CBF neurons which may be a potential mechanism for the selective loss of cholinergic neurons in aging and AD (Geula et al., 2003).

Glutamate receptors

Co-localization experiments reveal that ChAT- and p75NTR-positive CBF neurons express glutamate receptor 1 (GluR1) in aged humans (Fig. 1J; Ikonomovic and Armstrong, 1996, Ikonomovic et al., 2000). In contrast, the GluR2 subunit appears to undergo an age-related down-regulation within the CBF of elderly humans (Fig. 1K, L; Ikonomovic and Armstrong, 1996, Ikonomovic et al., 2000). It is interesting to note that the presence of the GluR1 subunit, and the absence of GluR2, is associated with

Steroid receptors in the CBF

CBF neurons may be influenced by sex hormones. Androgen receptors are present on CBF neurons and their expression is altered during aging (Ishunina and Swaab, 2001). Two estrogen receptors (ERs) have been identified and termed alpha (ERα; Koike et al., 1987) and beta (ERβ; Kuiper et al., 1996). Immunohistochemical studies indicate that ERα does not co-localize with ChAT, p75NTR or trkA containing CBF neurons, although scattered ERα positive nuclei were found within Ch2 and Ch4a in the monkey (

m2 muscarinic receptor neurons within the CBF

Several metabotropic muscarinic acetylcholine receptors (mAChRs) have been identified by differential affinities for antagonists, and it is established that five distinct genes m1–m5, encode highly related muscarinic receptor subtypes (Bonner et al., 1987, Hulme et al., 1990). It was assumed that m2 subtype of mAChR is the gene product which functions as a presynaptic autoreceptor to inhibit ACh release (Levey et al., 1991, Mash et al., 1985, Mash and Potter, 1986, Pohorecki et al., 1988) and

Non-cholinergic neurons within the CBF

In addition to the magnocellular cholinergic neurons located within the Ch4 subfields, there are many smaller non-cholinergic perikarya (see de Lacalle and Saper, 1997, Mufson and Kordower, 2000). Although the protein and mRNA for the inhibitory neuropeptide GAL are not found in human CBF neurons, small GAL-positive interneurons are admixed within CBF perikarya (Fig. 1O) (Benzing et al., 1993, Chan-Palay, 1988, Kordower et al., 1992, Kordower and Mufson, 1990, Mufson et al., 1993, Walker et

CBF dysfunction Alzheimer's disease

Several reports demonstrate a significant reduction in both CBF neurons (Whitehouse et al., 1982) and cortical ChAT activity (Wilcock et al., 1982, DeKosky et al., 1992, Perry et al., 1977) in human aging and AD. Most postmortem studies examining CBF system changes in AD were derived from end-stage patients with severe dementia. In contrast, there is a preservation of cortical ChAT activity (Fig. 2A) and NB neurons (Fig. 2B) in subjects with MCI and early AD (Davis et al., 1999, DeKosky et al.,

Acknowledgements

Supported by AG14449, AG16765, AG09466 and AG10161, AG10668, NS43939 and the Alzheimer's Association.

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