Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons

Nerve growth factor (NGF) has recently been implicated as a trophic agent in the survival and maintenance of basal forebrain cholinergic neurons. To test the hypothesis that NGF may play a role in the age-related degeneration of basal forebrain neurons and decline of cerebral cholinergic function, we have used a monoclonal antibody to the NGF receptor, 192 IgG, to immunocytochemically visualize and compare rat basal forebrain neurons responsive to NGF in aged (30 months) and young adult (10 months) rats. In a subpopulation of aged rats, NGF receptor-immunoreactive cells in the basal forebrain appear vacoulated and shrunken, and the neuropil staining is markedly reduced. While no substantial decline in cell density is apparent in Nissl-stained sections, the number of NGF receptor-positive cell profiles within the vertical limb of diagonal band nuclei is reduced by an average of 32% in aged rats. Marked reduction in the expression of NGF receptors in aged rats may signify loss of capacity of the basal forebrain neurons to bind and transport NGF from their terminals in the hippocampus and cortex, subsequent decrease in NGF delivered to the cell bodies, and eventual cellular dysfunction and death of neurons in aging.     

Nerve growth factor receptor-containing cholinergic neurons of the basal forebrain project to the thalamic reticular nucleus in the rat

The origin of nerve growth factor receptor-immunoreactive (NGFr-ir) fibers innervating the thalamic reticular nucleus (Rt) was here investigated in the rat using retrograde tracers in combination with immunocytochemistry. Neurons retrogradely labeled from Rt were scattered ipsilaterally throughout the medial septal nucleus and the other cell groups of the basal forebrain, which contained NGFr-ir cells; 10–20% of these retrogradely labeled neurons were also NGFr-ir. Furthermore, a few retrogradely labeled NGFr-ir cells were detected in the basal forebrain on the contralateral side. Retrograde tracing combined with a double immunocytochemical procedure revealed that all the NGFr-ir neurons labeled from Rt also displayed immunoreactivity for choline acetyltransferase. The present results demontrate that the NGFr-ir neurons of the basal forebrain which project to Rt are cholinergic. The possible functional implications of these findings are discussed.     

Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebrain neurons

Recent data indicate that the neurons of the cholinergic basal forebrain (CBF) respond to nerve growth factor (NGF) with increased survival under experimental conditions and have NGF receptors which mediate the binding and retrograde transport of NGF from axon terminals to somata. Focal intraparenchymal injections of retrograde tracing agents into neuropil demonstrate that the distribution of axons from cholinergic nuclei to cortex and hippocampus is topographically restricted and largely ipsilateral. Monoclonal antibody 192, a well-characterized antibody which recognizes only the rat NGF receptor, was labelled with 125I and injected into a lateral ventricle of adult rats. Highly specific bilateral transport to numerous neurons of the CBF system was demonstrated by autoradiography. This result directly demonstrates that suitably targeted antibodies can be taken up by specific neuronal populations following intraventricular injection and implies that CBF neurons may be influenced by relatively high molecular weight substances injected into cerebrospinal fluid.     

Hypertrophy of basal forebrain neurons and enhanced visuospatial memory in perinatally choline-supplemented rats

The effects of choline supplementation during two time-frames of early development on radial-arm maze performance and the morphology of basal forebrain neurons immunoreactive for the P75 neurotrophin receptor (NTR) in male and female Sprague–Dawley rats were examined. In the first experiment, rats were supplemented with choline chloride from conception until weaning. At 80 days of age, subjects were trained once a day on a 12-arm radial maze for 30 days. Compared to control littermates, supplemented rats made fewer working and reference memory errors; however, the memory enhancing effects of choline supplementation were greater in males than females. A morphometric analysis of NTR-immunoreactive cell bodies at three levels through the medial septum/diagonal band (MS/DBv) of these rats revealed that perinatal choline supplementation caused the somata of cells in the MS/DBv to be larger by 8–15%. In a second experiment, choline supplementation was restricted to embryonic days 12–17. A developmental profile of NTR immunoreactive cell bodies in the MS/DBv of 0-, 8-, 16-, 30- and 90-day old male and female rats again revealed that cell bodies were larger in choline-supplemented rats than controls. As in the behavioral studies, the effect of choline supplementation was greater in male than female rats. These data are consistent with the hypothesis that supplementation with choline chloride during early development leads to an increase in the size of cell bodies of NTR-immunoreactive cells in the basal forebrain and that this change may contribute to long-term improvement in spatial memory.     

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.     

Age-related decrease of nerve growth factor-like immunoreactivity in the basal forebrain of senescence-accelerated mice

The senescence-acceleratedmouse P10 (SAMP10) is a murine model of accelerated senescence characterized by the deterioration of learning and memory with advancing age. In the present study, we examined the distribution of nerve growth factor (NGF) immunohistochemically in SAMP10 mice and its control strain, SAMR1. In both strains, NGF-like immunoreactivity (NGF-IR) was observed in neurons throughout the entire forebrain and in glial cells in a particular location. In aged SAMP10 mice, each layer of the cerebral cortex retained its NGF-IR, although the thickness of the cortical mantle was markedly decreased in comparison with younger animals. There was an age-related decline in NGF-IR in the substantia innominata of SAMP10 mice at the age of 10 months, when compared to 2-month-old SAMP10. These results indicate age-related decrease of NGF in the basal forebrain in SAMP10 mice.     

Self-secretion of fibroblast growth factor-9 supports basal forebrain cholinergic neurons in an autocrine/paracrine manner

We examined the effect of fibroblast growth factor (FGF)-9 on primary cultures of rat basal forebrain cholinergic neurons (BFCN) obtained at embryonic day 17. FGF-9 enhanced survival of AChE-positive neurons, increased their mean soma size, and up-regulated their choline acetyltransferase (ChAT) activity. The ChAT-promoting effect of FGF-9 was approximately as potent as that of nerve growth factor (NGF) and was greater than those of basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glia-derived neurotrophic factor (GDNF). Simultaneous addition of FGF-9 and NGF induced extremely high ChAT levels, suggesting that FGF-9 and NGF may enhance cholinergic properties in BFCN via different pathways that can act synergistically. In immunocytochemical and in situ hybridization studies in cultured cells and also in sections of adult rat brain, BFCN showed cytoplasmic immunostaining for FGF-9 and expressed FGF-9 messenger RNA; thus, we concluded that FGF-9 acts on BFCN in an autocrine and/or paracrine manner. Although effective delivery of exogenous FGF-9 into the central nervous system remains a problem to be solved, FGF-9 may be a promising candidate for therapeutic trials in Alzheimer disease.     

Nerve growth factor response to excitotoxic lesion of the cholinergic basal forebrain is slightly impaired in aged rats

Nerve growth factor (NGF) promotes survival and function of basal forebrain cholinergic neurons. We studied NGF and choline acetyltransferase (ChAT) activity after partial quisqualic acid induced lesions of the basal forebrain in 3 and 27 months-old rats, in order to investigate whether NGF-related regeneration is disturbed in old age. 2 weeks post lesion, ChAT activity decreased by 25 to 32% in adult and old rats. 3 months post lesion, the ChAT deficit receded in adult rats, but remained unchanged in old rats. 2 weeks post lesion, NGF levels were reduced by 36 to 44%, but there was no significant difference between adult and old rats. 3 months post lesion, we found increased NGF levels by 44% in the posterior cortex of adult rats. These results indicate that the compensatory NGF increase in the posterior cortex after partial cholinergic lesion of the basal forebrain is slightly impaired in old age.     

Intense immunoreactivity for Mn-superoxide dismutase (Mn-SOD) in cholinergic and non-cholinergic neurons in the rat basal forebrain

The immunohistochemical localization of manganese (Mn)-superoxide dismutase (Mn-SOD) was studied in the rat basal forebrain using polyclonal antibodies to Mn-SOD. Neurons of the basal forebrain exhibit a high density of Mn-SOD immunoreactivity. Double immunostaining with a monoclonal antibody to choline acetyltransferase demonstrated that both cholinergic and non-cholinergic neurons in the basal forebrain are intensely immunoreactive for Mn-SOD.     

Therapeutic potential of CERE-110 (AAV2-NGF): targeted, stable, and sustained NGF delivery and trophic activity on rodent basal forebrain cholinergic neurons

Treatment of degenerating basal forebrain cholinergic neurons with nerve growth factor (NGF) in Alzheimer's disease has long been contemplated, but an effective and safe delivery method has been lacking. Towards achieving this goal, we are currently developing CERE-110, an adeno-associated virus-based gene delivery vector that encodes for human NGF, for stereotactic surgical delivery to the human nucleus basalis of Meynert. Results indicate that NGF transgene delivery to the targeted brain region via CERE-110 is reliable and accurate, that NGF transgene distribution can be controlled by altering CERE-110 dose, and that it is possible to achieve restricted NGF expression limited to but covering the target brain region. Results from animals examined at longer time periods of 3, 6, 9 and 12 months after CERE-110 delivery indicate that NGF transgene expression is stable and sustained at all time points, with no loss or build-up of protein over the long-term. In addition, results from a series of experiments indicate that CERE-110 is neuroprotective and neurorestorative to basal forebrain cholinergic neurons in the rat fimbria–fornix lesion and aged rat models, and has bioactive effects on young rat basal forebrain cholinergic neurons. These findings, as well as those from several additional non-clinical experiments conducted in both rats and monkeys, led to the initiation of a Phase I clinical study to evaluate the safety and efficacy of CERE-110 in Alzheimer's disease subjects, which is currently ongoing.     

Repeated episodic exposure to ethanol affects neurotrophin content in the forebrain of the mature rat

Chronic exposure to ethanol can cause deficits in learning and memory. It has been suggested that withdrawal is potentially more damaging than the ethanol exposure per se. Therefore, we explored the effect of repeated episodic exposure to ethanol on key regulators of cortical activity, the neurotrophins. Rats were exposed to ethanol via a liquid diet for 3 days per week for 6-24 weeks. Control rats were pair-fed an isocaloric liquid diet or ad libitum fed chow and water. The concentrations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were determined using enzyme-linked immunosorbant assays (ELISAs). Five telencephalic structures were examined: parietal cortex, entorhinal cortex, hippocampus, the basal nucleus, and the septal nuclei. All five areas expressed each of the three neurotrophins; BDNF was most abundant and NGF the least. The parietal cortex was susceptible to ethanol exposure, NGF and BDNF content increased, and NT-3 content fell, whereas no changes were detectable in the entorhinal cortex. In the hippocampus, the amount all three neurotrophins increased following episodic ethanol exposure. Neurotrophin content in the two segments of the basal forebrain was affected; NGF and NT-3 content in the basal forebrain was reduced and NGF and BDNF content in the septal nuclei was increased by ethanol exposure. In many cases where ethanol had an effect, the change was transient so that by 24 weeks of episodic exposure, no significant changes were evident. Thus, the effects of ethanol are site- and time-dependent. This pattern differs from changes caused by chronic ethanol exposure, hence, neurotrophins must be vulnerable to the effects of withdrawal. Furthermore, the ethanol-induced changes do not appear to fit a model consistent with retrograde regulation, rather they suggest that neurotrophins act through autocrine/paracrine systems.     

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.     

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.     

The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons

BDNF, through p75NTR, promotes apoptosis and inhibits axonal growth of sympathetic neurons, antagonizing the pro-survival and axon growth-promoting actions of NGF through TrkA. While the trafficking of the TrkA:NGF complex is well characterized, little is known about p75NTR:BDNF trafficking in these neurons. Here we show that BDNF binds to and appears inside sympathetic neurons relatively slowly, although the temperature-sensitive internalization step itself is rapid. P75NTR internalization is partially sensitive to disruption of clathrin- or raft-mediated internalization, while that of TrkA is entirely clathrin-mediated. P75NTR, but not Trk, associates with neurotrophins in lipid rafts and coimmunoprecipitates with the truncated beta-caveolin-1 isoform. Finally, we directly visualize the retrograde transport of p75NTR ligands to cell bodies, which is insensitive to inhibitors of Trk retrograde transport, suggesting mechanistic differences. We postulate that beta-caveolin-1-containing lipid rafts and possibly intracellular endosomes might be compartments to which p75NTR:BDNF complexes are trafficked separately from Trk.     

Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat

A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 IgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 IgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (ChAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 IgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKT1-saporin-injected controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 IgG-saporin. ChAT immunohistochemistry was confirmative of major cell loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the selectivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.     

Enhancement of choline acetyltransferase activity in coculture of rat septal and hippocampal neurons

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.     

Behavioural, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system

Use of the selective immunotoxin; 192 IgG-saporin, is helping to elucidate the role of the cholinergic system in cognition by overcoming the problems of interpretation associated with the use of non-specific lesioning agents. In separate studies, we have compared the long- and short-term effects of single site and combined saporin lesions of the nucleus basalis magnocellularis and medial septal area, on spatial learning and memory in radial arm and water maze tasks. At 11 months, only rats with combined lesions showed deficits in both radial and water maze tasks, although terminal cholinergic deafferentation was substantial and extensive tissue loss was seen at the injection sites in both single and combined lesions. However, the extensive tissue loss with long-term lesions suggested that behavioural deficits were not solely attributable to cholinergic deafferentation. In contrast, when rats with combined lesions were tested 5 months after lesioning, no deficits were apparent, although there was almost complete loss of choline acetyltransferase- and nerve growth factor receptor-immunoreactivity in the basal forebrain with no tissue damage at the injection sites. This study supports existing literature that selective loss of cholinergic neurons in the basal forebrain does not produce behavioural impairments in standard tasks of learning and memory, but deficits are apparent when damage is non-selective as occurs late after lesioning, confounding interpretation of behavioural data. It further highlights potential problems with this immunotoxin in long-term studies.     

Neurturin and persephin promote the survival of embryonic basal forebrain cholinergic neurons in vitro

The GDNF family ligands (GFLs) are a group of neurotrophic factors that influence the development, survival, and maintenance of specific populations of neurons in the central and peripheral nervous systems. The cholinergic neurons of the basal forebrain provide cholinergic innervation to cortical structures and their integrity is vital to normal cognitive function. GDNF, the original member of the GFL family promotes the survival of developing basal forebrain cholinergic neurons in vitro. We have now found that neurturin (NRTN) and persephin (PSPN) also promote the survival of basal forebrain neurons including both cholinergic neurons and a population of non-cholinergic neurons with an efficacy comparable to NGF. We also demonstrate that developing and mature basal forebrain cholinergic neurons (BFCN) express GFL receptors. Ret, the signaling component of the GFL-receptor complex, is expressed in most adult rat BFCN. In addition, Ret and the GFL co-receptors GFRalpha1 and GFRalpha2 are expressed in developing cholinergic neurons in cultures of embryonic basal forebrain. Our results suggest that the GFLs may be effective as neuroprotective agents for BFCNs in vivo.     

Co-expression of p75- and calbindin-immunoreactivity in cholinergic neurons of the raccoon basal forebrain

The cholinergic system of the basal forebrain is involved in the modulation of sensory information. This has previously been investigated in the raccoon, an animal especially interesting because of its highly developed somatosensory cortex. The present study focused on the co-expression of the low-affinity neurotrophin receptor p75^NTR and calbindin in cholinergic neurons of the raccoon basal forebrain and neostriatum. Carbocyanine immunofluorescence double labelling revealed the co-localization of choline acetyltransferase and p75^NTR as well as calbindin in a large portion of basal forebrain neurons, but not in the neostriatum. In contrast, immunolabelling of two other calcium-binding proteins, parvalbumin and calretinin, was found exclusively in non-cholinergic neurons.