Modulation of neuronal choline acetyltransferase activity by factors derived from cultures of non-neuronal cells from the CNS

We have found that cholinergic neurons in spinal cord-dorsal root ganglion cultures derived from E12-E13 mouse embryos are sensitive, as measured by changes in choline acetyltransferase activity, to factors secreted by non-neuronal cells derived from the same tissue at an identical developmental stage. Conditioned medium was produced by incubating non-neuronal cultures for 4 days in defined medium. The cholinotrophic activity present in the conditioned medium had a molecular weight of greater than 50,000 as determined by ultrafiltration and bound wheat germ lectin and heparin sepharose. Total RNA isolated from the non-neuronal cells, used to produce the conditioned medium, was translated in frog oocytes. Conditioned medium from the injected oocytes was also found to contain cholinotrophic activity. In contrast, the conditioned medium from water-injected oocytes was inactive. The interaction between the cholinotrophic activity in conditioned medium from frog oocytes and known second messengers was also examined. Dibutyryl cyclic AMP produced a concentration-dependent increase in choline acetyltransferase activity. If a maximal effective dose of dibutyryl cyclic AMP was added in conjunction with a maximal effective dose of conditioned medium from oocytes injected with total RNA a nearly additive response was noted. In contrast, the phorbol ester, phorbol 12-myristate 13-acetate, produced a biphasic change in the level of choline acetyltransferase activity; with lower doses stimulating and higher doses inhibiting the enzyme activity. When conditioned medium from oocytes injected with non-neuronal cell RNA was added in conjunction with the phorbol ester a decrease in the physiological response was noted.     

Endogenous and exogenous factors support neuronal survival and choline acetyltransferase activity in embryonic spinal cord cultures

Dissociated 4-day (stage 23) chick embryo lumbar cord cells were cultured at low or high cell densities for 1 or 5 days in the presence or absence of added spinal neuronotrophic factor (supplied as RN22 Schwannoma conditioned medium, RCM). In low density, 1-day cultures neuronal survival was dependent on added RCM whereas by 5 days no neurons survived, even in the presence of RCM. In high density 1-day cultures a substantial neuronal population could survive even without added RCM and a large proportion of this neuronal population would survive for 5 days. When conditioned media from high density lumbar cord cultures was supplied to low density unsupplemented cultures, a similar level of 5-day neuronal survival resulted. However, no neurons survived in RCM-supplemented 5-day high density cultures, indicating the presence in RCM of a material toxic for the neurons. Both the RCM and the high density lumbar culture-conditioned medium supported considerable choline acetyltransferase activity indicating the presence within these cultures of motoneurons.     

Axotomy-dependent stimulation of choline acetyltransferase activity by exogenous mouse nerve growth factor in adult rat basal forebrain

Transection of the adult rat dorsal fornix and fimbria (F-F) induced a sensitivity of the cholinergic neurons in the medial septum and diagonal band (MS/DB) to exogenous mouse nerve growth factor (mNGF). Continuous infusion of mNGF for two weeks after complete unilateral F-F aspiration resulted in a stimulation of choline acetyltransferase (ChAT)-specific activity in precise micro-dissections of the MS/DB ipsilateral to the transection to a level that was 200% higher than that measured in normal adult animals. This supranormal stimulation of ChAT activity reached plateau levels after 10 days of NGF infusion and was dose-dependent with an E.D.50 equal to 120 ng/day. Administration of mNGF had no effect on the ChAT activity in the MS/DB of normal animals or animals with a unilateral transection of only the supracallosal dorsal septo-hippocampal pathway. Partial transection experiments indicated that a predominent pathway for cholinergic neurons potentially sensitive to exogenous mNGF runs in the paramedian F-F. Administration of mNGF also induced a stimulation of ChAT activity in dissections of the caudate-putamen both ipsi- and contralateral to the infusion cannula. This indicates that unlike the cholinergic projection neurons of the MS/DB, adult cholinergic striatal interneurons are sensitive to exogenous NGF without prior axotomy.     

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.     

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 μM), or glutamate (75 μM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca²⁺ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca²⁺ were maximal in neurons from cerebellum and minimal in neurons from cortex. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15–35% was observed following 4 h of DDG pretreatment, increasing to 60–85% protection after 24 h of DDG pretreatment. Although the mechanism of DDG’s neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.     

Nerve growth factor levels and choline acetyltransferase activity in the brain of aged rats with spatial memory impairments

Nerve growth factor (NGF) and choline acetyltransferase (ChAT) activity levels were measured in 7 different brain regions in young (3-month-old) and aged (2-years-old) female Sprague-Dawley rats. Prior to analysis the spatial learning ability of the aged rats was assessed in the Morris' water maze test. In the aged rats a significant, 15–30%, increase in NGF levels was observed in 4 regions (septum, cortex, olfactory bulb and cerebellum), whereas the levels in hippocampus, striatum and the brainstem were similar to those of the young rats. The NGF changes did not correlate with the behavioral performance within the aged group. Minor 15–30%, changes in ChAT activity were observed in striatum, brainstem and cerebellum, but these changes did not correlate with the changes in NGF levels in any region. The results indicate that brain NGF levels are maintained at a normal or supranormal levels in rats with severe learning and memory impairments. Ther results, therefore, do not support the view that the marked atrophy and cell loss in the forebrain cholinergic system that is known to occur in the behaviorally impaired aged rats is caused by a reduced availability of NGF in the cholinergic target areas. The results also indicate that the slightly increased levels of NGF are not sufficient to prevent the age-dependent atrophy of cholinergic neurons, although they might be important for the stimulation of compensatory functional changes in a situation where the system is undergoing progressive degeneration.     

Nerve growth factor and choline acetyltransferase activity levels in the rat brain following experimental impairment of cerebral glucose and energy metabolism.

Intracerebroventricular (ICV) injection of streptozotocin (STZ) has been reported to impair cerebral glucose utilization and energy metabolism (Nitsch and Hoyer: Neurosci Lett, 128:199-202, 1991) and also to prejudice passive avoidance learning in adult rats (Mayer et al.: Brain Res 532:95-100, 1990). It is well established that the forebrain cholinergic system, whose integrity is essential for learning and memory functions, depends on the target-derived retrograde messenger nerve growth factor (NGF). Therefore, we measured NGF and choline acetyltransferase (ChAT) activity levels in the forebrain cholinergic system in adult rats that had received a single injection of either STZ or artificial cerebrospinal fluid into the left ventricle 1 or 3 weeks prior to sacrifice. One week after ICV STZ treatment, NGF content was significantly decreased (-32%) in the septal region, where NGF-responsive cell bodies are located and NGF exerts its neurotrophic action after retrograde transport from NGF-producing targets. In contrast, NGF levels in the cortex and hippocampus, which are target regions for the basal forebrain cholinergic neurons, and in the brainstem and cerebellum were increased (+12% to +47%) within 3 weeks after ICV STZ treatment. The alterations in NGF levels were not related to changes in ChAT activity that decreased in the hippocampus by only 15%. This might be due to masking effects exerted by compensatory NGF-mediated stimulation of ChAT activity in remaining functional neurons. It is suggested that impaired behavior which has been observed after STZ-induced impairment of cerebral glucose and energy metabolism may be at least partially related to a diminished capacity of central NGF-responsive neurons to bind and/or transport NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maternal glucocorticoid hormone influences nerve growth factor expression in the developing rat brain

Rat pups nursed from birth by mothers with increased plasma corticosterone show long-lasting biochemical and behavioral modifications. Here we have investigated nerve growth factor (NGF) concentrations in the basal forebrain, prefrontal cortex and hippocampus of both male and female offspring at 11 days of age. Maternal hypercorticosteronemia was achieved by giving corticosterone-enriched water (200 microg/ml) from delivery. There was a significant increase of NGF in the basal forebrain of both sexes and no changes in the prefrontal cortex. In the hippocampus, an increase in NGF was found in males. These results indicate that a moderate increase of corticosterone in the lactating mother modulates NGF in the developing rat. We propose that these effects contribute directly to the long-lasting behavioral and biochemical modifications in pups nursed by hypercorticosteronemic mothers.     

Acetylcholine-rich transplants in the hippocampus: influence of intrinsic growth factors and application of nerve growth factor on choline acetyltransferase activity

Three groups of rats received either unilateral fimbria-fornix lesions by aspiration through the overlying cingulate cortex (group I), a fimbria-fornix lesion followed by an intrahippocampal transplant of acetylcholine (ACh)-rich embryonic septal tissue (group II), or a similar septal transplant placed into the intact hippocampus, in the absence of the denervating lesion (group III). The 3 groups were subdivided into equal subgroups receiving 6 intrahippocampal injections of nerve growth factor (NGF) at 4-day intervals, control injections of cytochrome c, or no injections. On the 28th day all animals were sacrificed and the majority taken for biochemical analysis of hippocampal choline acetyltransferase (ChAT). The animals with intact hippocampi (group III) were given a denervating fimbria-fornix lesion 3 days prior to sacrifice in order to reveal graft-derived ChAT activity from intrinsic ChAT activity. The fimbria-fornix lesions (group I) depleted hippocampal ChAT activity to 15-20% of normal, which was not influenced by NGF injections. The ACh-rich grafts placed in the denervated hippocampus (group II) restored hippocampal ChAT activity to approximately 60% of the normal level, and this was promoted to approximately 84% of NGF, but not cytochrome c, injections into the hippocampus. Grafts placed into the intact hippocampus (group III) did not raise ChAT activity above the lesion-alone level, and this was not influenced by NGF injections. Acetylcholinesterase (AChE) histochemistry showed no difference in outgrowth from the grafts in the denervated hippocampus with or without NGF injections. The results are interpreted, in agreement with observations in tissue culture, as indicating that NGF enhances ChAT activity in grafted neurons, rather than promoting survival and growth per se.     

Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post-natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth-phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.     

Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions

Nerve growth factor (NGF) was injected intraventricularly during 4 weeks into adult rats with unilateral partial lesions of the cholinergic septo-hippocampal pathway. On the lesioned side, NGF treatment elevated choline acetyltransferase (ChAT) activity up to 60% above the activity measured on the lesioned side of cytochrome c-treated controls. On the unlesioned side, NGF treatment increased ChAT activity only to an insignificant degree. ChAT activity in the septum of NGF-treated animals was increased by 60% as compared to controls. The NGF-induced increases on the lesioned side and in the septum were not accompanied by elevations in acetylcholinesterase (AChE) activity. Furthermore, histochemical analysis revealed no difference in AChE staining pattern or intensity between NGF-treated and control animals. The lack of effect on AChE strongly suggests that the increases in ChAT activity in hippocampus and septum are due to an elevation of ChAT activity within cholinergic neurons surviving the lesion rather than to a promotion of sprouting of cholinergic fibers.     

Basic fibroblast growth factor in neuronal cultures of human fetal brain

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week-old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA-stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross-reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell-associated protein both in Western blot and in immuno-precipitation experiments. All the neurons showed bFGF immunoreactivity, as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.     

Distinct neuronal growth hormone receptor ligand specificity in the rat brain

A cerebral growth hormone axis is activated during recovery from brain injury and centrally administered growth hormone can rescue injured neurons. It remains unclear, however, whether this treatment effect occurs directly via neuronal growth hormone receptors. Immunohistochemistry confirmed growth hormone receptor protein on neuronal cell bodies in the rat cortex. Surprisingly, we found that central treatment with bovine growth hormone, which is equipotent to rat growth hormone in the rat periphery, failed to rescue cortical neurons following hypoxic ischemic injury. We further investigated the actions of rat and bovine growth hormone on primary neuron-enriched cultures of fetal rat cortex. In agreement with the in vivo treatment studies, rat but not bovine growth hormone rescued neurons from nutrient deprivation-induced cell death (p<0.05). This neuroprotective effect was inhibited by the selective growth hormone receptor antagonist G120D (p<0.001). Furthermore, rat but not bovine growth hormone had trophic effects on uninjured cultures (p<0.001). Immunocytochemistry showed growth hormone receptor on neurons within the neuron-enriched cultures. We show for the first time that the protective and trophic effects of rat growth hormone are mediated via growth hormone receptors on neurons and that the rodent neuronal growth hormone receptor exhibits unique ligand specificity.     

Alterations in cellular phenotypes differentiating from embryonic rat brain neurosphere cultures by immunoselection of neuronal progenitors

The neurosphere culture system is widely used to expand neural stem/progenitor cells in vitro and to provide a source of cells for transplantation approaches to CNS disorders. This study describes the populations of neurones, astrocytes and oligodendrocytes which differentiated from embryonic day (E) 14 rat cortical and striatal tissue grown as neurosphere cultures over three passages. The percentages of cells that adopted neuronal phenotypes decreased with passage, astrocytic percentages increased and oligodendrocytic percentages remained constant. In the second part of this study, immunomagnetic separation was used to positively select neuronal progenitor cells from E14 rat cortical and striatal tissue using an antibody, 2F7, which recognises an epitope on the cell surface of pre- and post-mitotic neurones. These immunomagnetically selected cells were grown as neurosphere cultures over three passages and gave rise to significantly different percentages of neurones, astrocytes and oligodendrocytes than those found in the baseline study. In particular, the percentage of neurones arising from the second and third passages was significantly higher following immunoselection. This indicates that neuronal progenitor cells can be isolated using immunomagnetic separation and then expanded using the neurosphere culture system, to generate enriched populations of neurones that can be used in CNS repair.     

Biological activity of a new neuronal growth factor from injured peripheral nerve

In response to transection injury, the distal nerve segment produces a soluble neurite promoting factor (SN). In this study, the ability to support neuronal survival and differentiation have been studied. Embryonic rat dorsal root ganglion (DRG) neurons were plated out on collagen substrates and incubated in medium containing either SN or nerve growth factor (NGF). The number of surviving neurons was counted after 1, 2, 4, 7, and 15 days in vitro. After fixation and staining, the diameter of the surviving neurons was measured. During the period of observation, 60.8 +/- 5.8% of plated neurons survived in the presence of NGF and 90.5 +/- 12.9% survived with SN (P less than 0.05). The mean of median neuronal cell diameter was 28 +/- 2.7 microns with NGF and 34.2 +/- 3.7 microns with SN, (P less than 0.01). This increased diameter was due to enhanced survival of 30-50 microns diameter neurons. In parallel experiments, the degree of myelination of DRG neurons by Schwann cells was assessed morphometrically. In the presence of SN there was an 86% increased in myelination compared with NGF which indicates that not only is the survival of neurons increased but they are able to become fully differentiated in the presence of SN.     

Distribution of neuronal receptors for nerve growth factor in the rat

To survey the distribution of neuronal receptors for NGF, sections of the rat brain, spinal cord, and peripheral ganglia were incubated in vitro with radioiodinated NGF and examined by autoradiography. NGF binds selectively with high affinity to most sympathetic neurons and many primary sensory neurons together with their intraspinal or intramedullary axons. In autoradiographs of the brain, labeled neuronal perikarya are seen in the basal forebrain, the caudate-putamen, the medulla oblongata, the ventral cochlear nucleus, and the dorsal nucleus of the lateral lemniscus. The distribution of neurons binding NGF resembles the distribution of cholinergic neurons in the forebrain, but these 2 systems overlap very little in the brain stem. In extracts of the brain or spinal cord enriched for plasma membranes, avid binding sites are regionally manifest with properties similar to those of fetal peripheral neurons. The localization of neurons expressing the high-affinity receptor for NGF defies simple correlation with neurotransmitter function or embryogenesis.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

Physical activity increases mRNA for brain-derived neurotrophic factor and nerve growth factor in rat brain

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) support the viability and function of many types of neurons, and are likely mediators of activity-dependent changes in the CNS. We examined BDNF and NGF mRNA levels in several brain areas of adult male rats following 0, 2, 4, or 7 nights with ad libitum access to running wheels. BDNF mRNA was significantly increased in several brain areas, most notably in the hippocampus and caudal13 of cerebral cortex following 2, 4, and 7 nights with exercise. Significant elevations in BDNF mRNA were localized in Ammon's horn areas 1 (CAI) and 4 (CA4) of the hippocampus, and layers II–III of the caudal neocortex and retrosplenial cortex. NGF mRNA was also significantly elevated in the hippocampus and caudal13 of the cortex, affecting primarily the dentate gyrus granular layer (DG) and CA4 of the hippocampus and layers II–III in caudal neocortex.