Compensatory increase in choline acetyltransferase activity in salivary glands and diaphragm muscle of the rat.

When the function of salivary glands was abolished by applying ligatures to their ducts and the function of one half of the diaphragm muscle was abolished by sectioning of its phrenic nerve, the choline acetyltransferase activity was found to be increased in not duct-ligated glands and in the intact hemidiaphragm 4 weeks later. The increase was not seen within the first week. The increase in activity appears to be particularly manifested in the nerve endings, since it was seen in the hemidiaphragm but not in the phrenic nerve. Increased stream of impulses in the efferent nerves is thought to be the cause of this increase in choline acetyltransferase activity.     

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.     

Choline acetyltransferase in transected nerves, denervated muscles and Schwann cells of the frog: correlation of biochemical electron microscopical and electrophysiological observations.

The activity of choline acetyltransferase was increased in the peripheral nerve stumps of transected sciatic nerves of frogs (Rana esculenta) 5–21 days after the transection but had fallen markedly by the beginning of the second month. The increase of choline acetyltransferase activity began at a time when large myelinated fibres were still unaffected by degeneration according to both ultrastructural and electrophysiological criteria, whereas the small fibres were undergoing degeneration. It preceded the appearance of the miniature end-plate potentials attributable to the Schwann cells in the denervated sartorius muscle, which occurred 22–25 days after nerve section. The steep fall of choline acetyltransferase activity in the degenerating nerve coincided with the final stages of destruction of the large myelinated axons. One-fifth of control choline acetyltransferase activity was found in the degenerated nerve even 3 months after nerve section, when all axonal remnants were completely resorbed and the peripheral nerve stump was almost exclusively composed of proliferated Schwann cells and of the Büngner bands, formed by their processes. The activity of choline acetyltransferase in denervated sartorius muscle diminished, starting from the fifth day after nerve section. It did not fall below 60% of control level even 3 months after denervation, but it could not be excluded that in the muscle some of the synthesis of acetylcholine observed during assays of choline acetyltransferase activity was due to non-specific acetylcholine-synthesizing activity of carnitine acetyltransferase.The observations strongly suggest that choline acetyltransferase is present in the Schwann cells of degenerated frog nerves and that the enzyme is newly synthesized, rather than taken up from degenerating axons. The synthesis of choline acetyltransferase in degenerating nerves is not restricted to the Schwann cells in direct contact with the muscle fibres; apparently, it depends on the relationship between the Schwann cells and axons, and not solely (if at all) on the relationship between the Schwann cells and muscle fibres.     

Development of neurons synthesizing noradrenaline and acetylcholine in the superior cervical ganglion of the rat in vivo and in vitro.

The development of neurons synthesizing noradrenaline and acetylcholine in the superior cervical ganglion of the rat has been examined after transection of the preganglionic nerve trunk in vivo and by maintenance of whole ganglia in modified Rose chambers in vitro. Temporal changes in the activities of two neurotransmitter enzymes, tyrosine hydroxylase and choline acetyltransferase, were measured as markers for neurons synthesizing noradrenaline and acetylcholine, respectively.In vivo, there was a small increase in choline acetyltransferase activity between birth and adulthood and this was comparable to the increase seen in ganglia from 2-day-old rats maintained in vitro for 14 days in the absence of nerve growth factor. Both in vivo and in vitro, high doses of nerve growth factor (10 μg/g and 1 μg/ml, respectively) resulted in increases in both tyrosine hydroxylase activity and choline acetyltransferase activity, the effect on choline acetyltransferase activity being greater in vitro than in vivo. Similar concentrations of nerve growth factor had no effect on the choline acetyltransferase activity in cultured ganglia taken from 3-week-old rats. It is concluded that increases in choline acetyltransferase activity occur only in ganglia from newborn rats because they contain neurons that still retain flexibility of development and can synthesize acetylcholine due to the influence of factors within the ganglion or in the culture medium.In vitro, following the elimination of endogenous nerve growth factor by the addition of anti-nerve growth factor, tyrosine hydroxylase activity decreased but choline acetyltransferase activity increased in cultured ganglia. High concentrations of guanethidine (100 μg/ml) caused decreases in both tyrosine hydroxylase and choline acetyltransferase activities. Following removal of guanethidine from the medium, choline acetyltransferase activity increased but tyrosine hydroxylase activity was unchanged. When guanethidine was replaced with nerve growth factor, tyrosine hydroxylase activity increased to a greater extent than choline acetyltransferase activity.Our results are discussed in terms of the presence in the ganglion of a multipotential cell type which can differentiate into either an adrenergic or a cholinergic neuron. Differentiation along cholinergic lines is accompanied by a loss of the ability to take up catecholamines and a release from dependence on nerve growth factor.     

Nerve growth factor affects uninjured, adult rat septohippocampal cholinergic neurons

The effect of nerve growth factor on the intact versus injured septohippocampal cholinergic system of adult rats was studied. Nerve growth factor was continuously infused into the lateral ventricle of adult uninjured rats or rats that had received unilateral partial transection of the fimbria. Controls (operated and unoperated) received intraventricular infusion of cytochrome c. After 2 weeks of nerve growth factor or cytochrome c treatments, choline acetyltransferase and acetylcholinesterase activities were measured in the septal area and in the hippocampus (divided into dorsal, medial and ventral parts). The continuous infusion of nerve growth factor resulted in a marked dose-dependent increase of choline acetyltransferase activity in both septum and hippocampus of adult unlesioned rats. In lesioned rats the nerve growth factor treatment was capable of inducing choline acetyltransferase activity in the hippocampus of not only the lesioned but also the unlesioned side, as well as in the septal area. In addition, nerve growth factor affected choline acetyltransferase activity differently in the hippocampus of the operated side with respect to the contralateral side or in unoperated animals. The chronic infusion of nerve growth factor did not affect acetylcholinesterase activity in the septum or in the hippocampus of either lesioned or unlesioned rats. The present findings indicate that nerve growth factor is capable of modulating the function of not only damaged but also normal adult forebrain cholinergic neurons. This suggests that nerve growth factor may modulate the function of these neurons in adulthood.     

Identification of the neural pathway underlying spontaneous crossed phrenic activity in neonatal rats

Cervical spinal cord hemisection at C2 leads to paralysis of the ipsilateral hemidiaphragm in rats. Respiratory function of the paralyzed hemidiaphragm can be restored by activating a latent respiratory motor pathway in adult rats. This pathway is called the crossed phrenic pathway and the restored activity in the paralyzed hemidiaphragm is referred to as crossed phrenic activity. The latent neural pathway is not latent in neonatal rats as shown by the spontaneous expression of crossed phrenic activity. However, the anatomy of the pathway in neonatal rats is still unknown. In the present study, we hypothesized that the crossed phrenic pathway may be different anatomically in neonatal and adult rats. To delineate this neural pathway in neonates, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA–HRP), a retrograde transsynaptic tracer, into the phrenic nerve ipsilateral to hemisection. We also injected cholera toxin subunit B–horseradish peroxidase (BHRP) into the ipsilateral hemidiaphragm following hemisection in other animals to determine if there are midline-crossing phrenic dendrites involved in the crossed phrenic pathway in neonatal rats. The WGA–HRP labeling was observed only in the ipsilateral phrenic nucleus and ipsilateral rostral ventral respiratory group (rVRG) in the postnatal day (P) 2, P7, and P28 hemisected rats. Bilateral labeling of rVRG neurons was shown in P35 rats. The BHRP study showed that many phrenic dendrites cross the midline in P2 neonatal rats at both rostral and caudal parts of the phrenic nucleus. There was a marked reduction of crossing dendrites observed in P7 and P28 animals and no crossing dendrites observed in P35 rats. The present results suggest that the crossed phrenic pathway in neonatal rats involves the parent axons from ipsilateral rVRG premotor neurons that cross at the level of obex as well as decussating axon collaterals that cross over the spinal cord midline to innervate ipsilateral phrenic motoneurons following C2 hemisection. In addition, midline-crossing dendrites of the ipsilateral phrenic motoneurons may also contribute to the crossed phrenic pathway in neonates.     

Effects of duct ligation on choline acetyltransferase activity in salivary glands of rats

Duct ligation was found to cause a decrease in the weights of submaxillary and parotid glands examined 3 weeks postoperatively. Choline acetyltransferase activity in ligated glands was compared with that in unligated contralateral glands. The enzyme activity was also measured in the glands from both sides of unoperated control animals. Interference in the assay of choline acetyltransferase by other acetylated compounds was avoided by introducing suitable control incubations. Ligated submaxillary glands showed a small decrease in the activity of choline acetyltransferase both when compared with contralateral glands and with glands of control rats. In parotid glands the enzyme activity was found to be lower only when ligated and contralateral glands were compared. Structural changes in the nerves and reduced traffic of impulses in them may have to be considered as explanations for the reduction in enzyme activity in duct–ligated glands.     

Effects of duct ligation on choline acetyltransferase activity in salivary glands of rats.

Duct ligation was found to cause a decrease in the weights of submaxillary and parotid glands examined 3 weeks postoperatively. Choline acetyltransferase activity in ligated glands was compared with that in unligated contralateral glands. The enzyme activity was also measured in the glands from both sides of unoperated control animals. Interference in the assay of choline acetyltransferase by other acetylated compounds was avoided by introducing suitable control incubations. Ligated submaxillary glands showed a small decrease in the activity of choline acetyltransferase both when compared with contralateral glands and with glands of control rats. In parotid glands the enzyme activity was found to be lower only when ligated and contralateral glands were compared. Structural changes in the nerves and reduced traffic of impulses in them may have to be considered as explanations for the reduction in enzyme activity in duct-ligated glands.     

The effect of reserpine treatment on choline acetyltransferase activity in rat submaxillary glands

Rats were treated daily with a low dose of reserpine (0.1 mg kg-1) injected subcutaneously for 3 weeks. In the submaxillary glands the noradrenaline content was reduced by about 95%. The total activity of the acetylcholine-synthesizing enzyme choline acetyltransferase remained unchanged. However, the activity of this enzyme was found to be increased when the reserpine treatment was followed by surgical sympathetic denervation and the glands were analysed 3 weeks post-operatively. The enzyme activity also increased in the glands when the surgical sympathetic denervation was performed on the day of the start of the reserpine treatment. The lack of effect of reserpine on choline acetyltransferase activity in the glands seems to exclude the possibility that it is the depletion of neuronal noradrenaline stores that initiates the events giving rise to increases in choline acetyltransferase activity after sympathetic denervation.     

Differential regulation of peptide and catecholamine characters in cultured sympathetic neurons

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

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

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

Choline acetyltransferase and calcitonin gene-related peptide immunoreactivity in motoneurons after different types of nerve injury

Summary This study examined changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity in hypoglossal motoneurons of rats at 1, 3, 7, 20 and 50 days after three types of nerve injury: crush, transection and resection. Peripheral reinnervation was assayed by retrograde labelling of the motoneurons after injections of the exogenous protein, horseradish peroxidase, into the tongue. Maximal reduction in choline acetyltransferase immunostaining occurred at seven days after nerve damage and the amount of the decrease was related to the nature of the injury. The recovery of choline acetyltransferase to normal levels was related to the timing of reinnervation after nerve crush, but not after transection or resection injuries. In contrast to these findings, a rapid increase in calcitonin gene-related peptide immunoreactivity preceded the decrease in choline acetyltransferase levels. A striking increase in calcitonin gene-related peptide immunoreactivity was observed at one day postoperative and was maximal at three days postoperatively for all injuries. Later changes in calcitonin gene-related peptide levels were dependent on the type of injury. Increased calcitonin gene-related peptide staining persisted to 20 days after nerve crush. After nerve transection or resection, calcitonin gene-related peptide immunoreactivity decreased to basal levels at seven days postoperatively. This declination was followed by a second rise in calcitonin gene-related peptide immunolabeling at 20 days for nerve transection or at 50 days after resection. Nearly complete reinnervation was established by 20 days after nerve crush. At 50 days after transection, less than half the number of normally-labelled neurons contained horseradish peroxidase. At this time only 1% of those whose axons had been resected were labelled. These observations suggest that different mechanisms regulate the responses of choline acetyltransferase and calcitonin gene-related peptide to nerve injury. The present results indicate that choline acetyltransferase levels in motoneurons can not be used to predict either the likelihood of or the timing of reinnervation after nerve transection or resection. However, our results strengthen the premise that an increase of calcitonin gene-related peptide immunoreactivity serves as a reliable index for predicting nerve regeneration/reinnervation after cranial nerve injury.     

Amphiphilic and hydrophilic forms of choline-O-acetyltransferase in cholinergic nerve endings of the Torpedo

In the purely cholinergic nerve endings isolated (i.e. synaptosomes) from the electric organ of the fish Torpedo, the enzyme choline acetyltransferase was found to exist not solely in its well-known soluble form but also in a form which is non-ionically bound to the plasma membrane; this activity could not be solubilized in solutions of high ionic strength (0.5 M NaCl). The non-ionic detergent Triton X-114 was used to solubilize synaptosomes isolated from either the electric organ of Torpedo or rat brain. This detergent allows to separate hydrophilic from amphiphilic proteins of cells or subcellular fractions. Twelve per cent of the synaptosomal choline acetyltransferase partitioned as amphiphilic and 80-97% as hydrophilic activity. The percentage of amphiphilic activity present in synaptosomes was significantly higher than that of the form of activity (4.4%) extracted from samples containing only the soluble form of choline acetyltransferase but was significantly lower than the percentage of amphiphilic enzyme present in preparations of synaptosomal plasma membrane (20-22%) which were enriched in the non-ionically membrane-bound form of choline acetyltransferase. These results indicate that the soluble and the non-ionically membrane-bound enzymes differ in their capacity to interact with non-ionic detergents. The preparations of synaptosomal plasma membranes contained significantly higher proportions of detergent-insoluble choline acetyltransferase activity than did the whole synaptosomes; the difference was more striking for the Torpedo than for the rat enzyme. This detergent-insoluble activity was not due to aggregates of the enzyme. Some properties of the hydrophilic and amphiphilic choline acetyltransferase of Torpedo were analyzed. The two forms of the enzyme did not exhibit different affinities for their substrates; they were found to differ with respect to their sensitivity to inhibition by increasing concentrations of the two products of the reaction, acetylcholine and coenzyme A and heat inactivation at 45 degrees C. Most probably the hydrophilic and amphiphilic activities correspond to what was referred to as soluble and non-ionically membrane-bound choline acetyltransferase, respectively. The amphiphilic form may be an integral enzyme of the plasma membrane of cholinergic nerve endings or may be tightly bound to a specific protein in this membrane which may act as a "receptor" for choline acetyltransferase.     

Analysis of the time course of changes in hippocampal acetylcholinesterase and choline acetyltransferase activities after various septal lesions in the rat: Return of enzymic activity after extensive medioventral lesions

Acetylcholinesterase and choline acetyltransferase activities in the hippocampus of the rat were estimated 2–360 days after three types of septal lesions: total (1), extensive medioventral (2), and small medioventral (3). A statistical model of multiple regression of enzymic activity on time was applied to the analysis of the results.After lesions of type (1) the enzymic activities decrease steadily reaching about 20% of the control values after some 10 days and remaining unchanged over a period of 1 yr. After lesions of type (2) the enzymic activities initially decrease as after type (1), but start to increase from the fifth postoperative day. The highest values for calculated daily rate of recovery were found between the sixth and seventh postoperative day. After 3 months the activity reached about 60% of control values. No further increase was observed over 1 yr. Lesions of type (3) reduced acetylcholinesterase and choline acetyltransferase activities in the hippocampus to about 60% of the control values. A very slight recovery was seen for choline acetyltransferase.The observed changes in activity of both enzymes after all three types of lesions were highly correlated.It is suggested that the recovery in acetylcholinesterase and choline acetyltransferase activities after lesions of type (2) could be due to a sprouting of cholinergic nerve terminals from intact parts of the septum.     

Neurochemical evidence for afferent GABAergic and efferent cholinergic neurotransmission in the frog vestibule

Glutamate decarboxylase and choline acetyltransferase activities with magnitudes similar to those of their homologous enzymes in frog nervous tissue were found in homogenates of the frog labyrinth. Transection of the vestibular nerve resulted in a gradual diminution of choline acetyltransferase activity until it reached an 88% decrease 6 weeks after surgery. In contrast, glutamate decarboxylase activity did not suffer any alteration at any time after nerve excision. The presence of their enzymes of synthesis is evidence of the neurotransmitter participation of GABA and acetylcholine in the frog vestibule; the observed decrease of choline acetyltransferase following vestibule nerve excision supports the efferent synaptic bouton localization of choline acetyltransferase. The suggestion that glutamate decarboxylase is located in a cell type (or compartment) that may well be the hair cell is supported by the fact that this enzyme does not suffer any modification after surgery. These results are in accordance with an efferent cholinergic neurotransmission and a putative afferent role of GABA in the frog vestibule.     

Acetylcholine synthesis in nerve endings to slow and fast muscles of developing chicks: Effect of muscle activity

The activity of the acetylcholine-synthesizing enzyme choline acetyltransferase was estimated in nerve terminals to fast, focally-innervated and slow, multiply-innervated muscles of chick embryos. Terminals in fast muscles contain considerably more choline acetyltransferase than terminals in slow muscles, and this could be related to factors determining the different patterns of innervation of the muscles. Neuromuscular blockade by curare increases the choline acetyltransferase content of both types of muscle, suggesting that more nerve terminals survive in inactive muscles.     

Selective induction by glucocorticoids of tyrosine hydroxylase in organ cultures of rat pheochromocytoma

Addition of physiological concentrations of glucocorticoids to organ cultures of a transplantable rat pheochromocytoma resulted in an increase in the specific activity of tyrosine hydroxylase. The increase was delayed, beginning after 12 h and reached its peak after 36 h, when the specific activity was twice that of controls. The activities of dopamine β-hydroxylase and dopa decar?ylase showed no significant changes. The tumour contains choline acetyltransferase and the specific activity of this enzyme was reduced by 25% following culture in the presence of glucocorticoids. Addition of cholinergic agonists and/or nerve growth factor to the culture medium had no effect on the specific activity of tyrosine hydroxylase. The effect of glucocorticoids on this enzyme was entirely abolished by inhibitors of ribonucleic acid or protein synthesis.These results are discussed in relation to the modulatory role of glucocorticoids in trans-synaptic and nerve growth factor-mediated enzyme induction in the peripheral sympathetic nervous system.     

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.     

Epidermal growth factor affects both glia and cholinergic neurons in septal cell cultures.

The effects of epidermal growth factor on high density primary cultures of fetal (embryonic day 17) rat septal cells were examined. Under serum-free conditions, the continuous exposure of these cultures to epidermal growth factor for seven days significantly decreased choline acetyltransferase (EC 2.3.1.6) activity in a dose-dependent manner. Maximal decreases were observed from 1 to 10 ng/ml epidermal growth factor. This effect was completely abolished by the addition of anti-epidermal growth factor antibodies. The epidermal growth factor-mediated decrease in choline acetyltransferase activity was culture-time dependent, being first detectable after five days of factor application and may likely represent an inhibition of the spontaneous increase in enzyme activity that occurs with time in culture. Concomitant with changes in enzyme activity, epidermal growth factor produced a significant and proportional decrease in the number of acetylcholinesterase-positive neurons. This decrease in acetylcholinesterase-positive cells did not reflect a decrease in cholinergic cell survival as nerve growth factor could restore the number of acetylcholinesterase-positive neurons in epidermal growth factor-treated cultures to control levels. Furthermore, in these high-density cultures, epidermal growth factor did not affect general neuronal survival, while it did produce an increase in the number and intensity of glial fibrillary acidic protein-immunoreactive astroglia as well as in the number of macrophage-like cells. The proliferative response of these non-neuronal cells to epidermal growth factor, as assessed by [3H]thymidine incorporation, was evident after three days of epidermal growth factor application, persisted thereafter, and could be antagonized by the inclusion of the antimitotic 5-fluorodeoxyuridine. Furthermore, 5-fluorodeoxyuridine completely blocked the epidermal growth factor-mediated decrease in choline acetyltransferase activity. However, when epidermal growth factor was tested in pure glial cultures, it only directly induced proliferation of astrocytes. These results suggest that the proliferative response of either one or both of these glial cell types in the mixed cultures may be indirectly affecting cholinergic cell expression.     

On the direct and crossed components of reflex responses to unilateral stimulation of the carotid body chemoreceptors in the dog

1. In dogs under chloralose-urethane anaesthesia the chemoreceptors of the two carotid bodies were separately stimulated.2. The distribution of three primary reflex responses to carotid body stimulation was studied: parasympathetic bradycardia, sympathetic vasoconstriction, and increase in somatic phrenic nerve activity.3. The reflex bradycardia evoked by either carotid body was mediated by both vagus nerves, but when either vagus was blocked a greater response could be obtained from the contralateral than from the ipsilateral carotid body.4. The reflex vasoconstriction evoked by either carotid body was seen in both hind limbs, with no predominance in either limb.5. The reflex increase in phrenic nerve activity evoked by either carotid body was seen in both phrenic nerves, with no predominance in either nerve.