Survival Of Septal Cholinergic Neurons Research Articles

Pituitary adenylate cyclase‐activating polypeptide promotes the survival of basal forebrain cholinergic neurons in vitro and in vivo: comparison with effects of nerve growth factor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. We therefore studied the effects of PACAP on septal cholinergic neurons. In primary cultures from septum of embryonic and postnatal rats, PACAP increased the number of neurons immunohistochemically stained for the low-affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also caused a corresponding increase in ChAT activity. In comparison, NGF had a greater effect than PACAP on the number of p75- and ChAT-positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons did not differ significantly from unoperated controls. In PACAP-treated rats the number was 48% of unoperated values, which represented a significant increase compared with vehicle-treated rats and a significant decrease compared with NGF-treated rats or unoperated controls. Double-staining experiments revealed that most ChAT-positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons.

Development of an antibody against a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein and identification of a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein in hippocampal neurons

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity. In this study, we have developed a monoclonal antibody against a brain injury-derived neurotrophic peptide-binding protein by immunizing mice with septal synaptosomes from five-week-old rat brain. Monoclonal antibodies were screened for inhibition of the binding of a 125I-labeled analogue of brain injury-derived neurotrophic peptide to rat brain synaptosomes. The monoclonal antibody 6A22 suppressed the biological activity of brain injury-derived neurotrophic peptide and abolished the protective effect of the neurotrophic peptide against glutamate-induced neurotoxicity. This monoclonal antibody recognized a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein, which was also identified by cross-linking experiments. Immunohistochemical studies showed that the 6A22 antibody bound to the cell surfaces of a subpopulation (about 60%) of hippocampal neurons in culture. These results are consistent with the possibility that the 40,000 mol. wt protein belongs to brain injury-derived neurotrophic peptide receptors.

Brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, insulin-like growth factor-I, and various antioxidants do not prevent the apoptotic death of developing septal cholinergic neurons following nerve growth factor withdrawal in vitro

The degree to which growth factors act alone or in combination to influence neuronal survival during the developmental of the central nervous system is not well understood. In this study, we investigated whether multiple growth factors might interact to regulate the survival of developing basal forebrain cholinergic neurons in vitro, in the rat. We have previously shown that most embryonic septal cholinergic neurons grown in sandwich cultures in serum-free, completely defined medium are dependent on nerve growth factor during a critical period of their development, such that nerve growth factor withdrawal during this period results in the protein synthesis-dependent, apoptotic death of most, but not all, of these neurons. Here we report that brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I applied individually in serum-free, completely defined medium, were not able either to support the development of septal cholinergic neurons from plating at embryonic day 16, or to prevent the cell death of these neurons induced by nerve growth factor withdrawal during days 14–18 after plating. We also found that the apoptotic death of developing septal cholinergic neurons induced by nerve growth factor withdrawal was not prevented by a number of antioxidants, with the exception of a high concentration (50mM) of ascorbic acid. However, this effect of ascorbic acid was prevented when pH was buffered, and is likely to have been mediated via a proton-induced sustained neuronal depolarization.These findings suggest that in the absence of serum and other additives, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I do not interact with nerve growth factor to regulate the survival of septal cholinergic neurons during the developmental period spanned by this in vitro model. In addition, the findings suggest that the apoptotic death of septal cholinergic neurons induced by nerve growth factor withdrawal is not mediated by oxidative stress or free radical generation.

A cyclic AMP analog and high potassium prevent the death of cultured septal cholinergic neurons after nerve growth factor withdrawal.

Survival, following the addition of a cAMP analog and high K+ to the medium, of cultured fetal septal cholinergic neurons was examined after nerve growth factor (NGF) deprivation. The number of acetylcholinesterase positive cells, which had progressively grown during 11-13 d of culture with NGF (50 ng/ml), was greatly reduced following 5 d of extended culture without NGF (55% of that with NGF). The degeneration of the cholinergic neurons was markedly reduced by addition of dibutyryl cAMP (dbcAMP, 1 mM), forskolin (10 microM) or KCl (15 mM) to the medium. K252b, which blocks the survival of NGF, had no effect on the action of dbcAMP. H-8 and nifedipine inhibited the survival of dbcAMP and high KCl, respectively. These results suggest that NGF, dbcAMP and high K+ promote the survival of septal cholinergic neurons acting via the receptor tyrosine kinase, protein kinase A and a Ca(2+)-dependent mechanism, respectively.

Atrophy but not death of adult septal cholinergic neurons after ablation of target capacity to produce mRNAs for NGF, BDNF, and NT3

The effect of unilateral excitotoxic ablation of hippocampal neurons was investigated on (1) the local production of mRNA for NGF and related neurotrophins, (2) the amount of NGF protein in the septal region, and (3) the viability and appearance of afferent septal cholinergic neurons in adult rats. After near complete ablation of hippocampal neurons, total levels of NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3) mRNA measured by quantitative Northern blot analysis in the hippocampal remnant fell significantly, to less than 25% of control values by 28 d and to less than 9% by 300 d. In the septal region ipsilateral to such lesions, NGF protein levels measured by ELISA fell significantly, to about 35% of control values, but the number of immunohistochemically detected cholinergic neurons did not decline significantly for up to 500 d. Instead, the cholinergic neurons persisted in an atrophied state, exhibiting severe shrinkage and reduced staining for the transmitter-synthesizing enzyme ChAT. The parameters of cell size and ChAT staining intensity correlated significantly with the amount of hippocampal tissue present. These findings indicate that in adult rats, target-derived NGF, BDNF, and NT3 do not regulate the survival of septal cholinergic neurons in proportion to the number of target neurons present. Moreover, the findings suggest that one or more of these target-derived neurotrophins regulate the structural and chemical phenotype of these neurons in the adult.

Trophic effect of erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in vivo

In vitro granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), and erythroid differentiation factor (EDF) augmented choline acetyltransferase (ChAT) activity in mouse embryonic primary septal neurons and in cholinergic hybridoma cell line, SN6.10.2.2. This is similar to the effects seen with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, in vivo GM-CSF and EPO promoted survival of septal cholinergic neurons in adult rats which had undergone fimbria-fornix transections. These results suggest that some of the hematopoietic factors act on cholinergic neurons as ‘neurotrophic factors’ to influence the differentiation, maintenance and regeneration of these neurons.

Culture of neuronal cells from postnatal rat brain: Application to the study of neurotrophic factors

1. The authors developed a primary culture technique for neuronal cells from postnatal rat brains and studied the effects of neurotrophic factors on the naturally developed neurons. 2. We demonstrated changes in the neurotrophic role of nerve growth factor (NGF) during the developmental stages of the rat: NGF was shown to act as a differentiation factor in the early stages and as a survival factor later. 3. It appeared that interleukin-6 (IL-6) supported the survival of septal cholinergic neurons obtained from 10-day-old rats. IL-6, however, did not induce the differentiation of embryonic rat septal cholinergic neurons. IL-6 improved the survival of mesencephalic catecholaminergic neurons from postnatal and embryonic rat brains, which have known not to be response to NGF.

The neurotrophic factor, n‐hexacosanol, reduces the neuronal damage induced by the neurotoxin, kainic acid

The long-chain fatty alcohol, n-hexacosanol, has been shown to possess neurotrophic properties in vitro on rat CNS cultures (Borg et al., 1987) and to promote the survival of septal cholinergic neurons after experimental axotomy (Borg et al., 1990). Long-chain alcohols have also been shown to be synthesized and metabolised by rat brain during development (Bishop and Hayra, 1981; Natarajan et al., 1984). The present study was undertaken in order to find out if a nonproteic neurotrophic factor like n-hexacosanol may be able to reduce the neuronal damages induced by the excitatory amino acid, kainic acid. When administered chronically by intraperitineal injection, hexacosanol (1 mg/kg) protected the pyramidal neurons of the hippocampus from the neurotoxic degeneration induced by an intracerebroventricular infusion of kainic acid in rats; the extent of the damage was limited to a small part of the CA3 region. Morphometric analysis showed that 72% of the neurons that would have died following kainic acid injection were spared by hexacosanol. Moreover the increased locomotor activity induced by the neurotoxin was also inhibited by hexacosanol and the behavioral effect was statistically correlated to the extent of neuronal loss. The present study suggests a possible role for nonproteic neurotrophic compounds against neurotoxic damages on central neurons. Moreover the peripheral administration of hexacosanol may lead to a significant breakthrough in the treatment of exicotoxin-related human diseases.

BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra

Brain-derived neurotrophic factor (BDNF), present in minute amounts in the adult central nervous system, is a member of the nerve growth factor (NGF) family, which includes neurotrophin-3 (NT-3). NGF, BDNF and NT-3 all support survival of subpopulations of neural crest-derived sensory neurons; most sympathetic neurons are responsive to NGF, but not to BDNF; NT-3 and BDNF, but not NGF, promote survival of sensory neurons of the nodose ganglion. BDNF, but not NGF, supports the survival of cultured retinal ganglion cells but both NGF and BDNF promote the survival of septal cholinergic neurons in vitro. However, knowledge of their precise physiological role in development and maintenance of the nervous system neurons is still limited. The BDNF gene is expressed in many regions of the adult CNS, including the striatum. A protein partially purified from bovine striatum, a target of nigral dopaminergic neurons, with characteristics apparently similar to those of BDNF, can enhance the survival of dopaminergic neurons in mesencephalic cultures. BDNF seems to be a trophic factor for mesencephalic dopaminergic neurons, increasing their survival, including that of neuronal cells which degenerate in Parkinson's disease. Here we report the effects of BDNF on the survival of dopaminergic neurons of the developing substantia nigra.

Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats

We have established a primary neuronal cell culture technique from the postnatal (P11 to P15) rat CNS to study the nerve growth factor (NGF) response to basal forebrain cholinergic neurons. The survival of septal cholinergic neurons in culture was monitored both by the determination of choline acetyltransferase activity and by counting acetylcholinesterase-positive cells. Cells obtained from postnatal septal regions were found to require a plentiful oxygen supply during the dissociation of the cells. NGF-mediated survival of the septal cholinergic neurons was similarly observed in the cultures by using different plating cell densities up to 12.5 × 10 5 cells/cm 2. These results suggest that the promotion by NGF of cell survival in culture is independent of plating cell density.

Hippocampal neurotrophic factors influence the cell size and survival of septal cholinergic neurons in culture

Hippocampal neurotrophic factors influence the cell size and survival of septal cholinergic neurons in culture

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)

Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor

The survival effect by nerve growth factor (NGF) on the cholinergic neurons of postnatal rat septal neurons in culture was examined. When the septal neurons from 10 to 12-day-old rats were cultured without NGF, the activities of choline acetyltransferase gradually decreased during the period of cultivation. The addition of NGF to the culture prevented the decline of activities. And, the number of acetylcholinesterase-positive neurons in culture with NGF was found to be more than that without NGF, after 5 days in culture. These results suggest that NGF promotes the survival of septal cholinergic neurons from postnatal rats in culture.

Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections

Several findings obtained in recent years suggest that NGF, aside from its well-established function as a neurotrophic factor for peripheral sympathetic and sensory neurons, also has trophic influence on the cholinergic neurons of the basal forebrain. The present study assessed whether NGF was able to affect survival of central cholinergic neurons after axonal transections in adult rats. The septo-hippocampal pathway was transected unilaterally by cutting the fimbria, and animals were implanted with a cannula through which NGF or control solutions were injected intraventricularly over 4 weeks. The lesions reduced the number of large cell bodies, as visualized by Nissl staining in the medial septal nucleus and in the vertical limb of the diagonal band of Broca. Furthermore, in the same nuclei, they reduced the number of cell bodies positively stained for AChE after pretreatment with diisopropylfluorophosphate (a method known to result in reliable identification of cholinergic neurons in the septal area). On lesioned sides, the number of cholinergic cells in medial septal nucleus and the vertical limb of the diagonal band was reduced by 50 +/- 4%, as compared to the number on contralateral sides. On lesioned sides of animals chronically treated with NGF, the number of AChE-positive cells in these areas was reduced only by 12 +/- 6%, as compared to control levels. These findings suggest that fimbrial transections resulted in retrograde degeneration of cholinergic septo-hippocampal neurons and that NGF treatment strongly attenuated this lesion-induced degeneration.(ABSTRACT TRUNCATED AT 250 WORDS)