Reversal of ethanol toxicity in embryonic neurons with growth factors and estrogen

Prenatal exposure to ethanol is the cause of fetal alcohol syndrome, which is characterized by brain abnormalities and decreased mental capacity. In the current study, cultured neurons from embryonic rat cortices were used to study the reversal of ethanol toxicity on neuronal survival and neurite outgrowth. Ethanol treatment followed by treatment with estrogen and certain growth factors were used to assess the potential of these growth factors and estrogen to reverse the effects of ethanol damage. Cortical neurons from embryonic day (E) 16 rats were grown in defined medium with a glial plane at a distance of 1mm from the neurons. Ethanol (45 mM) was administered on day in vitro 1 (DIV 1) and DIV 4. Insulin-like growth factor-I (IGF-I, 10 ng/ml), insulin-like growth factor-II (IGF-II, 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), nerve growth factor (NGF, 100 ng/ml), and estrogen (Es, 10 ng/ml) were administered on DIV 4 and DIV 5. Cell viability was determined on DIV 6 using the intravital dyes fluorescein diacetate and propidium iodide. IGF-I and bFGF reduced ethanol's toxic effect on neuronal survival. Estrogen, bFGF, and NGF increased total neurite length after ethanol treatment. Although none of the treatments had a statistically significant effect on the mean number of primary neurites, all caused a statistically significant increase in the mean number of secondary neurites per cell (a measure of neuritic branching) relative to the ethanol treatment alone.     

Reversal of ethanol toxicity in embryonic neurons with growth factors and estrogen

Prenatal exposure to ethanol is the cause of fetal alcohol syndrome, which is characterized by brain abnormalities and decreased mental capacity. In the current study, cultured neurons from embryonic rat cortices were used to study the reversal of ethanol toxicity on neuronal survival and neurite outgrowth. Ethanol treatment followed by treatment with estrogen and certain growth factors were used to assess the potential of these growth factors and estrogen to reverse the effects of ethanol damage. Cortical neurons from embryonic day (E) 16 rats were grown in defined medium with a glial plane at a distance of 1 mm from the neurons. Ethanol (45 mM) was administered on day in vitro 1 (DIV 1) and DIV 4. Insulin-like growth factor-I (IGF-I, 10 ng/ml), insulin-like growth factor-II (IGF-II, 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), nerve growth factor (NGF, 100 ng/ml), and estrogen (Es, 10 ng/ml) were administered on DIV 4 and DIV 5. Cell viability was determined on DIV 6 using the intravital dyes fluorescein diacetate and propidium iodide. IGF-I and bFGF reduced ethanol's toxic effect on neuronal survival. Estrogen, bFGF, and NGF increased total neurite length after ethanol treatment. Although none of the treatments had a statistically significant effect on the mean number of primary neurites, all caused a statistically significant increase in the mean number of secondary neurites per cell (a measure of neuritic branching) relative to the ethanol treatment alone.     

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.     

Influence of laminin on the responsiveness of early chick embryo neural tube neurons to nerve growth factor

Dissociated neurons from the neural tube containing the trigeminal motor nucleus from early chick embryos were cultured on laminin or collagen-polyornithine substrates, with and without nerve growth factor (NGF). Control cultures were grown in similar conditions with cytochrome-C. It was found that neuronal survival was not affected by NGF or cytochrome-C, but it was enhanced by laminin. The expression of neuritic processes, however, was significantly enhanced in the presence of NGF on both laminin and collagen-polyornithine surfaces, with the greatest number of neurons producing processes seen in the laminin-plus-NGF group. The length of processes was similarly enhanced by laminin and by NGF. Cytochrome-C did not influence any of these measures. The results indicate that while laminin potentiates the NGF effect on these early neuronal populations, NGF in conjunction with other substrata can have similar, though less dramatic, effects. These results, together with prior evidence of NGF receptors and specific NGF retrograde transport, suggest that this growth factor may play a significant role in the normal ontogeny of early motoneuron populations.     

Effects of insulin, insulin-like growth factor-II, and nerve growth factor on neurite formation and survival in cultured sympathetic and sensory neurons

Insulin and the insulin-like growth factors (IGFs) may directly affect the development of the nervous system. NGF, IGF-II, and insulin's effects on neurite formation and neuronal survival were studied in peripheral ganglion cell cultures from chick embryos. Neurite outgrowth was enhanced in a dose-dependent manner by insulin and IGF-II in sympathetic cell cultures. The half-maximally effective concentration, ED50, was about 0.4-0.6 nM for both polypeptides, and concentrations as low as 10 pM were active. However, in sensory neurons the ED50 for neurite outgrowth was about 30 nM for insulin and 0.1 nM for IGF-II, suggesting that these factors may have selective effects in different neuronal tissues. Neither serum nor the presence of non-neuronal cells was required for the response in sympathetic neurons. The specific anti-NGF antiserum inhibited the neurite outgrowth response to NGF but not to insulin nor IGF-II. Insulin and IGF-II additionally supported survival of sensory and sympathetic neurons; however, insulin was not as efficacious as NGF. The combination of high concentrations of NGF and insulin was no better than NGF alone in supporting sympathetic cell survival, or neurite outgrowth. This indicates that insulin acts on the same, or a subpopulation, of NGF-responsive neurons. These results support the hypothesis that insulin and its homologs belong to a broad family of neuritogenic polypeptides.     

Estrogen Regulates Neurofilament Gene Expression in Adult Female Rat Dorsal Root Ganglion Neurons

Recently, adult female dorsal root ganglion (DRG) neurons were shown to express the estrogen receptor gene and to bind estrogen. This gene expression and binding is hormone dependent. Moreover, in a subpopulation of DRG neurons, the estrogen receptor is colocalized with high-affinity (trkA) and low-affinity (p75^NGFR) receptors for nerve growth factor (NGF). In this NGF-responsive subpopulation of DRG neurons, estrogen regulates expression of the NGF receptor genes and may increase the sensitivity of these cells to the neurotrophin. The present study tested the hypothesis that neurofilament gene expression, which is regulated by NGF in these cells, is dependent on hormone status. In this study, ovariectomized (OVX) rats received either long-term physiological estrogen (conjugated estrogens; Premarin, Wyeth-Ayerst) replacement (low dose) or 10 times physiological levels (high dose). Quantitativein situhybridization with an RNA probe for the 68-kDa neurofilament mRNA revealed a significant dose-dependent effect of Premarin on DRG neurofilament gene expression. In OVX animals receiving low-dose Premarin replacement therapy the mean steady-state 68-kDa mRNA level was as high as 4 times that of untreated OVX rats. High-dose therapy increased the mean 68-kDa neurofilament steady-state mRNA level to as much as six-fold that observed in untreated OVX animals. The estrogen-dependent upregulation of neurofilament gene expression appeared to occur in all DRG neurons, rather than in a subpopulation of those cells. These data suggest an important role for estrogen in the maintenance and function of primary sensory neurons. Whether estrogen directly regulates neurofilament gene expression or indirectly regulates it by increasing DRG neuronal sensitivity to neurotrophins or other growth factors remains to be determined.     

Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses,enhanced morphological features,and decreased dependence on nerve growth factor

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons. © 1996 Wiley-Liss, Inc.     

Transplantation of fetal and early postnatal rat septal cholinergic neurons cultured in serum-free and serum-containing medium with nerve growth factor

Fetal rat (E17-E19) septal neurons were cultured in a defined, serum-free medium for 6-8 days with or without nerve growth factor (NGF) and transplanted into the hippocampus or the surrounding ventricle of 28 adult rats denervated of its septal input by a fimbria-fornix transection. The cholinergic septal neurons, which were visualized by acetylcholinesterase (AChE) histochemistry, always survived in transplantation to the adult brains from nearly pure neuronal cultures. Although choline acetyltransferase (ChAT) activity of septal neurons in culture was greatly increased (5.59-fold) by the addition of NGF to the defined medium, this ChAT induction appeared to have little effect on the subsequent survival or growth of the septal neurons after transplantation. These results demonstrate that survival of cultured fetal septal cholinergic neurons following transplantation is not dependent upon the presence of NGF or serum- or glia-derived factors during the preliminary culture. Postnatal rat (P4) septal neurons cultured for 5 days in serum-containing medium with NGF were also successfully transplanted in one of 3 cases.     

Human nerve growth factor prevents degeneration of basal forebrain cholinergic neurons in primates

Basal forebrain cholinergic neurons respond to nerve growth factor (NGF), and it has been suggested that the administration of NGF might prevent their degereration in patients with Alzheimer's disease. One major prerequisite to be fulfilled before the consideration of clinical trials of NGF in patients with Alzheimer's disease is the demonstration that human NGF affects basal forebrain cholinergic neurons in primates. In the present study, we used a recombunant human nerve growth facotr (rhNGF), which we previousl showed to be active on rat basa forebrain cholinergic neurons, in nonhuman primates with a unilateral transection of the fornix (a well-established model for the induction of retrograde degenerative changes in septal cholinergic neurons). After the lesion, one group of animals received rhNGF and a second group received vehicle solution for 2 weeks. In animals receiving vehicle, the medical septal nucleus ipsilateral to the lesion showed reductions in numbers (55°) and size of cell bodies immunoreactive for NGF receptor and choline acetyltransferase. In Sissl stains, many cells showed frduced size and basophilia. The rhNGF completely prevented alternation in the number and size of NGF receptor—and choline acetyltransferase—immunnoreactive neurons in the medical septal nucles and reversed atrophy in a subpopulation of large, basophilic medical which we have previously used in the same primate lesion paradigm. The restoration of the phenotype of injured acetylcholine-dependent memory impariments that occur in aged nonhuman primates. In concert, results of the present investigation provide critical information for the futrue use of NGF in in patients with neurological disorders that affect NGF-responsive cells in the peripherl and central nervous system.     

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of choline acetyltransferase activity in coculture of rat septal and hippocampal neurons

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.     

Reversibility of Nerve Growth Factor's Enhancement of Choline Acetyltransferase Activity in Cultured Embryonic Rat Septum

Choline acetyltransferase (ChAT) activity and survival of acetylcholinesterase (AChE)-positive neurons were measured in low-density cultures of embryonic (Day 14-15) rat septum exposed to various sequences of nerve growth factor (NGF) exposure and deprivation for up to 7 weeks in vitro. Most septal cultures grown 4-5 weeks with no exogenous NGF (including exposure to monoclonal or polyclonal antibodies against NGF) retained both a basal ChAT activity and the ability to increase ChAT activity in response to subsequently added NGF. When cultures were exposed to NGF (7S, 0.75 nM) for 2-3 weeks and then deprived of NGF for 2 weeks, ChAT activity fell gradually, but the number of AChE-positive neurons remained unchanged, and in many cases ChAT activity could be restored by subsequent re-exposure to NGF. Thus NGF's enhancement of ChAT activity in embryonic septal neurons in vitro is largely reversible and is not mediated by differential survival of cholinergic neurons.     

Loss of Developing Cholinergic Basal Forebrain Neurons Following Excitotoxic Lesions of the Hippocampus: Rescue by Neurotrophins

Previous studies have demonstrated that the viability of developing cholinergic basal forebrain neurons is dependent upon the integrity of neurotrophin-secreting target cells. In the present study, we examined whether infusions of nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) could prevent the loss of cholinergic septal/diagonal band neurons following excitotoxic lesions of their target neurons within the hippocampus. Postnatal Day 10 rat pups received unilateral intrahippocampal injections of ibotenic acid. Rats then received intracerebroventricular (icv) injections of nerve growth factor (30 μg/injection), brain-derived neurotrophic factor (60 μg/injection), or saline immediately following the lesion and continuing every third day for 27 days. Both saline- and BDNF-treated rats displayed a significant loss of septal/diagonal band neurons expressing the protein and mRNA for choline acetyltransferase (ChAT) and p75 low-affinity nerve growth factor receptor ipsilateral to the lesion. The magnitude of this loss was significantly attenuated in BDNF-treated rats. Many remaining neurons were atrophic with stunted dendritic processes. In contrast, NGF treatment completely rescued these cells and prevented the shrinkage of remaining cholinergic septal neurons. In addition, both NGF and BDNF induced a sprouting of cholinergic processes within the residual hippocampal remnant ipsilateral to the infusions. The present study demonstrates that icv injections of NGF, and to a lesser extent BDNF, prevent the loss of developing basal forebrain neurons which occurs following removal of normal target cells. Diffusion studies revealed relatively poor penetration of BDNF into brain parenchyma. Thus, it remains to be determined whether the failure of BDNF to provide optimal trophic support for these cells is biological or due to restricted bioavailability of this trophic factor.     

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.     

Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulin-like growth factors I and II

To study the selectivity of neurotrophic actions in the brain, we analyzed the actions of several known growth factors on septal cholinergic, pontine cholinergic, and mesencephalic dopaminergic neurons in culture. Similar to nerve growth factor (NGF), basic fibroblast growth factor (bFGF) stimulated choline acetyltransferase activity in septal cultures. In contrast to NGF, bFGF also enhanced dopamine uptake in mesencephalic cultures and stimulated cell proliferation in all 3 culture types. Insulin and the insulin-like growth factors I and II stimulated transmitter-specific differentiation and cell proliferation in all culture types. Epidermal growth factor (EGF) produced a small increase in dopamine uptake by mesencephalic cells and stimulated cell proliferation in all culture types. In septal cultures, bFGF was most effective when given at early culture times, NGF at later times. The stimulatory actions of bFGF and insulin did not require the presence of glial cells and were not mediated by NGF. In mesencephalic cultures, the stimulation of dopamine uptake by bFGF and EGF was dependent on glial proliferation. The results suggest different degrees of selectivity of the neurotrophic molecules. NGF and, very similarly, bFGF seem to influence septal cholinergic neurons directly and rather selectively, whereas the neurotrophic actions of insulin and the insulin-like growth factors appear to be more general.     

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.     

Long-Term Estrogen Replacement Coordinately Decreases trkA and β-PPT mRNA Levels in Dorsal Root Ganglion Neurons

A population of adult dorsal root ganglion (DRG) neurons bind NGF with high affinity and express the trkA gene. In these cells, NGF regulates gene expression and function. Recently, a number of laboratories reported the presence of estrogen receptors in DRG neurons and profound effects of estrogen on DRG gene expression. Our laboratory, for example, has reported a significant and coordinate decrease in DRG trkA and beta-preprotachykinin (beta-PPT) mRNA levels following 90 days of daily estrogen injections to ovariectomized (OVX) rats. These data suggest, as has been suggested for medial septal cholinergic neurons, that estrogen may collaborate with NGF in the regulation of DRG neuronal gene expression and function. The current study examined further this potential collaboration in the DRG by determining the effect of short-term estrogen replacement in OVX rats on DRG trkA mRNA levels following sciatic nerve transection and the resulting removal of a vital source of NGF for those cells. In OVX rats, about 40% of lumbar DRG neurons contained trkA mRNA. Short-term estrogen replacement had no effect on the percentage of neurons containing trkA mRNA, but increased the mean trkA mRNA level in uninjured DRGs of OVX rats by 23%. Axotomy in OVX rats reduced the mean trkA mRNA level by 55% but did not significantly decrease the percentage of neurons containing the mRNA. Estrogen replacement, 7 days after axotomy, partially and significantly restored the mean trkA mRNA level. It was 49% greater than that of the untreated axotomized DRGs. It did not, however, significantly increase the percentage of DRG neurons containing trkA in axotomized DRGs. These observations show that short-term estrogen has an opposite effect on DRG neuronal trkA mRNA levels as compared to that of long-term estrogen demonstrated in our previous study. Moreover, the current data show that estrogen regulates trkA mRNA levels in the absence of target-derived NGF. These data suggest that estrogen may collaborate with NGF in the maintenance of normal adult DRG gene expression and function. Furthermore, these data suggest that loss of estrogen, such as that associated with menopause, may contribute to a decline in DRG neuronal function and an exacerbation of ongoing neuropathic processes.     

Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor.

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.     

Interferon-γ inhibits DNA synthesis and insulin-like growth factor-II expression in human neuroblastoma cells

Interferon-γ (IFN-γ) is known to be an antiproliferative, differentiation agent in many cell types, including neuroblastoma. In this study, we determined the effects of IFN-γ on cellular growth and expression of insulin-like growth factor II (IGF-II) and IGF receptors in the human neuroblastoma cell line SH-SY5Y. Incubation of SH-SY5Y cells in IFN-γ (20–100 U/ml) induced the formation of long neuritic processes. IFN-γ treatment also induced decreases in [³H]TdR incorporation, as well as serum-dependent changes in cell number. Treatment with IFN-γ reduced cell number 33% in the presence of serum but had no effect on cell number in the absence of serum. IGF-II mRNA content was 60% inhibited by IFN-γ, and was not serum dependent. The concentration of immunoreactive IGF-II in SH-SY5Y conditioned medium was also reduced in the presence of IFN-γ, to less than half of control levels. In contrast, type I IGF receptor mRNA content was increased more than three-fold after treatment with IFN-γ and serum. Co-incubation in IFN-γ (20–100 U/ml) and IGF-II on (3–10 nM) prevented the inhibitory effects of IFN-γ on [³H]TdR ncorporation in serum-free media. Our results suggest that IFN-γ may inhibit DNA synthesis and cell growth by interfering with an IGF-II/type I IGF receptor autocrine growth or survival mechanism.