Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

Abstract

Background: Septal cholinergic neurons account for most of the cholinergic innervations of the hippocampus, playing a key role in the regulation of hippocampal synaptic activity. Disruption of the septo-hippocampal pathway by an experimental transection of the fimbria-fornix drastically reduces the target-derived trophic support received by cholinergic septal neurons, mainly nerve growth factor (NGF) from the hippocampus. Axotomy of cholinergic neurons induces a reduction in the number of neurons positive for cholinergic markers in the medial septum. In several studies, the reduction of cholinergic markers has been interpreted as analogous to the neurodegeneration of cholinergic cells, ruling out the possibility that neurons lose their cholinergic phenotype without dying. Understanding the mechanism of cholinergic neurodegeneration after axotomy is relevant, since this paradigm has been extensively explored as an animal model of the cholinergic impairment observed in neuropathologies such as Alzheimer's disease.The principal aim of this study was to evaluate, using modern quantitative confocal microscopy, neurodegenerative changes in septal cholinergic neurons after axotomy and to assess their response to delayed infusion of NGF in rats.

Results: We found that there is a slow reduction of cholinergic cells labeled by ChAT and p75 after axotomy. However, this phenomenon is not accompanied by neurodegenerative changes or by a decrease in total neuronal number in the medial septum. Although the remaining axotomized-neurons appear healthy, they are unable to respond to delayed NGF infusion.

Conclusions: Our results demonstrate that at 3 weeks, axotomized cholinergic neurons lose their cholinergic phenotype without dying and down-regulate their NGF-receptors, precluding the possibility of a response to NGF. Therefore, the physiological role of NGF in the adult septal cholinergic system is to support phenotypic differentiation and not survival of neurons. This evidence raises questions about the relationship between transcriptional regulation of the cholinergic phenotype by retrograde-derived trophic signaling and the transcriptional changes experienced when retrograde transport is impaired due to neuropathological conditions.

Figure 1

Effect of septo-hippocampal pathway axotomy in cholinergic septal neurons. A, Light microscopy of coronal sections stained for AChE 3 or 21 days after lesion procedure, showing the early effect of axotomy in cholinergic projections towards the hippocampus. Twenty-one days after axotomy, AChE-positive terminals are strongly reduced in the ipsilateral side of the lesion, as compared to the contralateral side (scale bar 1 mm). Inferior panels, magnification inset of AChE-positive neurites in dentate gyrus (scale bar 200 μm). B, Light microscopy of immunohistochemistry anti-ChAT in coronal sections, showing the effects of the axotomy in cell bodies of cholinergic septal neurons, 3 or 21 days after axotomy. Twenty-one days after the lesion, the number of ChAT-positive neurons is clearly reduced on the ipsilateral side of the medial septum (CL, contralateral; IL, ipsilateral. Scale bar: 1 mm). C, Time course of ChAT- and p75-positive neuron loss after axotomy. The graph shows the quantification of ChAT- or p75-immunopositive neurons in the medial septum from serial sections of brains at 3, 7, 14 and 21 days after the lesion. Note that the numbers of ChAT- and p75-immunopositive cells decay with similar kinetics but with a different slope between 7-14 days.

Figure 2

The number of neurons that are positive only for p75 is increased in the ipsilateral side after axotomy. A, Confocal microscopy of double-immunofluorescence anti-ChAT/anti-p75 reveals an increase in the number of neurons that are positive only for p75 at different times after the axotomy. The superior panel is a panoramic view of the medial septum at 14 days after axotomy (scale bar: 1 mm). The central and inferior panels are magnification insets (scale bar: 200 μm) of neurons contralateral and ipsilateral to the lesioned side, showing p75-positive and ChAT-negative cells as indicated by the white arrows. B, The graph shows the percentage of total septal neurons expressing p75 that are p75-positive and ChAT-negative at 1, 3, 7 or 14 days after axotomy. Black bars represent the control side of the septum and white bars represent the side ipsilateral to the lesion. Asterisk indicates significance level p < 0.05.

Figure 3

p75-positive and ChAT-negative neurons are not GABAergic. Confocal microscopy of triple-immunofluorescence anti-ChAT/anti-p75/anti-parvalbumin shows no significant colocalization of GABAergic marker and p75. Left, a panoramic view of the medial septum (scale bar: 1 mm) and panel showing a magnification inset of a representative group of neurons from triple-labeled sections (scale bar: 300 μm).

Figure 4

Cholinergic septal neurons do not undergo apoptosis after axotomy. A, Triple-immunofluorescence against Neu-N (neuronal marker), GFAP (astroglial marker) and activated Caspase-3 shows no colocalization of the apoptotic marker with neurons at different time points (1, 3, 7, 14 days) after axotomy. There is an increase in the number of cells that are immunopositive for activated caspase-3 with time. The correlation between GFAP and activated Caspase-3 suggests that astrocytes are undergoing apoptosis (scale bar: 50 μm). B, Triple-immunofluorescence against ChAT, p75 and p53, shows no colocalization of p53 (an early apoptotic marker) with p75- or ChAT-immunopositive neurons in the brains of axotomized rats 14 days after the axotomy. Confocal microscopy (scale bar: 50 μm). Triple-immunofluorescence against ChAT, p75 and activated caspase-3 shows no colocalization of activated Caspase-3 and p75 or ChAT immunopositive neurons after 14 days of axotomy. Confocal microscopy (scale bar 50 μm). C, Axotomized septal neurons are not labeled with Fluorojade C (a specific staining for degenerating neurons). Superior panel, a brain section from a rat injected in medial septum with 100 mM H₂O₂ was stained with Neurotrace and Fluorojade C as a positive control. The arrow indicates a degenerating neuron. Center panel, double-labeling with Neurotrace and Fluorojade in a brain section from an untreated rat. Inferior panel shows no colocalization of Neurotrace and Fluorojade C in medial septal neurons 14 days after axotomy (scale bar 50 μm).

Figure 5

There is no difference between the number of septal neurons when comparing the ipsilateral and contralateral regions of brains 21 days after axotomy. A, Left, confocal microscopy of triple labeling for Neurotrace (red), NeuN (green) and GFAP (blue) from the septal region of a lesioned brain. In order to assure that we were counting only neurons, we used two neuronal markers and an astrocytic marker (GFAP). Right, inset magnification of a neuron (scale bar: 80 μm) showing the Neurotrace and NeuN labeling profiles. B, Diagram illustrating the area of the medial septum (MS) that was considered for the quantification of cholinergic or total number of neurons (adapted from Rat Brain Atlas [65]). Each side of the total area was divided to 4 fields and then photographed and manually quantified. The anatomical landmarks used to define the MS are also indicated (see Experimental Methods). cc, corpus callosum; LV, lateral ventricle; aca, anterior commissure. C, Quantification of p75-positive, ChAT-positive and total septal neurons. Comparison of the number of septal neurons on contralateral and ipsilateral sides shows differences in the numbers of p75- and ChAT-immunopositive neurons, but no significant difference in total number of neurons (n = 5; Student's t-test, p > 0.001, ± SD) 21 days after axotomy.

Figure 6

Effect of intracerebroventricular infusion of NGF on the loss and recovery of the cholinergic phenotype. A, The septohippocampal path was axotomized and NGF-infusion was performed for two weeks. Left, quantification of septal cholinergic neurons in the lesioned side as compared to the contralateral side in untreated animals (control), lesioned animals (lesion) and lesioned animals brain-infused with NGF (lesion + NGF sim.). Right, light microscopy of anti-NGF immunohistochemistry showing the wide distribution of infused NGF and uptake of NGF by septal neurons. Exogenous NGF infused right after axotomy protects cholinergic cells from ChAT-loss. B, The septohippocampal path was axotomized and 3 weeks after the axotomy (delayed NGF-infusion), NGF was infused for two additional weeks. This procedure did not protect cholinergic cells from ChAT-loss, contrary to the protection observed with simultaneous infusion. C, Confocal microscopy of double immunofluorescence against TrkA and ChAT showing the colocalization of these markers in septal neurons 21 days after axotomy (inferior panel). There are no ChAT-negative cells positive for TrkA, suggesting that neurons cannot respond to NGF 3 weeks post-axotomy due to down-regulation of NGF receptors.