Regional decreases of cortical choline acetyltransferase after lesions of the septal area and in the area of nucleus basalis magnocellularis

Choline acetyltransferase and [³H]choline uptake have been measured in neocortical regions and hippocampus one week after lesions which destroyed the septum bilaterally, and after unilateral lesions in the area of nucleus basalis magnocellularis. Lesions of the septal area, which severely decreased choline acetyltransferase in hippocampus, only moderately decreased choline acetyltransferase in a posterior cortical region and had no effect in frontal and parietal regions. In contrast, lesions which included nucleus basalis magnocellularis decreased choline acetyltransferase markedly in frontal and parietal regions and had less of an effect in the posterior cortical regions. Lesion-induced decreases of [³H]choline uptake paralleled those of choline acetyltransferase. Lesions which included nucleus basalis magnocellularis had no effect on choline acetyltransferase in hippocampus, nucleus accumbens, olfactory tubercle, midbrain or pons-medulla.These results suggest the existence of topographically distinct cholinergic inputs to neocortex. In agreement with previous studies, cholinergic projections from the peripallidal region of nucleus basalis magnocellularis are predominantly to frontal and parietal neocortex. In contrast to previous suggestions, cholinergic projections to neocortex from the septal area are limited to the posterior regions of neocortex.     

Guidance of acetylcholinesterase-containing fibres by target tissue in co-cultured brain slices

Slices of various brain regions were prepared from newborn and from 7-day old rats and co-cultured in different combinations. In the majority of co-cultures of septal and hippocampal slices, acetylcholinesterase-positive fibres originating in the septal nuclei invaded the adjacent hippocampal slice. A similar pattern of hippocampal ingrowth by acetylcholinesterase-positive fibres occurred with slices prepared from the nucleus basalis of Meynert and from spinal cord. Septal neurones also projected to cortical slices, an effect which even occurred in the presence of their natural target tissue. In contrast to these massive projections to brain areas which in situ receive cholinergic inputs, no significant acetylcholinesterase-positive fibre ingrowth was observed in tissues which lack major cholinergic afferents in situ (hypothalamus, substantia nigra and cerebellum). These results indicate that under our culture conditions, acetylcholinesterase-positive fibres selectively invade cholinergic target areas. This effect is independent of the brain area from which the cholinergic neurones were derived.     

Localization and axonal transport of immunoreactive cholinergic organelles in rat motor neurons—An immunofluorescent study

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated α-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors.The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: (1) Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. (2) Deafferentation did not notably influence accumulations of immunoreactive material. (3) Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. (4) Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots.It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.     

Acetylcholine metabolism by cultured neurons from rat nodose ganglia: regulation by a macromolecule from muscle-conditioned medium

Acetylcholine metabolism has been studied in primary cultures of neurons dissociated from newborn rat nodose ganglia. Neither nerve growth factor nor muscle-conditioned medium had any detectable effect on the long-term survival of these neurons, which appeared well differentiated upon phase-contrast and electron microscopic examination. [3H]Acetylcholine synthesis and accumulation by 2-3-week old nodose cultures and choline acetyltransferase activity were increased (2.0 +/- 0.15)-fold and (2.0 +/- 0.48)-fold, respectively, upon growth with muscle-conditioned medium, whereas acetylcholinesterase was decreased (1.5 +/- 0.1)-fold (means +/- SEM, n = 5-9). The same effects were observed when comparing nodose cultures grown in the presence of proteins purified from conditioned medium in four fractionation steps. This purified material had no effects on the protein content of the cultures, or on lactate dehydrogenase activity, and thus did not affect the overall growth of the cultures. We demonstrate that this factor copurifies with a factor(s) involved in the regulation of choline acetyltransferase and acetylcholinesterase in neuron cultures from newborn rat superior cervical ganglia [Swerts, Le Van Thai, Vigny and Weber (1983) Devl Biol. 100, 1-11] and from embryonic day 13 rat embryo spinal cord [Giess and Weber (1984) J. Neurosci. 4, 1442-1452]. Although the cholinergic factor from muscle-conditioned medium has not been purified to homogeneity, the data suggest that the same extracellular, macromolecular factor may be involved in the regulation of acetylcholine metabolism in derivatives from the neural crest, the neural tube and the epibranchial placodes.     

Genetic evidence that chloroadenosine increases the specific activity of choline acetyltransferase in PC12 cells via modulation of an adenosine-dependent adenylate cyclase

Both chloroadenosine (EC50 = 3 X 10(-7) M) and cholera toxin, like nerve growth factor, increase the specific activity of choline acetyltransferase in PC12 cells over a period of several days. The increase in choline acetyltransferase activity in response to chloroadenosine appears to be caused by the ability of chloroadenosine to increase adenosine 3':5'-phosphate synthesis by binding to an adenosine receptor that activates adenylate cyclase. To test this hypothesis we determined if chloroadenosine can cause an increase in choline acetyltransferase activity in adenosine kinase-deficient PC12 cells. We have previously shown that adenosine analogues are significantly less effective at regulating adenosine 3':5'-phosphate in adenosine kinase-deficient PC12 cells than in wild type cells [Erny and Wagner (1984) Proc. natn. Acad. Sci. U.S.A. 81, 4974-4978]. Adenosine kinase-deficient PC12 cells are resistant to the induction of choline acetyltransferase in response to chloroadenosine, but not cholera toxin, supporting the role of adenosine 3':5'-phosphate in mediating the effects of chloroadenosine. The increase in choline acetyltransferase activity in wild type cells was accompanied by an increase in acetylcholine levels, demonstrating that chloroadenosine also regulates storage of acetylcholine. Acetylcholine levels were quantitated using an assay based on the ability of acetylcholine to compete with [125I]bungarotoxin for binding to the acetylcholine receptor.     

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12-16 day old embryos) were much more responsive than similar neurons from young embryos (8 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor in treatment and pathogenesis of Alzheimer's disease

The etiology of Alzheimer's disease (AD) is still unknown. In addition, this terrible neurodegenerative disease will increase exponentially over the next two decades due to longer lifespan and an aging "baby-boomer" generation. All treatments currently approved for AD have moderate efficacy in slowing the rate of cognitive decline in patients, and no efficacy in halting progression of the disease. Hence, there is an urgent need for new drug targets and delivery methods to slow or reverse the progression of AD. One molecule that has received much attention in its potential therapeutic role in AD is nerve growth factor (NGF). This review will demonstrate data from humans and animals which promote NGF as a potential therapeutic target by (1) outlining the hypothesis behind using NGF for the treatment of AD, (2) reviewing both the normal and AD altered signaling pathways and effects of NGF in the central nervous system (CNS), and (3) examining the results of NGF treatment obtained from animal models of AD and AD patients.     

Differential regulation of peptide and catecholamine characters in cultured sympathetic neurons

Mechanisms regulating peptidergic, noradrenergic and cholinergic development were compared in dissociated cell cultures of neonatal rat sympathetic ganglia. The majority of cultured neurons contained at least two neurotransmitters and many neurons contained three or more. These studies were undertaken to determine whether co-existing transmitters were co-ordinately regulated by the environment. Co-culture of sympathetic neurons with ganglion non-neuronal cells increased substance P and choline acetyltransferase activity but decreased somatostatin and tyrosine hydroxylase activity. Conversely, elimination of non-neuronal cells virtually abolished neuronal expression of substance P and choline acetyltransferase and increased somatostatin and tyrosine hydroxylase. Consequently, under these conditions, somatostatin and tyrosine hydroxylase were similarly regulated, whereas substance P was associated with choline acetyltransferase. By contrast, stimulation of adenylate cyclase or treatment with membrane-permeable adenosine 3',5'-phosphate analogs increased tyrosine hydroxylase and decreased choline acetyltransferase, but had no effect on substance P or somatostatin levels. Moreover, potassium- or veratridine-induced membrane depolarization increased tyrosine hydroxylase but decreased substance P, somatostatin and norepinephrine levels. However, inhibition of neurotransmitter release with magnesium or calcium-free medium prevented the decrease in norepinephrine levels but not the decrease in substance P and somatostatin. Consequently, the effects of membrane depolarization on peptide levels cannot be ascribed to release and subsequent depletion of substance P and somatostatin and must result from decreased net synthesis (synthesis minus catabolism) of the transmitters. Nerve growth-factor treatment also differentially regulated transmitter metabolism; nerve growth factor increased protein-specific activities of tyrosine hydroxylase and choline acetyltransferase but did not increase the protein-specific content of substance P and somatostatin. Quantitative transmitter expression was also influenced by neuron density; increasing density elevated substance P and choline acetyltransferase activity but decreased somatostatin and tyrosine hydroxylase activity per neuron. Finally, culture of sympathetic neurons in a defined (serum-free) medium also altered some but not all traits, decreasing substance P, somatostatin and choline acetyltransferase without any change in tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of neural and adrenal medullary surface membrane glycoproteins recognized by antisera to cultured rat sympathetic neurons and PC12 pheochromocytoma cells

The object of this paper is to identify and compare the glycoproteins in the nervous system which are recognized by antisera prepared against cultured rat sympathetic neurons and PC12 pheochromocytoma cells cultured with or without nerve growth factor. Each of the three antisera was preabsorbed with rat liver, kidney, spleen and thymus to remove activity directed against common rat antigens and then used for indirect immunoprecipitation of solubilized membrane antigens from fucoselabeled cultures of neurons and of PC12 cells before and after treatment with nerve growth factor. Electrophoresis of immunoprecipitated material in sodium dodecylsulfate polyacrylamide gradient gels and fluorography revealed that all three antisera recognized fucosylated proteins of apparentM_r = 230,000; 180,000; 160,000; and 140,000. The antisera against PC12 cells also showed weak recognition of four additional components at apparentM_r = 290,000; 118,000; 55,000; and 25,000. While the patterns of glycoproteins recognized by the three antisera were qualitatively similar, they did show marked quantitative differences that tended to reflect the composition of the cells against which the antisera were prepared. Mild treatment with trypsin and lactoperoxidase-catalyzed iodination of intact living cells indicated that the 230,000-, 180,000-, and 140,000-dalton components are exposed on the cell surface. The properties of the 230,000-dalton material suggest that it is identical to the nerve growth factor-inducible large external (NILE) glycoprotein previously described in PC12 cultures. Immunoprecipitations carried out after absorption of each antiserum with brain and adrenal membranes indicated that the 230,000-, 180,000- and 140,000-dalton components are present in brain and that the 180,000-and 140,000-dalton components are present in the adrenal medulla.The present experiments have identified several neural-specific, antigenic surface glycoproteins. Antisera against these glycoproteins should be useful for determining their cellular localizations and functions.     

Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice

Trans-3-(3′4′-dimethoxyphenyl)-4-[(E)-3″,4″-dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3′4′-dimethoxyphenyl)-4-[(E)-3″,4″-dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10–30 μM) or Comp.2 (30 μM) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03–3 μM) or Comp.2 (0.3–3 μM) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 μM) and Comp.2 (3–30 μM) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.), an antidepressant, were administrated once a day on days 15–28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2′-deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia.     

Eosinophil peroxidase induces expression of cholinergic genes via cell surface neural interactions

Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-l-Glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-l-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced its effects via the activation of NF-κB. MEK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.     

Involvement of brain-derived neurotrophic factor (BDNF) in MP4-induced autoimmune encephalomyelitis

The role of brain-derived neurotrophic factor (BDNF) in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) is still unclear. Here we investigate the clinical course, CNS histopathology and peripheral antigen-specific immunity in MP4-induced EAE of BDNF (−/+) mice. We demonstrate that these mice displayed less severe disease compared to BDNF (+/+) mice, reflected by decreased inflammation and demyelination. In correspondence to diminished frequencies of T and B cells in CNS infiltrates, the peripheral MP4-specific T_H1/T_H17 response was attenuated in BDNF (−/+), but not in wild-type animals. In contrast, immunization with ovalbumin triggered similar frequencies of IFN-γ- and IL-17-secreting T cells in both groups. The cytokine secretion and proliferative activity upon mitogen stimulation did not reveal any global defect of T cell function in BDNF (−/+) mice. By influencing the antigen-specific immune response in autoimmune encephalomyelitis, BDNF may support and maintain the disease in ways that go beyond its alleged neuroprotective role.