Neurite development in vitro. I. The effects of adenosine 3′5′-cyclic monophosphate (cyclic AMP)

Elevated levels of 3′5′ adenosine monophosphate (cyclic AMP) stimulate a wide variety of cellular events including aggregation, differentiation, morphological expression, pigment migration, and secretion. The role of cyclic AMP in these events prompted our present study of embryonic chick dorsal root ganglia. Test substances were applied to cultures during the routine feeding procedure. Their development was quantitatively evaluated on the basis of explant size, length of glial-like outgrowth, distribution of growth, neurite number, length, diameter, and degree of arborization. These parameters were all shown to be independent of each other. The high variability of in vitro neurite development necessitated the use of over 100 cultures per treatment group. Cultures treated with 5′ AMP exhibited no significant differences from controls. Those treated with cyclic AMP, dibutyryl cyclic AMP, or Nerve Growth Factor (NGF) exhibited statistically significant increases in area of outgrowth, the number of neurites per culture, and in diameters, lengths, and degree of neurite arborization. The growth promoting activity of dibutyryl cyclic AMP and NGF were greater than those of cyclic AMP. Electron microscopic study shows neurites formed under the influence of cyclic AMP or its dibutyryl derivative to resemble those grown in NGF. These studies suggest the possibility that cyclic AMP stimulates neurite growth by mediating the process of microtubule (MT) assembly. They further prompt us to speculate that one way NGF enhances neurite development is by stimulating MT assembly via a “Second Messenger System”.     

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia.

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.     

EFFECTS OF NERVE GROWTH FACTOR ON CYCLIC AMP LEVELS IN EMBRYONIC CHICK DORSAL ROOT GANGLIA FOLLOWING FACTOR DEPRIVATION

Nerve growth factor (NGF) at 10 B.U./ml produced a 5-fold increase in the concentration of cyclic AMP 10min after addition to freshly excised embryonic chick dorsal root ganglia (DRG). The presence of NGF at 10 B.U./ml, but not 1 B.U./ml, over a 6 h incubation in nutrient-free medium prevented this cyclic AMP increase when the DRG were again challenged with NGF (10 B.U./ml) after the 6 h. Incubation of DRG for 6 h at 37°C without NGF did not prevent the cyclic AMP response from occurring when NGF was presented at this time. The basal cyclic AMP concentration, however, decreased by 65–75% during the course of the 6 h incubation, and the continuous presence of NGF (10 B.U./ml) was unable to prevent this. When NGF was added to 6-h NGF-deprived DRG, the time for maximum cyclic AMP increase decreased from 15min to 6min with increasing NGF concentrations from 1 to 50 B.U./ml, although the relative magnitude of the cyclic AMP increase was essentially the same (approx 3-fold) at these NGF concentrations. Attempts were made to correlate the cyclic AMP response in NGF-deprived DRG with another rapid response resulting from administration of NGF to NGF-deprived DRG, namely, the reactivation of hexose uptake. The cyclic AMP and permeation responses both occurred on the same time scale (5-15 min), and both responded to increasing concentrations of NGF with a decreasing time to achieve maximal effect. A temporal relationship between cyclic AMP and membrane permeability was noted, but it could not be ruled out that it might reflect difficulties in methodology and/or inadequacy in current knowledge of the underlying mechanisms.     

Effects of Nerve Growth Factor and Phorbol Derivative on Reactivation of Herpes Simplex Virus Type 1 in Cultured Cells of Latently Infected Adult Mouse Trigeminal Ganglia

Reactivation of herpes simplex virus type 1 (HSV-1) occurred rapidly in cells of latently infected adult mouse trigeminal ganglia which were cultured in serum-free medium in the presence of sufficient nerve growth factor (NGF). However, HSV-1 reactivation was delayed significantly in ganglionic cultures in the absence of exogenous NGF or in cultures treated with 2-aminopurine in the presence of NGF. The delayed viral reactivation in ganglionic cultures without NGF was accelerated by treatment with phorbol myristate acetate or dibutyryl cyclic AMP. Culture conditions which affected HSV-1 reactivation did not affect replication of HSV-1 in normal ganglionic cultures.     

3′,5′‐Cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na⁺‐channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine‐induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 281–288, 2001     

Amitriptyline-Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.     

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Pineal gland cells in culture : Morphology, biochemistry, differentiation, and co-culture with sympathetic neurons

We present here the initial report of a method for reproducibly obtaining primary cell cultures from pineal glands of 2-day-old rats. During culture, the putative pinealocytes became associated with each other in “nests”. Cells in these nests displayed vesicle-crowned rodlets and cilia, which are fine structural features in vivo of pinealocytes from neonatal rats. Treatment of the cultured cells with either norepinephrine or dibutyryl-cyclic AMP (db-cAMP) resulted in an increase in the activity of serotonin N-acetyltransferase, a marker activity for pineal function. This stimulation could be blocked by either cycloheximide or actinomycin D, and norepinephrine stimulation was also blocked by -propranolol. Further, the pineal cell cultures were able to support the growth of dispersed cells of rat superior cervical ganglia and to allow neurite outgrowth in these co-cultures, though the presence of nerve growth factor (NGF) in the medium of these cultures could not be detected.     

Nerve Growth Factor Stimulates Phospholipid Methylation in Target Ganglionic Neurons Independently of the Cyclic AMP and Sodium Pump Responses

Suspensions of neurons prepared from embryonic day 12 (E12) chick sympathetic ganglia were incubated with [methyl-3H]methionine in the absence of nerve growth factor (NGF). Presentation of the factor for different periods of time resulted in an approximate three-fold stimulation of radioactivity incorporated into total phospholipid, followed by a rapid decline thereafter. Both the magnitude and the time of the response were dependent on the NGF concentration used. Also examined were possible relationships of phospholipid methylation to two other short-latency responses to NGF, i.e., control of the Na+,K+-pump and elevation of cyclic AMP content. Incubation of E12 sympathetic neurons with known transmethylase inhibitors (shown to be active in the present system) failed to prevent reactivation of the Na+,K+-pump in response to NGF administration. E16 sympathetic neurons and E15 sensory neurons, which do not depend on exogenous NGF for control of their Na+,K+-pump, still show a stimulation of phospholipid methylation when challenged with the factor. Blockage of the pump with ouabain also fails to prevent a methylation response. Thus, the pump and methylation responses to NGF occur independently of each other. Intact E8 chick dorsal root ganglia, but not E12 sympathetic ganglia, display a rapid and transient rise in their cyclic AMP content when presented with NGF. At a concentration of 10 biological units/ml, NGF elicits a peak of phospholipid methylation at 4 min, and a peak of cyclic AMP at 10 min. Methylation inhibitors prevent the methylation response, but not that of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the neurotrophins nerve growth factor,neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 395–410, 1997     

Changes in the Expression of β and Actins During Differentiation of PC 12 Cells

Cells of the rat pheochromocytoma line PC12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of beta and gamma isoforms of nonmuscle actin in extracts of these differentiating cells showed that the beta:gamma ratio decreased from 1.30 +/- 0.05 to 0.99 +/- 0.05 after 6 days of NGF treatment. Cells treated with N6,O2-dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of beta:gamma isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the beta:gamma actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension-grown PC12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a "second messenger" for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC12 cells.     

A cAMP-activated pathway, including PKA and PI3K, regulates neuronal differentiation

Neuronal differentiation is a complex process in which many different signalling pathways may be involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to induce neuronal differentiation and also to cooperate with NGF to induce PC12 neurite outgrowth in a Ras-dependent manner. However, the neuritogenic activities associated with cAMP are still not well understood. The purpose of this study was to investigate the potential neuritogenic activities mediated by cAMP. For this purpose, we used the human neuroblastoma cell line SH-SY5Y. These neuroblastoma cells respond to cAMP by forming neurite-like extensions. We tried to identify some essential pathways involved in the cAMP-induced neurite elongation of these cells. Our results indicated that PKA is transiently activated in this elongation model. When we blocked PKA activity, elongation did not take place. Similarly, PI3K also plays an essential role because when we blocked this kinase activity, there was no neurite elongation. Indeed, over-expression of the p110-catalytic subunit or an activating form of the p85-regulatory subunit (p65) is able to induce some degree of neurite extension. Moreover, our results showed that when elongation is initiated, PI3K is still essential for maintenance of the neuronal morphology, whereas PKA or MAPK (ERKs or p38) activation does not appear to be necessary during this process.     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: concentrations in Morris hepatomas of different growth rates

Cyclic AMP and cyclic GMP levels were examined in Morris hepatoma explants in vivo. All eight tumor lines examined had significantly elevated cyclic AMP and cyclic GMP levels when compared to normal liver from tumor-bearing rats. No apparent correlation was observed between the rates of tumor growth and cyclic nucleotide levels; however, two tumor lines (3924A and 7288ctc) had very high levels of cyclic GMP.     

Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.     

The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in Stentor coeruleus.

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N⁶, 2′-0-dibutyryl adenosine cyclic 3′:5′-monophosphate (DBC) or theophylline begun at the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions. To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.