Experimental immune-mediated damage of septal cholinergic neurons

Degeneration of cholinergic neurons in the medial septum and the diagonal band of Broca is a frequent neuropathological feature of Alzheimer's disease. To determine whether an immune process can injure these basal forebrain cholinergic neurons, we serially immunized guinea pigs with septal cholinergic hybrid cells (SN-56). Following immunization, a relatively selective damage of septal cholinergic neurons, reduction in septal choline acetyltransferase (ChAT) activity and decrease in acetylcholine release in hippocampus were detected. Serum IgG from guinea pigs immunized with SN-56 cells and stereotactically injected into the medial septal region of rats produced a loss of ChAT activity in the medial septum, frontal cortex and hippocampus, together with impairment of learning and long term spatial memory. These data suggest that relatively selective damage to septal cholinergic neurons can be caused by an immune-mediated process in experimental animals.     

Degeneration of the cholinergic innervation of the locus ceruleus in Alzheimer''s disease

Choline acetyltransferase (Acetyl-CoA: choline O-acetyltransferase: EC 2.3.1.6) (ChAT) enzyme activity and neuron density were measured in the locus ceruleus (LC) of autopsied brains of neurologically normal individuals and patients who had Alzheimer's disease. Neuron density in the LC of individuals with Alzheimer's was significantly reduced to approximately 50% of normal values. ChAT activity was also reduced by about 50%. Furthermore, the number of pigmented neurons in the LC was highly correlated with presynaptic ChAT activity. These findings were specific for the LC, since deficits in ChAT and neuron density were not found in two adrenergic brainstem nuclei (C1 and C2). We measured mitogen activity in LC extracts in order to determine whether loss of cholinergic afferents to the LC, as evidenced by loss of ChAT, was related to putative trophic factors. Mitogen activity was significantly reduced (50%) in the Alzheimer's group as compared to normals. Mitogen activity was significantly correlated with ChAT activity and the density of neurons in the LC. The loss of cholinergic nerve terminals in the LC in Alzheimer's disease may be functionally significant, since acetylcholine has important effects on LC physiology. The highly significant relationships between ChAT, neuron density and mitogen activity has important implications for our understanding of mechanisms of neurodegeneration in Alzheimer's disease.     

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.     

Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (−83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (−80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 μM and −63% at 100 μM) and in co-cultures (+25% at 0.1 μM and −28% at 100 μ M). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.     

Changes in the septohippocampal cholinergic system following removal of molar teeth in the aged SAMP8 mouse

We investigated the effect of dysfunctional teeth on age-related changes in the septohippocampal cholinergic system by assessing acetylcholine (ACh) release and choline acetyltransferase (ChAT) activity in the hippocampus and ChAT immunohistochemistry in the medial septal nucleus and the vertical limb of the diagonal band in young-adult and aged SAMP8 mice after removal of their upper molar teeth (molarless condition). Aged molarless mice showed decreased ACh release and ChAT activity in the hippocampus and a reduced number of ChAT-immunopositive neurons in the medial septal nucleus compared to age-matched control mice, whereas these effects were not seen in young-adult mice. The results suggest that the molarless condition in aged SAMP8 mice may enhance an age-related decline in the septohippocampal cholinergic system.     

Neurotrophic effect of hematopoietic cytokines on cholinergic and other neurons in vitro

We examined the effects of interleukin-3 (IL-3) and other hematopoietic cytokines on the neurotransmitters, neurite formation, and differentiation in cholinergic and other types of neurons. IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, granulocyte colony-stimulating factor and erythropoietin (Epo) elevated choline acetyltransferase (ChAT) activity in septal cholinergic cell line SN6 as well as in primary cultured septal neurons without increasing protein contents of the cells. These effects were dose-dependent and the optimal doses were not different from those for blood cells. IL-3 had neurite-promoting activity but GM-CSF had no such effect. Both IL-3 and GM-CSF decreased intracellular acetylcholine concentration, and elevated glutamic acid decarboxylase and intracellular GABA in septal neuronal cultures. Epo elevated monoamines in PC12 cells. These effects are thought to result from direct action through their specific receptors in neurons, because (i) anti-IL-3-receptor antibody abolished the ChAT activity in septal neurons increased by IL-3; (ii) mRNA and immunoreactivity for β subunits of IL-3 receptors were expressed in septal cholinergic neurons and (iii) presence of receptos for GM-CSF and Epo in neurons has been reported. Our observation and others strongly support that neural-immune interactions are important not only in the defense mechanism in the nervous system but also in the development, differentiation and function of neurons.     

Effect of thyroid hormone analogs on the activity of choline acetyltransferase in cultures of dissociated septal cells

Thyroid hormones influence the expression of transmitter-specific enzymes by central cholinergic neurons. Based on the fact that these cholinergic neurons degenerate selectively in human Alzheimer's disease, it was hypothesized that thyroid hormones might be beneficial in its treatment. However, since thyroid hormones influence the function of most peripheral organs, derivatives selective for central cholinergic neurons are necessary. The structural requirements of the receptor mediating the effects of the thyroid hormones on central cholinergic neurons were therefore compared with those of the receptors mediating actions on peripheral organs. Cultures were prepared of dissociated neurons from the septal area of fetal rat brains, and the differentiation of cholinergic neurons was assessed by measuring the activity of choline acetyltransferase (ChAT). Triiodothyronine (T3) was found to stimulate ChAT activity in a dose-dependent manner. The effect of T3 was additive to that of nerve growth factor. The potency of derivatives of T3 in elevating ChAT activity in the cultures was compared with their known anti-goiter activity determined in vivo and their binding affinity to the hepatic nuclear receptor measured in vitro. The findings indicate that the structural requirements of central and peripheral receptors are similar and that it therefore appears unlikely that analogs of thyroid hormones can be developed which selectively affect cholinergic neurons.     

The basal forebrain cholinergic system in aging and dementia. Rescuing cholinergic neurons from neurotoxic amyloid-β42 with memantine

The dysfunction and loss of basal forebrain cholinergic neurons and their cortical projections are among the earliest pathological events in the pathogenesis of Alzheimer's disease (AD). The evidence pointing to cholinergic impairments come from studies that report a decline in the activity of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE), acetylcholine (ACh) release and the levels of nicotinic and muscarinic receptors, and loss of cholinergic basal forebrain neurons in the AD brain. Alzheimer's disease pathology is characterized by an extensive loss of synapses and neuritic branchings which are the dominant scenario as compared to the loss of the neuronal cell bodies themselves. The appearance of cholinergic neuritic dystrophy, i.e. aberrant fibers and fiber swelling are more and more pronounced during brain aging and widely common in AD. When taking amyloid-β (Aβ) deposition as the ultimate causal factor of Alzheimer's disease the role of Aβ in cholinergic dysfunction should be considered. In that respect it has been stated that ACh release and synthesis are depressed, axonal transport is inhibited, and that ACh degradation is affected in the presence of Aβ peptides. β-Amyloid peptide 1-42, the principal constituent of the neuritic plaques seen in AD patients, is known to trigger excess amount of glutamate in the synaptic cleft by inhibiting the astroglial glutamate transporter and to increase the intracellular Ca²⁺ level. Based on the glutamatergic overexcitation theory of AD progression, the function of NMDA receptors and treatment with NMDA antagonists underlie some recent therapeutic applications. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist interacts with its target only during states of pathological activation but does not interfere with the physiological receptor functions. In this study the neuroprotective effect of memantine on the forebrain cholinergic neurons against Aβ42 oligomers-induced toxicity was studied in an in vivo rat dementia model. We found that memantine rescued the neocortical cholinergic fibers originating from the basal forebrain cholinergic neurons, attenuated microglial activation around the intracerebral lesion sides, and improved attention and memory of Aβ42-injected rats exhibiting impaired learning and loss of cholinergic innervation of neocortex.     

Galanin-like immunoreactivity within Ch2 neurons in the vertical limb of the diagonal band of Broca in aging and Alzheimer's disease

Summary The neuropeptide galanin is known to inhibit the evoked release of acetylcholine in ventral hippocampus of the rat. Co-localization of this peptide with choline acetyltransferase in neurons of the cholinergic septal nuclei has been demonstrated in the rat and non-human primate. The severe deficiency of the cholinergic hippocampal projection system arising mainly from the vertical limb nucleus of the diagonal band of Broca, also referred to as Ch2 region, is a constant finding in Alzheimer's disease, a disorder which is neuropathologically characterized by the appearance of senile plaques, neurofibrillary tangles and congophilic angiopathy in neo- and archicortical structures. In the present study for the first time galanin immunoreactivity in the human Ch2 region is morphologically investigated and related to the severity of hippocampal plaques and neurofibrillary tangles in Alzheimer's disease. An inverse relationship between decreasing galanin immunoreactivity in the Ch2 region and amounts of senile plaques and neurofibrillary tangles in the hippocampus is indicated. Considering the cholinergic deficiency in Alzheimer's disease as a secondary phenomenon to primary cortical and hippocampal lesions, and realizing the inhibitory effect of galanin upon acetylcholine release in hippocampus, this preliminary study suggests that a decreased galanin immunoreactivity in Ch2 in Alzheimer's disease reflects a possible negative feedback mechanism to a degenerating cholinergic projection system.Supported fully by a research grant from the JANIVO Foundation     

Galanin microinjected into the medial septum inhibits scopolamine-induced acetylcholine overflow in the rat ventral hippocampus

Galanin-like immunoreactive terminals hyperinnervate the basal forebrain cholinergic neurons in Alzheimer's disease. To investigate the hypothesis that galanin acts directly on basal forebrain cell bodies, in vivo microdialysis studies were conducted in awake rats which analyzed the actions of galanin on acetylcholine release. Microinjection of galanin into the cholinergic cell body region of the medial septum-diagonal band (MS-DBB) inhibited acetylcholine release in the ventral hippocampus. These results are consistent with an interpretation that galanin terminals synapsing on cholinergic cell bodies of the basal forebrain may serve to inhibit the release of acetylcholine in the terminal fields of the cholinergic neurons.     

Death of subcortical cholinergic neurons in certain neurodegenerative disorders may not be due to an overstimulation of N-methyl-D-aspartate receptors

Stimulation of N-methyl-D-aspartate (NMDA) receptors increases the activity of enzymes involved in the synthesis of nerve cell specific neurotransmitters. In the present study this phenomenon has been used to identify the neurons in the septal-diagonal band region having NMDA receptors. Exposure of cultures relatively enriched in subcortical cholinergic neurons to a depolarizing concentration of K+ (40 mM) significantly enhanced the expression of choline acetyltransferase (ChAT) activity. In contrast, when these septal cells were treated with as much as 100 microM NMDA no significant increase was observed in the activity of choline acetyltransferase, although there was a marked enhancement in glutamate decarboxylase activity. The results would indicate that subcortical cholinergic neurons do not possess excitatory amino acid receptors of the NMDA subtype, and that therefore neurotoxicity mediated through NMDA receptors may not be involved in the death of cholinergic neurons in degenerative disorders of the brain, such as Down's syndrome and Alzheimer's disease, which entail major losses of these neurons.     

Distribution of polysialylated neural cell adhesion molecule in rat septal nuclei and septohippocampal pathway: transient increase of polysialylated interneurons in the subtriangular septal zone during memory consolidation

During memory consolidation neuroplastic events in the mediotemporal corticohippocampal pathway are accompanied by transient increases in the frequency of neurons expressing polysialylated neural cell adhesion molecule (NCAM PSA), a posttranslational modification associated with morphofunctional change. As a bidirectional pathway between the hippocampus and the septal nuclei also influences memory processing, we have determined the distribution of NCAM PSA within this system before and after learning in the adult Wistar rat. The most intense NCAM PSA immunoreactivity was observed in the medial and triangular septal nuclei, regions that regulate hippocampal theta rhythm during memory consolidation. Within the fimbria, NCAM PSA was expressed only in a subpopulation of fibres, most likely cholinergic projections from the medial septum to the hippocampus. Grey level analysis or direct cell counting revealed no learning-specific change in NCAM PSA expression in these septal subregions after avoidance conditioning or spatial training. A population of discrete polysialylated neurons in the subtriangular septal zone, however, exhibited a transient twofold frequency increase at 12 hr after training in either task. Immunohistochemical analysis revealed these cells to be gamma-aminobutyric acid (GABAergic) interneurons co-expressing vasoactive intestinal peptide. The unique location of these interneurons is proposed to provide a natural plexus by which bidirectional communication between the septum and hippocampus may be modified during memory consolidation.     

Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes

To characterize the role of NGF in the development of forebrain cholinergic neurons, we established primary cell culture systems to grow these cells under controlled in vitro conditions. Cultures of dissociated cells were prepared from the septal area of fetal (E17) rats, which contained part of the group of basal forebrain cholinergic neurons. Cultures were treated either with NGF (100 ng/ml) or with an antiserum against NGF (1:500 dilution). To assess the influence of non-neuronal cells, 2 types of high-density cultures were prepared: mixed neuronal-glial cultures and pure neuronal cultures. Cholinergic neurons were identified using choline acetyltransferase (ChAT) immunocytochemistry and AChE cytochemistry. Receptors for NGF (NGF-R) were located immunocytochemically using monoclonal antibodies against rat NGF-R. We report that, first, NGF-R are exclusively localized on cholinergic neurons in septal cultures. All neurons labeled with antibodies against NGF-R also contained AChE. Twenty-one percent of all AChE-positive neurons were not stained in NGF-R immunocytochemistry (AChE has earlier been shown to be colocalized with ChAT in septal cultures). Second, NGF treatment increases and anti-NGF treatment reduces the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF promotes survival of septal cholinergic neurons in these cultures. In cultures of high plating density, NGF increased the number of NGF-R and ChAT-positive neurons without affecting the number of AChE-positive neurons in these cultures. These results suggest that exogenous NGF is not required for survival of cholinergic neurons in high-density cultures but stimulates the expression of ChAT and NGF-R. Third, NGF stimulates fiber growth of septal cholinergic neurons, as assessed by computerized image analysis of AChE-positive neurons. Fourth, NGF specifically increases ChAT and AChE activities in septal cultures. These NGF-mediated increases in enzyme activities are more pronounced when neurons are grown together with glial cells. In pure neuronal cultures, NGF increased ChAT and AChE activities by 101 and 16%, and in mixed neuronal-glial cultures by 318 and 87%, respectively. Anti-NGF blocked the effects of NGF but failed to reduce ChAT and AChE activities below control levels in cultures of high plating density. Fifth, astrocytes attenuate the expression of ChAT and AChE by septal neurons in the absence of NGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neural cell adhesion molecule ablation in mice causes hippocampal dysplasia and loss of septal cholinergic neurons

The neural cell adhesion molecule (NCAM) is implicated in nervous system development and plasticity. In humans, abnormal NCAM expression has been linked to the pathogenesis of neuropsychiatric and neurodegenerative disorders accompanied by cognitive dysfunctions. Impaired cognition is also observed in NCAM-deficient (NCAM(-/-) ) mice. Considering the importance of the septal cholinergic nuclei and the hippocampus for cognition, we performed quantitative morphological analyses of these areas in young and adult (2 and 13 months old, respectively) NCAM(-/-) mice and wild-type (NCAM(+/+) ) littermates. In young mice, we found lower numbers of septal cholinergic neurons in NCAM(-/-) than in NCAM(+/+) mice. Despite deficient numbers of neurons, total lengths of cholinergic axons and choline acetyltransferase protein levels in the hippocampus of NCAM(-/-) mice were normal. The hippocampus of NCAM(-/-) mice was atrophic, notably in the CA3 subfield and the dentate gyrus (DG). The atrophy appeared to have different primary causes in the two subfields: loss of pyramidal cells in CA3 and reduced branching and volume of granule cell dendrites in the DG. The frequency of dendritic spines on dentate granule cells in NCAM(-/-) mice was normal. Numbers of parvalbumin-positive (PV(+) ) interneurons were reduced in NCAM(-/-) mice in proportion to subfield volume loss, and the ratios of principal cells to PV(+) interneurons were similar to those of NCAM(+/+) mice. None of the observed abnormalities was exaggerated or alleviated in adult NCAM(-/-) mice. Our observations indicate that NCAM ablation causes structural abnormalities in the hippocampus and the forebrain cholinergic system in adult mice, which may contribute to impaired cognition in NCAM(-/-) mice.     

Trophic actions of recombinant human nerve growth factor on cultured rat embryonic CNS cells

NGF is a neurotrophic factor for basal forebrain cholinergic neurons and may serve to counteract the cholinergic deficits that are observed in Alzheimer's disease. Prior to the introduction of clinical trials, it is essential that recombinant human NGF (rhNGF) be produced and that its actions on target cells in the CNS be demonstrated. We prepared rhNGF and examined its actions on fetal rat brain neurons in culture including, in particular, the cholinergic neurons of the basal forebrain. rhNGF was more potent in increasing choline acetyltransferase (ChAT) activity in septal cultures than NGF purified from mouse salivary glands (mNGF). ED50s of the beta-NGF dimers were 4.9 pM for rhNGF and 12.4 pM for mNGF. The maximal ChAT activity response was achieved at approximately 35 pM with both NGFs and their efficacies were not significantly different. The two NGFs were not additive in effect. Identical to the results with mNGF, rhNGF strongly enhanced the intensity of ChAT immunostaining in septal cultures. Neither rhNGF nor mNGF affected the appearance of the cultures under phase-contrast illumination. Survival of cells at very low plating density on polyornithine/laminin-coated culture dishes was not affected by rhNGF or mNGF. Protein content and the uptake of GABA were also unaffected. At concentrations of up to 10 micrograms/ml, rhNGF did not significantly increase uptake of dopamine into cultures of ventral mesencephalon. We conclude that rhNGF produces potent and selective actions on cholinergic neurons of the basal forebrain as previously shown for mNGF.     

Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease.

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimer's disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT- (210,000) and VAChT- (174, 000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT- and VAChT-immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini-Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD.     

Dopamine D5 receptor localization on cholinergic neurons of the rat forebrain and diencephalon: a potential neuroanatomical substrate involved in mediating dopaminergic influences on acetylcholine release

The study of dopaminergic influences on acetylcholine release is especially useful for the understanding of a wide range of brain functions and neurological disorders, including schizophrenia, Parkinson's disease, Alzheimer's disease, and drug addiction. These disorders are characterized by a neurochemical imbalance of a variety of neurotransmitter systems, including the dopamine and acetylcholine systems. Dopamine modulates acetylcholine levels in the brain by binding to dopamine receptors located directly on cholinergic cells. The dopamine D5 receptor, a D1-class receptor subtype, potentiates acetylcholine release and has been investigated as a possible substrate underlying a variety of brain functions and clinical disorders. This receptor subtype, therefore, may prove to be a putative target for pharmacotherapeutic strategies and cognitive-behavioral treatments aimed at treating a variety of neurological disorders. The present study investigated whether cholinergic cells in the dopamine targeted areas of the cerebral cortex, striatum, basal forebrain, and diencephalon express the dopamine D5 receptor. These receptors were localized on cholinergic neurons with dual labeling immunoperoxidase or immunofluorescence procedures using antibodies directed against choline acetyltransferase (ChAT) and the dopamine D5 receptor. Results from this study support previous findings indicating that striatal cholinergic interneurons express the dopamine D5 receptor. In addition, cholinergic neurons in other critical brain areas also show dopamine D5 receptor expression. Dopamine D5 receptors were localized on the somata, dendrites, and axons of cholinergic cells in each of the brain areas examined. These findings support the functional importance of the dopamine D5 receptor in the modulation of acetylcholine release throughout the brain.     

An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain

Culture conditions have been established for growing neurons from the medial frontal part of the forebrain, containing the septum and the diagonal band of Broca, of 17-day-old rat embryos in a chemically defined medium. At 10 days in vitro, the cultures contained more than 96% nerve cells of which about 18% were cholinergic neurons, while the proportion of astrocytes was less than 1%. The majority of the cells that stained for acetylcholinesterase were bipolar but with different sizes and shapes. During development both the specific activity of choline acetyltransferase (ChAT) and the amount of protein increased markedly in the cholinergic cultures, ChAT activity rising much more than the protein content. Exposure of the cultures to nerve growth factor (NGF) or 3,3',5-triiodo-L-thyronine (T3) enhanced the expression of ChAT activity in a dose-dependent manner. The elevation of ChAT activity was due to an increase in the amount of enzyme per cholinergic cell, since, during the experimental period studied, neither treatment with NGF nor with T3 had significant effects on the total protein content of the cultures or on the number of cells, including the cholinergic neurons. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT activity was much greater than the sum of the individual effects. The observations indicate that subcortical cholinergic neurons, which are affected in Alzheimer's disease and in Down's syndrome, are subject to regulation by an interaction between thyroid hormone and local humoral factors such as NGF.     

Coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in mouse clonal hybrid neurons derived from the septal region.

Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum-containing medium without extra differentiating agents, one-third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200-kDa neurofilament protein, neuron-specific enolase, microtubule-associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy-1.2, saxitoxin-binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis.     

Characterization of a neurotrophic factor produced by cultured astrocytes involved in the regulation of subcortical cholinergic neurons.

When dissociated subcortical cells were cultured in the presence of conditioned medium of relatively differentiated astrocytes (ACM), a marked increase was observed in the expression of choline acetyltransferase (ChAT), an enzyme required for the synthesis of the neurotransmitter acetylcholine. Astrocytes from the target regions of subcortical neurons, the hippocampus and the cerebral cortex, produced neurotrophic factor consistently more than those derived from the nontarget region, the cerebellum. The production of cholinergic trophic activity was increased with the maturation of astrocytes. Even though, nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are known cholinergic trophic compounds produced by astrocytes in vitro, a large part of the neurotrophic activity in our ACM was not related to either of these 2 factors. This is because (i) ACM and NGF produced an additive effect on ChAT activity, (ii) only a small proportion of the cholinergic trophic activity in ACM was abolished by anti-NGF antibody, and (iii) treatment with CNTF had no effect on ChAT activity of basal forebrain cholinergic neurons. On the other hand, when cholinergic neurons are cultured on a preformed layer of astrocytes, addition of basal fibroblast growth factor (bFGF) failed to increase further the ChAT activity. Similarly the effects of ACM and bFGF were not additive. A large proportion of the cholinergic trophic activity in ACM was neutralized by anti-bFGF antibody. These findings would suggest that the trophic activity on septal cholinergic neurons in our ACM was due to bFGF or a bFGF-like compound.