Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca²⁺) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K⁺). Changes in [Ca²⁺]; across time were measured in cultures of neurons treated without ethanol or with 100, 2110, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca²⁺]_i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca²⁺]_i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca²⁺]_i with the greatest increase in basal [Ca²⁺]_i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca²⁺]_i; during stimulation with high K⁺. The greatest increases in [Ca⁺]_i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K⁺ stimulated changes in [Ca²⁺]_i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca²⁺ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca²⁺]_i, following ethanol treatment are greater when NGF is present.     

Chronic ethanol treatment alters AMPA-induced calcium signals in developing Purkinje neurons

Cerebellar Purkinje neurons developing in culture were treated chronically with 30 mM (140 mg%; 3-11 days in vitro) ethanol to study the actions of prolonged ethanol exposure on responses to exogenous application of AMPA, a selective agonist at the AMPA subtype of ionotropic glutamate receptors. There was no consistent difference between control and chronic ethanol-treated neurons in resting membrane potential, input resistance, or the amplitude or duration of the membrane responses to AMPA (1 or 5 microM applied by brief microperfusion) as measured using the nystatin patch method of whole cell recording. In additional studies, the Ca2+ signal to AMPA was examined using the Ca2+ sensitive dye fura-2. The mean peak Ca2+ signal elicited by 5 microM AMPA was enhanced in the dendritic region (but not the somatic region) of chronic ethanol-treated Purkinje neurons compared to control neurons. In contrast, there was no difference between control and chronic ethanol-treated neurons in the peak amplitude of the Ca2+ signal to 1 microM AMPA, whereas the recovery of the Ca2+ signals was more rapid in both somatic and dendritic regions of ethanol-treated neurons. Resting Ca2+ levels in the somatic and dendritic regions were similar between control and ethanol-treated neurons. These data show that the membrane and Ca2+ responses to AMPA in Purkinje neurons are differentially affected by prolonged ethanol exposure during development. Moreover, chronic ethanol exposure produces a selective enhancement of AMPA-evoked dendritic Ca2+ signals under conditions reflecting intense activation (i.e., 5 microM AMPA), whereas both somatic and dendritic Ca2+ signals are attenuated with smaller levels of activation (i.e., 1 microM AMPA). Because Ca2+ is an important regulator of numerous intracellular functions, chronic ethanol exposure during development could produce widespread changes in the development and function of the cerebellum.     

Cultured postnatal rat septohippocampal neurons change intracellular calcium in response to ethanol and nerve growth factor

Ethanol exposure affects cellular mechanisms involved in the regulation of calcium (Ca²⁺) homeostasis. Neurotrophins, such as nerve growth factor (NGF), stabilize intracellular Ca²⁺([Ca²⁺]_i) during a variety of neurotoxic insults. In this study, changes in [Ca²⁺]_i during treatment with ethanol and NGF were measured at the cell body of neurons using the Ca²⁺ indicator indo-1. Cultured postnatal day-of-birth (P0) septohippocampal (SH) neurons that were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI), increased [Ca²⁺]_i in response to ethanol. This response was dose-related. P0 SH neurons treated with NGF had lower [Ca²⁺]_i than neurons withdrawn from NGF, implying that NGF may modulate Ca²⁺ homeostasis in these neurons. NGF also prevented the dose-related increase in [Ca²⁺]_i in ethanol-treated SH neurons. The SH neurons increased [Ca²⁺]_i when they were stimulated with 30 mM potassium chloride (KCl). Ethanol inhibited the potassium-stimulated change in [Ca²⁺]_i but the combination of ethanol and NGF caused [Ca²⁺]_i to increase with 100 mg% and 400 mg% ethanol and to decrease to a lower level with 200 mg% ethanol. These data were compared to data from previously published similar aged medial septal (MS) neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Clin. Exp. Res. 20 (1996) 1385–1394) and with embryonic gestational day 21 (E21) SH neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Brain Res. 729 (1996) 176–189). Differences in [Ca²⁺]_i responses were observed in ethanol and NGF-treated postnatal SH neurons compared with P0 MS neurons and E21 SH neurons. Of these differences, most occurred during the combined treatment with ethanol and NGF compared with either treatment alone.     

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)     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Nerve growth factor receptor-immunoreactive neurons within the developing human cortex.

A monoclonal antibody recognizing the p75 receptor for nerve growth factor (NGF) was used to assess the immunohistochemical expression of NGF receptors within the developing human neo-, limbic, and paralimbic cortices as well as the hippocampal complex. Between embryonic weeks 16 and 26, a transient population of neurons located within the upper and lower subplate zones of the neo-, limbic, and paralimbic cortices expressed the receptor for NGF. In contrast, NGF receptor-immunoreactive neurons were only observed in the upper subplate zone of the entorhinal cortex at embryonic week 40 (term), a staining pattern not observed in a 5-year-old specimen. The expression of NGF receptor-immunoreactive neurons within the upper subplate zone between embryonic weeks 16 and 40 was characterized by a dense band of immunoreactive neurons and neuropil. These neurons were bipolar with basal and apically directed neurites. NGF receptor-immunoreactive neurons were also scattered throughout the lower subplate zone and underlying white matter between embryonic weeks 19 and 26. These neurons were multipolar, with less apically directed neurites. NGF receptor-immunoreactive subplate neurons displayed a topographic distribution with the heaviest concentration found within limbic and paralimbic cortices as well as association neocortex. In contrast, light to moderate NGF receptor-immunoreactivity was seen in sensory-motor cortex. Within the hippocampal complex, only a few lightly stained NGF receptor-immunoreactive neurons were seen within the fimbria, hilar region of the dentate gyrus, and subiculum. The expression of NGF receptor-immunoreactivity increased within the subplate zone of the pre- and parasubiculum culminating in intense entorhinal cortex staining. As the entorhinal cortex merged with the developing inferior temporal association cortex, there was a marked reduction in staining intensity. In contrast to those in the subplate zone, neurons within the germinal zone and cortical plate were NGF receptor immunonegative at all times examined. The presence of NGF receptors in the subplate zone suggests that neurotrophins such as NGF play an important role in the transient viability of these neurons as well as in the guidance of cortical afferent inputs into topographically organized regions of the cerebral cortex.     

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.     

Nerve growth factor regulates synaptophysin expression in developing trigeminal ganglion neurons in vitro

The role of neuronal growth factors in synaptic maturation of sensory neurons, including trigeminal ganglion (TG) neurons, remains poorly understood. Here, we show that nerve growth factor (NGF) regulates the intracellular distribution of the synaptic vesicle protein synaptophysin (Syp) in newborn rat TG neurons in vitro. While reducing the number of Syp-positive cell bodies, NGF dramatically increases Syp immunoreactivity in both proximal and distal segments of the neurite. Intriguingly, the increase in Syp immunoreactivity occurs only in neuron-enriched cultures, in which the number of non-neuronal cells is significantly reduced. Together, our data indicate that NGF is a candidate molecule involved in early postnatal maturation of TG neurons, including control of presynaptic assembly, and thereby formation of synaptic connections.     

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca²⁺) homeostasis in these neurons was investigated in this study. Changes in intracellular-free Ca²⁺ concentration ([Ca²⁺]_i) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCl⁺). SH neurons were treated with 0, 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (−NGF). NGF treatment decreased, while ethanol did not affect basal [Ca²⁺]_i. The combination of ethanol and NGF treatment led to increases in basal [Ca²⁺]_i. While [Ca²⁺]_i was lower during stimulation with KCl⁺ following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca²⁺]_i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca²⁺]_i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca²⁺ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca²⁺ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity.     

Rescue of lesioned septal cholinergic neurons by nerve growth factor: specificity and requirement for chronic treatment

We earlier reported that chronic intraventricular injections of NGF into adult rats with partial transection of the fimbria prevent the lesion-induced disappearance of cholinergic neurons in the medial septal nucleus and the diagonal band of Broca (Hefti, 1986). The present study assessed the specificity and treatment requirements of this effect of NGF. Immunohistochemical visualization of NGF receptors (NGF-R) revealed that these molecules are selectively located in forebrain cholinergic neurons of unlesioned brains. Fimbrial transection resulted in transient accumulation of NGF-R in proximal stumps of lesioned axons but failed to induce the expression of NGF-R by other cells in the septal area or near the lesion. Two to three weeks after lesioning, the number of septal neurons expressing NGF-R was reduced by approximately 50% in parallel with the reduction of the number of neurons expressing cholinergic marker enzymes. Repeated intraventricular NGF injections during 4 weeks prevented the disappearance of these cells. Fimbrial transections also reduced the number of septal GABAergic neurons visualized by glutamate decarboxylase immunohistochemistry. The loss of GABAergic neurons was not prevented by NGF. These findings suggest that NGF prevents the lesion-induced degeneration of cholinergic neurons by directly acting on NGF-R expressed by cholinergic cells and that NGF does not affect any neuron with an axonal lesion. Delayed start of the NGF treatment failed to prevent the disappearance of lesioned cholinergic neurons, providing evidence that NGF treatment indeed promotes the survival of these cells rather than simply upregulating the expression of transmitter-specific enzymes. A single injection of NGF at the time of the lesion was not sufficient to prevent the lesion-induced degeneration of cholinergic neurons. Furthermore, termination of chronic NGF treatment after 4 weeks was followed by loss of septal cholinergic neurons after an additional 4 weeks. These findings suggest that the continuous presence of NGF during more than 4 weeks is required to prevent the degeneration of cholinergic cells. The data are discussed in the context of a possible physiological role of NGF in the function of adult forebrain cholinergic neurons.     

Nerve growth factor treatment in dementia

Millions of people are affected by Alzheimer disease. As longevity increases, so will the number of patients with dementia. This has led to an intense search for successful treatment strategies. One area of interest is neurotrophic factors. Brain development and neuronal maintenance, as well as protective efforts, are mediated by a large number of different neurotrophic factors acting on specific receptors. In neurodegenerative disorders, there may be a possibility of rescuing degenerating neurons and stimulating terminal outgrowth with use of neurotrophic factors. The first neurotrophic factor discovered was nerve growth factor (NGF). A wealth of animal studies have shown that cholinergic neurons are NGF sensitive and NGF dependent, which is especially interesting in cognitive disorders, in which central cholinergic projections are important for cognitive function. In Alzheimer disease, cholinergic neurons have been shown to degenerate. This suggests that NGF may be used to pharmacologically counteract cholinergic degeneration and/or induce terminal sprouting in Alzheimer disease. Data from animal studies, as well as from the author's recent clinical trial, in which NGF was infused to the lateral ventricle in patients with Alzheimer disease, will be presented. Effects of NGF on cognition, as well as issues regarding dosage, side effects, and alternative ways of administering NGF, will be discussed.     

Transient elevation of nerve growth factor content in the rat hippocampus and frontal cortex by chronic ethanol treatment

Abstract The nerve growth factor (NGF) content in the hippocampus and frontal cortex of chronic ethanol-treated rats was measured and compared with that of control rats, using a two-site enzyme immunoassay (EIA) system. The different time periods of chronic ethanol treatment caused transient elevation of the NGF content in both the hippocampus and frontal cortex. The NGF content in the hippocampus was significantly elevated in rats undergoing ethanol treatment of 2 weeks and 1 month. Nerve growth factor content of the 1 month treatment was higher than that of the 2 week treatment. However, a 3 month administration of ethanol reduced the NGF content to the control level. The NGF content in the frontal cortex increased significantly in the 2 week administration, but decreased to the control level in the 1 month administration. The increase of NGF may be caused by the proliferation of glial cells or the enhancement of neuronal production of NGF.     

Transforming growth factor α differentially affects GABAergic and cholinergic neurons in rat medial septal cell cultures

The effects of transforming growth factor α (TGFα) on low and high density cultures of fetal (embryonic day 17) rat medial septal cells were investigated and in some instances, compared to those of epidermal growth factor (EGF). In high density cultures, TGFα induces a significant increase in the number of astroglia and microglia. While the effects of TGFα on the astroglia are more pronounced when compared to EGF, those on the microglia are less notable. In addition, TGFα produces a time- and dose-dependent decrease in the activity of choline acetyltransferase (EC 2.3.1.6) and a proportional decrease in the number of acetylcholinesterase-positive neurons in these high density cultures. However, although both EGF and TGFα decreased choline acetyltransferase activity maximally at the same concentration (10 ng/ml), the latter was consistently more potent. TGFα does not affect cholinergic cell survival but the expression of their chemical phenotype and does so indirectly via the glial cells. On the other hand, TGFα directly induces a dose and time-dependent increase in glutamic acid decarboxylase activity in these high density cultures without affecting the number of glutamic acid decarboxylase immunoreactive neurons. In low density cultures, TGFα acts as a general neuronal survival factor, affecting both cholinergic and GABAergic neurons. Here TGFα's neurotrophic activity is more evident than its effects on their chemical phenotype. These results suggest that TGFα exerts distinct and differential effects on the biochemical expression of two neuronal populations in the developing medial septum maintained in high density culture. Finally, as TGFα acts as a general neuronal survival factor in low density cultures, cell to cell interactions appear to be important in the ultimate response of these cells to this growth factor.     

Nerve growth factor sensitivity is broadly distributed among myenteric neurons of the rat colon

Nerve growth factor (NGF) acts on the two-receptor system of trkA and p75 to mediate neuroprotection and influence phenotype and function in the peripheral nervous system, but the effects of NGF on the enteric nervous system (ENS) are virtually unknown. To establish a basis for enteric responsiveness to NGF, we studied the presence and distribution of NGF-sensitive receptors in the myenteric neurons of the normal rat colon and examined their activation via trkA phosphorylation. Fluorescent immunocytochemistry on wholemounts showed that the two NGF receptors were abundantly present in the ENS, with 71% of all neurons positive for trkA and 78% for p75. More thanr 60% of the myenteric neurons expressed both receptors, and exogenous application of NGF resulted in trkA phosphorylation, evidence for high NGF sensitivity within the ENS. trkA was co-expressed with choline acetyltransferase (61% of trkA-positive neurons), neuronal nitric oxide synthase (22%), or calbindin (10%), suggesting widespread potential for NGF action. We conclude that functional receptors for NGF are widely distributed among the diverse enteric phenotypes and argue for a novel NGF-mediated regulatory system within the ENS.     

Nerve growth factor and gastric hyperalgesia in the rat

We recently demonstrated an association between the development of hyperalgesia and an increase in nerve growth factor (NGF) during gastric inflammation. We hypothesized that block of NGF signalling will blunt injury-induced hyperalgesia. Male Sprague-Dawley rats (300-400 g) were anaesthetized, the stomach was exposed and placed in a circular clamp. Acetic acid (60%) or saline (control) was injected into this area and aspirated 45 s later, resulting in kissing ulcers. A balloon was surgically placed into the stomach and electromyographic responses to gastric distension (GD) were recorded from the acromiotrapezius muscle. Animals received a daily injection of neutralizing NGF antibody or control serum for 5 days. NGF in the stomach wall was measured with an ELISA. The severity of gastric injury was assessed macroscopically and by determination of myeloperoxidase (MPO) activity. Gastric injury enhanced the visceromotor response to GD and increased NGF content. Anti-NGF significantly blunted the development of hyperalgesia and led to a decrease in gastric wall thickness and MPO activity. Increases in NGF contribute to the development of hyperalgesia after gastric injury. This may be partly mediated by direct effects on afferent nerves and indirectly by modulatory effects on the inflammatory response.     

Nerve growth factor receptors on normal and injured sensory neurons

The density and binding properties of receptors for nerve growth factor (NGF) in normal and injured sensory neurons have been analyzed by quantitative radioautography following incubation of tissue sections with radioiodinated NGF. The technique is designed to study binding sites that are half-maximally saturated by picomolar concentrations of NGF: Additional sites of lower affinity have not been emphasized. In normal adult rats, approximately half of lumbar sensory neurons have high-affinity receptors for NGF. One month after the sciatic nerve is cut, the mean number of high-affinity sites on heavily labeled neurons in the fifth lumbar dorsal root ganglion falls to less than 20% of normal values because of reduced receptor density and cell volume. Neurons with high-affinity receptors are more liable to atrophy after injury than those lacking such receptors. Receptors are lost not only in the cell bodies of sensory neurons but also on their peripheral and central processes. Delayed administration of NGF to the sciatic nerve 3 weeks after it is cut restores the receptor density to normal values and partially restores the neuronal cell volume. As part of the response to axonal injury and possibly because the cell body is deprived of NGF, fewer high-affinity receptors are displayed by sensory neurons. For at least 3 weeks after nerve transection, neurons that are atrophic and depleted of NGF receptors can be resuscitated by exogenous NGF.     

Nerve growth factor modulates myelin-associatedglycoprotein binding to sensory neurons

Myelin-associated glycoprotein (MAG) is a molecule expressed by myelinating cells at the myelin/axon interface, which binds to an as yet unidentified molecule on neurons. We have used a MAG-immunoglobulin Fc fusion protein to examine the expression and regulation of the MAG binding molecule on sensory neurons in culture. Binding of the MAG-Fc reached a maximum at 24-48 h and was higher on neurons which expressed high levels of neurofilament. Nerve growth factor (NGF) upregulated expression of the MAG binding molecule in a dose dependent manner. Schwann cells co-cultured with sensory neurons in serum-free medium stimulated maximal expression of the MAG binding molecule, which was decreased by addition of anti-NGF to the co-cultures. This indicated that Schwann cells can modulate expression of the MAG binding molecule via production of NGF and may represent a physiological mechanism for regulation of MAG-MAG binding molecule interactions during myelination and remyelination.     

Nerve growth factor induces Fos-like immunoreactivity within identified cholinergic neurons in the adult rat basal forebrain

Immunocytochemical techniques were used to examine and compare the effects of intracerebroventricular administration of nerve growth factor (NGF) on Fos expression within identified cholinergic and non-cholinergic neurons located in different regions of the adult rat basal forebrain. Animals were killed 1, 3, 6, and 12 h after receiving NGF (0.5 or 5.0 μg) or vehicle into the left lateral ventricle and sections through the medial septum, diagonal band of Broca, nucleus basalis magnocellularis, and striatum were processed for the combined immunocytochemical detection of Fos and choline acetyltransferase (a marker for cholinergic neurons), or Fos and parvalbumin (a marker for gamma aminobutyric acid (GABA)-containing neurons). NGF produced a significant increase in the percentage of cholinergic neurons containing Fos-like immunoreactivity within all four regions examined. The largest increases were detected in the medial septum (47.8%) and the horizontal limb of the diagonal band of Broca (67.7%). In these areas, NGF-mediated induction of Fos-like immunoreactivity was detected as early as 3 h, peaked at 6 h, and was reduced by 12 h, postinfusion. Small but significant increases in the percentage of cholinergic neurons containing Fos-like immunoreactivity were also detected in the striatum (4.2%) and in the nucleus basalis magnocellularis (19.2%) 3–12 h following administration of the higher dose of NGF. No evidence for an NGF-mediated induction of Fos within parvalbumin-containing neurons was detected in any of the four regions at any of the time-points examined; however, evidence for an NGF-mediated induction of Fos within epithelial cells lining the lateral ventricle was observed. These data demonstrate that NGF induces Fos expression within cholinergic, and not parvalbumin-containing (GABAergic), neurons in the basal forebrain, and furthermore that intracerebroventricular administration of NGF influences the different subgroups of basal forebrain cholinergic neurons to different degrees. ©1977 Elsevier Science B.V. All rights reserved.     

Nerve growth factor responsiveness of cultured major pelvic ganglion neurons from the adult rat.

The bladder and other pelvic viscera are innervated in the rat by the major pelvic ganglion (MPG), a mixed sympathetic/parasympathetic population of neurons that participates in lower urinary pathophysiology. Neurons from the MPG of adult females were removed, dissociated and cultured in order to test retention of the neuronal phenotype and whether they responded to Nerve Growth Factor (NGF). The bladder-specific subset of MPG neurons were distinguished by retrograde labeling prior to culture. The adult ganglionic neurons adapted to culture with greater than 80% survival in the best cases. The cultured neurons retained excitability, as determined by measuring voltage-activated ionic currents. They were positive for neuron-specific beta-tubulin and many retained immunoreactivity for characteristic peptides and transmitter synthetic enzyme. The proportion of neurons in the different categories tested varied somewhat from that in vivo, but there was no evidence of selective death of a particular population. The cultured MPG neurons were responsive to NGF and anti-NGF antibody. NGF supported neuronal survival and expression of tyrosine hydroxylase. Added NGF also affected the expression of neuropeptide Y. Hypertrophied neurons from animals with experimental bladder outlet obstruction demonstrated increased responsiveness to NGF. The data suggest that NGF participates in adult neural plasticity due to continued responsiveness to the factor. Furthermore, questions concerning regulation of MPG neurons may be addressed in vitro.