Research report
p75NGFR and cholinergic neurons in the developing forebrain: a re-examination

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Abstract

The low-affinity nerve growth factor receptor (p75NGFR) apparently can mediate apoptosis in a variety of cells in vitro and in vivo. Previously, our laboratory suggested that p75NGFR induced apoptosis in a subpopulation of cholinergic forebrain neurons during postnatal development, i.e., the number of choline acetyltransferase (ChAT)-positive neurons in a control strain of mice decreased whereas it remained higher in p75NGFR-deficient (−/−) mice. Discrepancies with subsequent data sets in our laboratory caused us to thoroughly re-analyze the fate of these cholinergic medial septum and neostriatal neurons in new sets of p75NGFR −/− and two DNA control strains of mice during development. Between postnatal day (P)6 and P15 the number of ChAT-positive neurons detected in the medial septum of 129/Sv mice and Balb/c mice increased by ∼64% and ∼62%, respectively. This increase is contrary to previous reports from our laboratory and indicative of normal postnatal development (including an increase in ChAT-enzyme) of the cholinergic forebrain neurons. In p75NGFR −/− mice the number of ChAT-positive neurons in the medial septum remained constant between P6 and P15 and was ∼31% and ∼56% higher at P6 than 129/Sv and Balb/c mice, respectively. At P15 and adulthood, p75NGFR −/− mice had similar numbers of cholinergic neurons as control mice. In the developing neostriatum, the number of ChAT-positive neurons increased by ∼56% between P6 and P15 and did not differ between p75NGFR −/− and control mice at any time. Analyses for apoptotic DNA fragmentation (TUNEL labeling) at P8 revealed no differences between p75NGFR −/− and control mice in 12 forebrain regions, including the septum and neostriatum. At all times, all mice had similar levels of acetylcholinesterase-positive cholinergic innervation of the molecular layer in the dorsal dentate gyrus. These findings suggest that the p75NGFR does not necessarily mediate apoptosis in medial septum or neostriatal cholinergic neurons during the postnatal time period. The discrepant results of the previous study are most likely due to a less rigorous application of criteria for data acquisition, including anatomical boundaries that define the nucleus.

Introduction

The family of neurotrophins includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4). Traditionally, neurotrophins have been recognized for promoting cell survival, neurite outgrowth and phenotypic maturation 22, 26through the activation of specific high-affinity transmembrane tyrosine kinase (Trk) receptors [5]. NGF binds to and activates TrkA, BDNF and NT-4 activate TrkB, and NT-3 activates TrkC. All neurotrophins bind with equal affinity to another receptor, p75NGFR, which is functionally involved in mediating retrograde transport, providing ligand binding specificity, and enhancing Trk functioning 4, 8, 9.

Several reports now provide evidence suggesting an apoptosis-inducing role for p75NGFR in various developing cell types 6, 7, 14, 29. In the presence of NGF, chick retinal neurons expressing p75NGFR in the absence of TrkA underwent apoptosis, and such cell death was prevented following treatment with antibodies against NGF, p75NGFR or a p75NGFR-interfering peptide (dc28–36) 13, 14. Similarly, BDNF activation of p75NGFR caused cultured sympathetic neurons to die, and in BDNF-deficient mice, more peripheral sympathetic neurons were observed than in control littermates [3]. The interactions and balance between the signaling cascades of TrkA and p75NGFR may predict the final fate of the cell 23, 41, such that p75NGFR is able to induce cell death in the absence of a Trk-initiated signaling cascade [39].

The cholinergic neurons of the developing and adult medial septum contain TrkA and p75NGFR 24, 27, 35. The cholinergic interneurons of the neostriatum contain TrkA, and until the end of the first postnatal week also p75NGFR. These regions provide a model system in which to study the in vivo interactions of TrkA and p75NGFR. The use of transgenic mice containing a genetic deletion for each of these receptors has allowed for further detailed study of the role of TrkA and p75NGFR in cholinergic cell survival, and target innervation of the septo-hippocampal and striatal systems during development and in adulthood. In control mice, the number of detectable cholinergic neurons in the medial septum, neostriatum and the cholinergic innervation of the hippocampal formation, increases with age, reaching mature levels between P14 and P21 11, 16, 28, 34. TrkA-deficient mice had significantly reduced numbers of cholinergic neurons in the medial septum and neostriatum, reportedly caused by enhanced cell death, and reduced cholinergic innervation of the hippocampal formation [11].

The reports about the effects of deleting p75NGFR have been conflicting. Our laboratory and others have suggested that p75NGFR −/− mice have more 18, 38, 40, and yet others have suggested they have fewer 32, 33cholinergic medial septal neurons than control mice. In the neostriatum, p75NGFR −/− mice may have more cholinergic neurons [37], although others have not observed this 32, 40.

Inconsistencies between results describing the normal postnatal developmental process for medial septal cholinergic neurons have been reported, i.e., decreasing cholinergic cell numbers due to death [38]vs. increasing cell numbers 11, 36. The various discrepancies led us to re-examine the cholinergic septo-hippocampal system and the neostriatum during postnatal development and in adulthood in new sets of two DNA control strains (129/Sv and Balb/c) and in p75NGFR −/− mice.

Section snippets

Animals

All animal protocols were approved by the Dalhousie University Animal Care Committee and conformed to Canadian Council on Animal Care guidelines. Deep anesthesia was achieved by i.p. injection of 6.5 mg/kg sodium pentobarbitol. Breeding pairs of mice homozygous (−/−) for the p75NGFR gene [25](Jackson Laboratory number, JR2124) and their DNA control strains (129/Sv, JR2448; Balb/c, JR0651) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Histological procedures

At P6, P15 or adulthood (3–4 months),

p75NGFR −/− mice have more detectable basal forebrain ChAT-positive neurons than control mice at P6 but not later

Analyses of the overall effects revealed that the number of ChAT-positive neurons in the medial septum differed significantly between mouse strains (p<0.0001, ANOVA, F2,53=19.45) and with age (p<0.004, ANOVA, F2,53=6.02). Post-hoc analyses of the main effect of strain indicated that p75NGFR −/− and 129/Sv mice had more ChAT-positive medial septum neurons than Balb/c mice (p<0.004 and 0.02, respectively). Post-hoc analyses of the main effect of age indicated that P15 and adult mice had more

Discussion

We detected inconsistencies between results in our laboratory describing the normal development of forebrain cholinergic neurons and the role of p75NGFR in this process. This led to our thorough re-examination of the cholinergic septo-hippocampal system and the neostriatum during postnatal development and in adulthood in two control strains and p75NGFR −/− mice. In contrast to previous reports from our laboratory 37, 38, our current results suggest that (i) the number of ChAT-positive forebrain

Conclusions

In summary, these results suggest that p75NGFR does not affect the number of cholinergic neurons in the adult forebrain, induce apoptotic cell death in a subpopulation of cholinergic neurons, or affect the cholinergic innervation of the adult hippocampal formation. p75NGFR appears to regulate ChAT and AChE expression and neuron size during development of the cholinergic septo-hippocampal system.

The discrepancies between various groups regarding the number of cholinergic neurons in adult p75NGFR

Acknowledgements

The authors would like to thank members (past and present) of the Vision 2000 Laboratory for Molecular Neurobiology at Dalhousie University for helpful comments and suggestions. We are grateful for the generous gift of p75NGFR antibodies from Dr. L.F. Reichardt (University of California, San Francisco) and Dr. P.A. Barker (Montreal Neurological Institute). This work was supported by a scholarship from the Izaak Walton Killam Foundation (NLW), and a doctoral award (NLW), grant (MT14456) and

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