Electron microscopic evidence for microglial phagocytic activity and cholinergic cell death after administration of the immunotoxin 192IgG-saporin in rat

192IgG-saporin represents a novel cholinergic immunotoxin which selectively and specifically destroys cholinergic cells in rat basal forebrain. Activated microglial cells are known to play an important role in phagocytosis in regions of neuronal loss. To study the immunotoxin-induced phagocytic events in the basal forebrain activated microglial cells were visualized by lectin cytochemistry using Griffonia simplicifolia agglutinin and analyzed by electron microscopy. Three and 7 days following an intracerebro-ventricular injection of 4 μg 192IgG-saporin, increased numbers of activated microglial cells were observed at both survival times, but the number was strikingly increased at day 7 postlesion. Three days after immunotoxin application microglial cells displayed features similar to those of resting microglia. Only translucent vacuole-like hollows were found intracellularly beneath the plasma membrane of microglial cells and in the adjoining extracellular space. Most neurons in the vicinity of microglial cells did not show any signs of degeneration. However, 7 days after injection of the immunotoxin microglial cells revealed different stages of phagocytosis. The majority of microglial cells were localized in perineuronal positions attached by processes to large areas of neuronal soma or dendrites, which in general showed signs of severe degeneration. The present study provides electron microscopic evidence for phagocytic microglial reactions in the rat basal forebrain after cholinergic lesion by 192IgG-saporin. J. Neurosci. Res. 48:465–476, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Expresgions of nerve growth factor and p75 low affinity receptor after transient forebrain ischemia in gerbil hippocampal CA1 neurons

Expressions of nerve growth factor (NGF) and low affinity p75 NGF receptor (p75 NGFR) in gerbil hippocampal neurons after 3.5-min transient forebrain ischemia were studied. Most hippocampal CAI neurons were lost (neuronal density = 44 ± 12/mm) at 7 days after recirculation, while no cell death was found in the sham-control neurons (220 ± 27/min). NGF immunoreactivity was normally present in the sham-control hippocampal neurons. However, it decreased in hippocampal CAI neurons, and slightly decreased in the neurons of CA3 and dentate gyrus areas from 3 hr after recirculation. By 7 days, NGF immunoreactivity returned almost completely to the sham-control level in the CA3 and dentate gyrus neurons but decreased markedly in the CAI neurons. In contrast, p75 NGFR immunoreactivity was scarcely present in the sham-control hippocampal neurons but was induced from 1 hr after recirculation in the CAI and CA3 neurons and from 3 hr in the dentate gyrus. At 7 days, p75 NGFR immunoreactivity was expressed greatly in the surviving CAI neurons and the reactive astrocytes but was not seen in the other hippocampal neurons. The markedly decreased NGF and greatly induced p75 NGFR immunoreactivity found in the CAI neurons after transient forebrain ischemia suggests that NGF and p75 NGFR may be involved in the mechanism of delayed neuronal death. © 1995 Wiley-Liss, Inc.     

Effects of estrogen on basal forebrain cholinergic neurons vary as a function of dose and duration of treatment

Studies suggest that estrogen replacement can influence learning and memory processes via effects on cholinergic neurons located in specific regions of the basal forebrain. In the present study, immunocytochemical techniques were used to examine the effects of estrogen on basal forebrain cholinergic neurons as a function of the dose and duration of estrogen treatment. Ovariectomized rats received 2, 10, 25, or 100 μg estradiol every other day for a period of 1, 2, or 4 weeks. Sections through the basal forebrain were then processed for the detection of choline acetyltransferase (ChAT) or the low-affinity nerve growth factor receptor (p75NGFR), and the number of immunoreactive cells in the medial septum (MS), the horizontal limb of the diagonal band of Broca (HDB) and the nucleus basalis magnocellularis (NBM) were counted. The effects of dose and duration of estrogen treatment were evaluated by analysis of variance and individual group means were compared with ovariectomized controls using a two-tailed Dunnets test. Administration of 2, 10, or 25 μg estradiol for 1 week produced a dose-related increase in the number of ChAT-like immunoreactive (IR) cells detected in the MS. Likewise treatment with 10 μg estradiol for 1 week, or with 2 μg estradiol for 2 weeks resulted in a significant increase in the number of ChAT-IR cells detected in the NBM. These effects were not observed following treatment with higher doses of estradiol. Nor were they maintained following repeated administration of estradiol for longer periods of time. In contrast, repeated administration of estradiol for 2 or 4 weeks resulted in significant decreases in the number of p75NGFR-IR cells detected in the MS, with the greatest effects observed following treatment with the higher doses of estradiol for longer periods of time. These findings demonstrate that (1) estrogen replacement produces regionally selective effects on basal forebrain cholinergic neurons which vary as a function of both the dose and duration of estrogen treatment, and (2) estrogen has both short-term and longer-term effects on basal forebrain cholinergic neurons, each of which may contribute to the effects of estrogen on learning and memory process and the development of age- and disease-related cognitive decline.     

Effects of intraventricular transplantation of NGF-secreting cells on cholinergic basal forebrain neurons after partial immunolesion

The aim of the present study was to examine the effects of nerve growth factor on brain cholinergic function after a partial immunolesion to the rat cholinergic basal forebrain neurons (CBFNs) by 192 IgG-saporin. Two weeks after intraventricular injections of 1.3 μg of 192 IgG-saporin, about 50% of CBFNs were lost which was associated with 40–60% reductions of choline acetyltransferase (ChAT) and high-affinity choline uptake (HACU) activities throughout the basal forebrain cholinergic system. Two groups of lesioned animals received intraventricular transplantations of mouse 3T3 fibroblasts retrovirally transfected with either the rat NGF gene (3T3^NGF+) or the retrovirus alone (3T3^NGF−) and were sacrificed eight weeks later. In vivo production of NGF by 3T3^NGF+ cells was confirmed by NGF immunohistochemistry on the grafts and NGF immunoassay on cerebrospinal fluid (CSF) samples. Both ChAT and HACU activities returned to normal control levels in the basal forebrain and cortex after 3T3^NGF+ transplants, whereas no recovery was observed in 3T3^NGF− transplanted animals. There was a 25% increase in the size of remaining CBFNs and an increased staining intensity for NGF immunoreactivity in these cells after NGF treatments. Acetylcholinesterase (AChE) histochemistry revealed that the optical density of AChE-positive fibers in the cerebral cortex and hippocampus were reduced by about 60% in immunolesioned rats which were completely restored by 3T3^NGF+ grafts. In addition, decreases in growth-associated protein (GAP)-43 immunoreactivity after immunolesion and increases in synaptophysin immunoreactivity after 3T3^NGF+ grafts were observed in the hippocampus. Our results further confirm the notion that transfected NGF-secreting cells are useful in long-term in vivo NGF treatment and NGF can upregulate CBFN function. They also highly suggest that NGF induces terminal sprouting from remaining CBFNs. © 1996 Wiley-Liss, Inc.     

Pathway for interferon-gamma to promote the differentiation of cholinergic neurons in rat embryonic basal forebrain/septal nuclei

BACKGROUND: The supernatant of interferon-gamma (IFNγ) co-cultured with neonatal rat cortical glia can promote the cells in embryonic basal forebrain/septal nuclei to differentiate into cholinergic neurons, but the mechanism is still unclear. OBJECTIVE: To analyze the pathways for IFNγ to promote the differentiation of primarily cultured cholinergic neurons in rat embryonic basal forebrain/septal nuclei through culture in different conditioned medium. DESIGN: A controlled experiment taking cells as the observational target. SETTINGS: Department of Biochemistry and Molecular Biology, Youjiang Medical College for Nationalities; Department of Cell Biology, Beijing University Health Science Center. MATERIALS: Sixty-four pregnant Wistar rats for 16 days (250–350 g) and 84 Wistar rats (either male or female, 5–7 g) of 0–1 day after birth were provided by the experimental animal department of Beijing University Health Science Center. Rat IFNγ were provided by Gibco Company; Glial fibrillary acidic protein by Huamei Company. METHODS: The experiments were carried out in the Department of Cell Biology, Beijing University Health Science Center and Daheng Image Company of Chinese Academy of Science from July 1995 to December 2002. ① Interventions: The nerve cells in the basal forebrain/septal nuclei of the pregnant Wistar rats for 16 days were primarily cultured, and then divided into four groups: Blank control group (not any supernatant and medium was added); Control group (added by mixed glial cell or astrocyte conditioned medium); IFNγ group (added by mixed glial cell or astrocyte conditioned medium+IFNγ). Antibody group (added by mixed glial cell or astrocyte conditioned medium+IFNγ+Ab-IFNγ). Mixed glial cell or astrocyte conditioned medium was prepared using cerebral cortex of Wistar rats of 0–1 day after birth. ② Evaluation: The immunohistochemical method was used to perform the choline acetyltransferase (ChAT) staining of cholinergic neurons. The ChAT positive cells were counted. MAIN OUTCOME MEASURES: Comparison of ChAT positive cells in rat basal forebrain and septal nuclei in different conditioned medium. RESULTS: ① ChAT positive cells in mixed glial cell conditioned medium: The ChAT positive cells in the IFNγ group and antibody group were significantly more than those in the control group (P < 0.01). ② ChAT positive cells in astrocyte conditioned medium: The ChAT positive cells in the IFNγ group were significantly more than those in the control group, but there was no significant difference between the antibody group and control group (P > 0.05). CONCLUSION: IFNγ cannot directly promote the differentiation of cholinergic neurons, but plays a role through activating glial cells (except astrocytes) to produce IFNγ like molecules.     

Rabbit forebrain cholinergic system: morphological characterization of nuclei and distribution of cholinergic terminals in the cerebral cortex and hippocampus

Although the rabbit brain, in particular the basal forebrain cholinergic system, has become a common model for neuropathological changes associated with Alzheimer's disease, detailed neuroanatomical studies on the morphological organization of basal forebrain cholinergic nuclei and on their output pathways are still awaited. Therefore, we performed quantitative choline acetyltransferase (ChAT) immunocytochemistry to localize major cholinergic nuclei and to determine the number of respective cholinergic neurons in the rabbit forebrain. The density of ChAT-immunoreactive terminals in layer V of distinct neocortical territories and in hippocampal subfields was also measured. Another cholinergic marker, the low-affinity neurotrophin receptor (p75(NTR)), was also employed to identify subsets of cholinergic neurons. Double-immunofluorescence labeling of ChAT and p75(NTR), calbindin D-28k (CB), parvalbumin, calretinin, neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase, or substance P was used to elucidate the neuroanatomical borders of cholinergic nuclei and to analyze the neurochemical complexity of cholinergic cell populations. Cholinergic projection neurons with heterogeneous densities were found in the medial septum, vertical and horizontal diagonal bands of Broca, ventral pallidum, and magnocellular nucleus basalis (MBN)/substantia innominata (SI) complex; cholinergic interneurons were observed in the caudate nucleus, putamen, accumbens nucleus, and olfactory tubercule, whereas the globus pallidus was devoid of cholinergic nerve cells. Cholinergic interneurons were frequently present in the hippocampus and to a lesser extent in cerebral cortex. Cholinergic projection neurons, except those localized in SI, abundantly expressed p75(NTR), and a subset of cholinergic neurons in posterior MBN was immunoreactive for CB and nNOS. A strict laminar distribution pattern of cholinergic terminals was recorded both in the cerebral cortex and in CA1-CA3 and dentate gyrus of the hippocampus. In summary, the structural organization and chemoarchitecture of rabbit basal forebrain may be considered as a transition between that of rodents and that of primates.     

Evidence that toxicity of lipopolysaccharide upon cholinergic basal forebrain neurons requires the presence of glial cells in vitro

The cholinergic system of the basal forebrain is affected in brains of dementia patients and during neuroinflammation. The aim of this study was to establish a method to cultivate basal forebrain cholinergic neurons in dissociated, pure neuronal cultures and to apply this method to study the effect of acute and chronic experimentally-induced inflammation using lipopolysaccharide. Purity of the cultures, degrees of neuronal dissociation, connectivity and neuronal survival were investigated by immunocytochemistry for microtubule-associated protein-2 (neurons), glial fibrillary acidic protein (astroglia), complement receptor 3 (microglia), choline acetyltransferase and the neurotrophin receptor p75 (cholinergic neurons). Neuronal cultures only contained <7% astrocytes and <1% microglia when using a "sandwich-technique". Acute (1, 10 microg/ml) as well as chronic (0.1, 1 microg/ml) treatment with lipopolysaccharide did neither affect total number of neurons, nor number of p75-positive neurons or enhance expression of major histocompatibility complex I or II. Our results suggest that lipopolysaccharide-induced degeneration of both microtubule-associated protein-2-like immunoreactive as well as specific killing of cholinergic forebrain neurons in vitro are mediated by glial cells.     

Selective in vivo fluorescence labelling of cholinergic neurons containing p75 in the rat basal forebrain

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimer's disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75^NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75^NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75^NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75^NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75^NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.     

Maternal choline supplementation differentially alters the basal forebrain cholinergic system of young‐adult Ts65Dn and disomic mice

Down syndrome (DS), trisomy 21, is a multifaceted condition marked by intellectual disability and early presentation of Alzheimer's disease (AD) neuropathological lesions including degeneration of the basal forebrain cholinergic neuron (BFCN) system. Although DS is diagnosable during gestation, there is no treatment option for expectant mothers or DS individuals. Using the Ts65Dn mouse model of DS that displays age‐related degeneration of the BFCN system, we investigated the effects of maternal choline supplementation on the BFCN system in adult Ts65Dn mice and disomic (2N) littermates at 4.3–7.5 months of age. Ts65Dn dams were maintained on a choline‐supplemented diet (5.1 g/kg choline chloride) or a control, unsupplemented diet with adequate amounts of choline (1 g/kg choline chloride) from conception until weaning of offspring; post weaning, offspring were fed the control diet. Mice were transcardially perfused with paraformaldehyde, and brains were sectioned and immunolabeled for choline acetyltransferase (ChAT) or p75‐neurotrophin receptor (p75^NTR). BFCN number and size, the area of the regions, and the intensity of hippocampal labeling were determined. Ts65Dn‐unsupplemented mice displayed region‐ and immunolabel‐dependent increased BFCN number, larger areas, smaller BFCNs, and overall increased hippocampal ChAT intensity compared with 2N unsupplemented mice. These effects were partially normalized by maternal choline supplementation. Taken together, the results suggest a developmental imbalance in the Ts65Dn BFCN system. Early maternal‐diet choline supplementation attenuates some of the genotype‐dependent alterations in the BFCN system, suggesting this naturally occurring nutrient as a treatment option for pregnant mothers with knowledge that their offspring is trisomy 21. J. Comp. Neurol. 522:1390–1410, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Effects of estrogen replacement therapy on cholinergic basal forebrain neurons and cortical cholinergic innervation in young and aged ovariectomized rhesus monkeys

Estrogen modulates the function of cholinergic basal forebrain neurons in aged female rats. The present study tested the hypothesis that estrogen enhances the phenotype of cholinergic basal forebrain neurons and their cortical cholinergic innervation in young adult and aged ovariectomized rhesus monkeys. Sixteen monkeys (9 young and 7 aged) received two injections of estradiol cypionate or vehicle separated by 3 weeks. All monkeys were killed 1 day after the last injection. Quantitative immunofluorescence in the vertical limb of the diagonal band (VLDB) revealed enhanced optical density for choline acetyltransferase (ChAT) in both young and aged monkeys treated with estrogen. In contrast, optical density for low-affinity p75 neurotrophin receptor immunoreactivity in the VLDB did not change after estrogen treatment in either aged or young animals. Quantitative immunofluorescence for either ChAT or the low-affinity p75 neurotrophin receptor in the nucleus basalis Meynert failed to reveal differences between vehicle and estrogen treatment in either age group. Quantitative estimates of acetylcholinesterase (AChE) fiber density revealed that estrogen-treated aged monkeys but not their younger counterparts had decreased numbers of AChE-positive fibers in layer II of frontal, insular, and cingulate cortices. These data indicate that estrogen administered in a manner simulating natural hormonal cyclicity produces modest age-specific chemical phenotypic and regional changes in select neuronal subfields of the cholinergic basal forebrain and their cortical projection sites in nonhuman primates.     

GABAergic interneurons containing calbindin D28K or somatostatin are major targets of GABAergic basal forebrain afferents in the rat neocortex.

The arborization pattern and postsynaptic targets of the GABAergic component of the basal forebrain projection to neo- and mesocortical areas have been studied by the combination of anterograde tracing and pre- and postembedding immunocytochemistry. Phaseolus vulgaris leucoagglutinin (PHAL) was iontophoretically delivered into the region of the diagonal band of Broca, with some spread of the tracer into the substantia innominata and ventral pallidum. A large number of anterogradely labelled varicose fibres were visualized in the cingulate and retrosplenial cortices, and a relatively sparse innervation was observed in frontal and occipital cortical areas. Most of the labelled axons were studded with large en passant varicosities (Type 1), whereas the others (Type 2) had smaller boutons often of the drumstick type. Type 1 axons were distributed in all layers of the mesocortex with slightly lower frequency in layers 1 and 4. In the neocortex, layer 4, and to a smaller extent upper layer 5 and layer 6 contained the largest number of labelled fibres, whereas only a few fibres were seen in the supragranular layers. Characteristic type 2 axons were very sparse but could be found in all layers. Most if not all boutons of PHAL-labelled type 1 axons were shown to be GABA-immunoreactive by immunogold staining for GABA. Altogether 73 boutons were serially sectioned and found to make symmetrical synaptic contacts mostly with dendritic shafts (66, 90% of total targets), cell bodies (6, 8.2% of total), and with one spine. All postsynaptic cell bodies, and the majority of the dendritic shafts (44, 60.3% of total targets) were immunoreactive for GABA. Thus at least 68.5% of the total targets were GABA-positive, but the majority of the dendrites not characterized immunocytochemically for technical reasons (15.1%) also showed the fine structural characteristics of nonpyramidal neurons. The target interneurons included some of the somatostatin- and calbindin-containing subpopulations, and a small number of parvalbumin-containing neurons, as shown by double immunostaining for PHAL and calcium-binding proteins or neuropeptides. We suggest that the innervation of inhibitory interneurons having extensive local axon arborizations may be a mechanism by which basal forebrain neurons-most notably those containing GABA--have a powerful global effect on the majority of principal cells in the entire cortical mantle.     

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei

The degree of lesion produced by 192 IgG-saporin relative to controls was compared using three independent methods. Microdialyzed acetylcholine (ACh), choline acetyltransferase (ChAT) activity, and the rate of ACh synthesis were compared in the frontal cortex and hippocampus. Microdialysis of rats was performed 1 and 15 weeks post-lesion. In week 16, the rats were sacrificed after an injection of deuterated choline (Ch) for determination of the rate of ACh synthesis. ChAT activity was determined at the same timepoints in a separate set of rats. At 1 week, ChAT activity and microdialyzed ACh showed similar degrees of depletion. At 15 weeks, microdialyzed ACh was significantly lower than the synthesis rate in cortex, but not in hippocampus. A small increase in ChAT activity between 1 and 15 weeks was found in the cortex, but not hippocampus. In the hippocampus, however, the rate of ACh synthesis was significantly greater than ChAT activity. This was true for two doses of immunotoxin; the greater compensation occurring with the lesser lesion. Microdialyzed ACh levels were not different from the other measures in hippocampus. Residual cholinergic terminals in the hippocampus, but not frontal cortex, compensate for a selective cholinergic lesion by increasing the rate of synthesis and may thereby alleviate hippocampus-dependent behavioral deficits.     

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.     

Laser scanning and electron microscopic evidence for rapid and specific in vivo labelling of cholinergic neurons in the rat basal forebrain with fluorochromated antibodies

Recently developed methods for the selective labelling of cholinergic basal forebrain neurons containing the low-affinity neurotrophin receptor p75 (p75(NTR)) in vivo and in vitro are based on carbocyanine 3 (Cy3)-tagged antibodies directed against p75(NTR). The present study focuses on the maintenance of this neuronal label after injection of such fluorescent antibodies into the cerebral ventricle. One, 3, and 10 days after injection this marker exclusively stains neurons immunoreactive for the cholinergic markers choline acetyltransferase and vesicular acetylcholine transporter in the rat medial septum, diagonal band and nucleus basalis. Thirty days after injection the in vivo labelling was nearly abolished. Predominant labelling of lysosomes was shown by electron microscopic analysis following photoconversion of the Cy3-label to an electron-dense reaction product. The pre-labelling of cholinergic neurons might facilitate pharmacological and electrophysiological approaches in living slices and cell culture systems as well as detailed investigations focused on the transport of neurotrophins in vivo and in animals with experimentally altered p75(NTR) expression.     

Effects of amyloid-beta on cholinergic and acetylcholinesterase-positive cells in cultured basal forebrain neurons of embryonic rat brain

The neurotoxic effects of amyloid-beta(1-42) and amyloid-beta(25-35) (A beta) on cholinergic and acetylcholinesterase-positive neurons were investigated in primary cultures derived from embryonic 18-day-old rat basal forebrain. After various time intervals, the cultures were treated with 1, 5, 10 or 20 microM A beta for different time periods. The cholinergic neurons and their axon terminals were revealed by vesicular acetylcholine transporter immunohistochemistry and the cholinoceptive cells by acetylcholinesterase histochemical staining. To assess the toxic effects of these A beta peptides on the cholinergic neurons, image analysis was applied for quantitative determination of the numbers of axon varicosities/terminals and cells. The results demonstrate that, following treatment with 1 or 5 microM A beta for 5, 10, 30, 60 or 120 min, no changes in vesicular acetylcholine transporter immunohistochemical staining were observed. However, after treatment for 30 min with 10 or 20 microM A beta, the number of stained axon varicosities was reduced, and treatment for 2 h they had disappeared. In contrast, vesicular acetylcholine transporter-positivity could be seen in some of the neuronal perikarya even after 3 days after treatment. The acetylcholinesterase staining was homogeneously distributed in the control neurons. After A beta treatment, the histochemical reaction end-product was detected in some of the neuronal perikarya or in the dendritic processes near to the soma. It is concluded that the neurotoxic effects of A beta appear more rapidly in the cholinergic axon terminals than in the cholinergic and acetylcholinesterase-positive neuronal perikarya.     

Sequential upregulation of cell adhesion molecules in degenerating rat basal forebrain cholinergic neurons and in phagocytotic microglial cells

To study the functional role of adhesion molecules in neurodegenerative events in vivo, the basal forebrain cholinergic lesion-induced expression of the intercellular adhesion molecule (ICAM)-1 and leukocyte function-associated antigen (LFA)-1 was studied by double immunocytochemistry and Western blot analysis. A single intracerebroventricular application of the cholinergic immunotoxin, 192IgG-saporin, produced a selective cholinergic cell loss in rat basal forebrain nuclei detectable by gradual loss of choline acetyltransferase (ChAT)-immunoreactive cells starting 3 days but being nearly complete 7 days after injection of the toxin. The degeneration of cholinergic neurons was accompanied by a striking appearance of activated microglial cells in the lesioned areas. Four days following injection of 192IgG-saporin, ICAM-1 immunoreactivity was predominantly observed in ChAT-positive neurons and partly in activated microglia in the basal forebrain nuclei, while LFA-1 expression at this time point was restricted to neurons. However, 7 days after cholinergic lesion, only a few, shrunken neuronal somata were found to be immunoreactive for ICAM-1 and LFA-1, while activated microglial cells demonstrated strong immunoreactivity for ICAM-1 and LFA-1 in the lesioned forebrain areas, persisting up to 14 days after lesion while no immunoreactivity was observed in neurons at this time point. Western blot analysis demonstrated increased ICAM-1 level in the basal forebrain already detectable 4 days after surgery but being more pronounced 7 days post lesion. The data suggest that ICAM-1 and LFA-1 may act as intercellular recognition signals by which degenerating cholinergic neurons actively participate in the sequence of events leading to their targeting and elimination by phagocytotic microglia.     

Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells

Notch-1 receptor signaling pathway is involved in neuronal and glial differentiation. Its involvement in microglial functions, however, has remained elusive. This study reports the localization of Notch-1 receptor immunoreactivity in the amoeboid microglial cells (AMC) in the postnatal rat brain. By immunofluorescence, Notch-1 receptor was colocalized with its ligands, Jagged-1 and Delta-1, in the AMC in the corpus callosum and subventricular zone. Notch-1 immunopositive cells were confirmed to be microglia labeled by OX42 and lectin. Immunoexpression of Notch-1 receptor was progressively reduced with age. Western blot analysis showed that Notch-1 protein level in the corpus callosum in which the AMC were heavily populated was concomitantly decreased. In postnatal rats challenged with lipopolysaccharide (LPS), Notch-1 receptor immunofluorescence in AMC was noticeably enhanced. Furthermore, Notch-1 protein level in the corpus callosum was increased as revealed by Western blotting analysis. In primary microglial culture treated with LPS, mRNA expression of Notch-1 and its ligand Jagged-1 was upregulated but that of Delta-1 was reduced. The expression pattern of Notch-1 and its ligands was confirmed in murine BV-2 cells. Furthermore, Notch-1 neutralization with its antibody reduced its protein expression. More importantly, neutralization of Notch-1 concomitantly suppressed the mRNA expression of IL-6, IL-1, M-CSF, and iNOS; TNF-alpha, mRNA expression, however, was enhanced. Western blot confirmed the changes of protein level of the above except for IL-6, which remained relatively unaltered. It is concluded that Notch-1 signaling in the AMC and LPS-activated microglia/BV-2 cells modulates the expression of proinflammatory cytokines and nitric oxide.