Regulation of electrical activity and neuronal excitability in Helisoma trivolvis by carbon monoxide.

Carbon monoxide (CO), like other gaseous neuromodulators, has a dual nature as both a toxic gas and a physiologically relevant signaling molecule. In the nervous system, high concentrations of CO can lead to neuronal injury while lower concentrations are found to be neuroprotective. The number of cellular targets affected by physiological concentrations of CO is rapidly growing and includes ion channels in various cell types. The modulation of ion channels by CO in neurons, however, and the effect it has on neural activity are incompletely understood. Here, the well-characterized buccal neurons, B5 and B19, of the freshwater snail, Helisoma trivolvis, were used to investigate the role that CO plays in regulating spontaneous firing activity and neuronal excitability. Neurons were studied in single-cell culture, thereby removing other signals normally present in the intact nervous system and allowing for the optimal characterization of physiological effects of CO. We found that the CO donor molecule, carbon monoxide releasing molecule-2 (CORM-2), hyperpolarized the resting membrane potential of B5 neurons and silenced their spontaneous firing activity. These effects were mediated through the inhibition of a persistent sodium current. CORM-2 also inhibited neuronal excitability. This effect was mediated by the inhibition of voltage-gated calcium channels by CO. The general findings of CO acting as a hyperpolarizing signal and an inhibitor of neuronal excitability extended to B19 neurons. Taken together, these findings suggest that CO is a potent modulator of ion channels with broad implications for the modulation of neural activity in a wide range of neuron-types.

Antibacterial activity and chemical compounds of leaves and branches of Protium hebetatum / Atividade anti bacteriana e compostos químicos das folhas e ramos de Protium hebetatum

ABSTRACT The extracts and fractions of leaves and branches of Protium hebetatum D. C. Daly (Burseraceae) were investigated for their antibacterial activity and chemical composition. The methanol extract of branches (EMG) was considered active against the Escherichia coli and the Proteus vulgaris, showing an inhibition zone of 13 mm, and was selected for bioassay-guided phytochemical fractionation. From the technique of broth microdilution, the extract was considered a moderate inhibitor against Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis, with a minimum inhibitory concentration (MIC) of 1 mg/mL. The dichloromethane fraction was considered a moderate inhibitor against S. aureus (MIC of 1 mg/mL) and a potent inhibitor against E. faecalis (MIC of 0.5 mg/mL). F1, F2, F5 and F6 from chromatographic column of dichloromethane fraction were considered moderate inhibitors against S. aureus (MIC of 1 mg/mL). Through analysis by a gas chromatography mass spectrometry, eighteen compounds were identified, from which thirteen (isoeugenol, p-vinylguaiacol, metoxyeugenol, coumarin, 5-hydroxy-scopoletin, 4,7-dihydroxy-6-metoxicromam-2-one, 4[(1E]-3-hydroxy-1-propenyl)-2-methoxyphenol, piperonal, scoparon, o-guaiacol, spathulenol, seringol and antiarol) are unprecedented in these species. We also identified the triterpenes α-amyrin and β-amyrin, the steroids stigmasterol and sitosterol and the coumarin scopoletin, which was closely linked to the antibacterial activity of the samples.

RESUMO Atividade antibacteriana e compostos químicos de folhas e galhos de Protium hebetatum. Extratos e frações de folhas e galhos de Protium hebetatum D. C. Daly (Burseraceae) foram investigados quanto sua atividade antibacteriana e composição química. O extrato metanólico dos galhos (EMG) foi considerado ativo contra Escherichia coli e Proteus vulgaris, apresentando um halo de inibição de 13 mm, sendo selecionado para um fracionamento fitoquímico biomonitorado. A partir da técnica de microdiluição em caldo o EMG foi considerado um inibidor moderado contra Staphylococcus aureus, Pseudomonas aeruginosa e Enterococcus faecalis, apresentando uma concentração inibitória mínima (CIM) de 1mg/mL. A fração diclorometânica foi considerada inibidora moderada contra S. aureus (CIM de 1 mg/mL) e inibidora potente contra E. faecalis (CIM de 0,5 mg/mL). F1, F2, F5 e F6 provenientes da fração diclorometânica foram consideradas inibidoras moderadas contra S. aureus (CIM de 1 mg/mL). Através da análise por cromatografia gasosa acoplada a espectrometria de massa, foram identificados dezoitos compostos, dos quais treze (isoeugenol, p-vinilguaiacol, metoxieugenol, cumarina, 5-hidroxi-escopoletina, 4,7-dihidroxi-6-metoxicromam-2-ona, 4[(1E]-3-hidroxi-1-propenil)-2-methoxifenol, piperonal, escoparona, o-guaiacol, espatulenol, seringol e antiarol) foram identificados pela primeira vez nesta espécie. Foram também identificados os triterpenos α-amirina e β-amirina, os esteroides estigmasterol e sitosterol e a cumarina escopoletina, que estão intimamente ligados à atividade antibacteriana da espécie.

Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels.

Dopamine (DA) plays fundamental roles as a neurotransmitter and neuromodulator in the central nervous system. How DA modulates the electrical excitability of individual neurons to elicit various behaviors is of great interest in many systems. The buccal ganglion of the freshwater pond snail Helisoma trivolvis contains the neuronal circuitry for feeding and DA is known to modulate the feeding motor program in Helisoma. The buccal neuron B5 participates in the control of gut contractile activity and is surrounded by dopaminergic processes, which are expected to release DA. In order to study whether DA modulates the electrical activity of individual B5 neurons, we performed experiments on physically isolated B5 neurons in culture and on B5 neurons within the buccal ganglion in situ. We report that DA application elicited a strong hyperpolarization in both conditions and turned the electrical activity from a spontaneously firing state to an electrically silent state. Using the cell culture system, we demonstrated that the strong hyperpolarization was inhibited by the D2 receptor antagonist sulpiride and the phospholipase C (PLC) inhibitor U73122, indicating that DA affected the membrane potential of B5 neurons through the activation of a D2-like receptor and PLC. Further studies revealed that the DA-induced hyperpolarization was inhibited by the K channel blockers 4-aminopyridine and tetraethylammonium, suggesting that K channels might serve as the ultimate target of DA signaling. Through its modulatory effect on the electrical activity of B5 neurons, the release of DA in vivo may contribute to a neuronal output that results in a variable feeding motor program.

Effects of undecylenic acid released from denture liner on Candida biofilms.

Denture liners (DL) are easily colonized by Candida spp. In an attempt to prevent biofilm colonization, manufacturers have incorporated undecylenic acid (UDA) into DL. In this in vitro study, the effects of UDA released from DL on Candida biofilms were investigated. The concentrations of UDA released from commercial DL were determined by gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) and minimum fungistatic concentration (MFC) tests were performed for C. albicans or C. glabrata, with UDA for comparison with the concentrations released from DL. Specimens of DL with (experimental group) and without UDA (control group) were fabricated, and Candida biofilms were developed on DL surfaces. Biofilms were evaluated by cell counts, metabolic activity, structure, and secretion of proteinase or phospholipase. The concentrations of UDA released were within the MIC and MFC ranges. In the presence of UDA, C. albicans biofilms were thinner and had lower numbers of viable and active cells, although no significant enzymatic changes were observed relative to the control group (p > 0.05). In contrast, C. glabrata biofilms exhibited higher cell counts and greater metabolic activity and also increased proteinase activity in the presence of UDA relative to the control group (p < 0.05). Overall, UDA did not prevent Candida biofilm formation.

Action of Coriandrum sativum L. Essential Oil upon Oral Candida albicans Biofilm Formation.

The efficacy of extracts and essential oils from Allium tuberosum, Coriandrum sativum, Cymbopogon martini, Cymbopogon winterianus, and Santolina chamaecyparissus was evaluated against Candida spp. isolates from the oral cavity of patients with periodontal disease. The most active oil was fractionated and tested against C. albicans biofilm formation. The oils were obtained by water-distillation and the extracts were prepared with macerated dried plant material. The Minimal Inhibitory Concentration-MIC was determined by the microdilution method. Chemical characterization of oil constituents was performed using Gas Chromatography and Mass Spectrometry (GC-MS). C. sativum activity oil upon cell and biofilm morphology was evaluated by Scanning Electron Microscopy (SEM). The best activities against planktonic Candida spp. were observed for the essential oil and the grouped F(8-10) fractions from C. sativum. The crude oil also affected the biofilm formation in C. albicans causing a decrease in the biofilm growth. Chemical analysis of the F(8-10) fractions detected as major active compounds, 2-hexen-1-ol, 3-hexen-1-ol and cyclodecane. Standards of these compounds tested grouped provided a stronger activity than the oil suggesting a synergistic action from the major oil constituents. The activity of C. sativum oil demonstrates its potential for a new natural antifungal formulation.

Effects of Mikania genus plants on growth and cell adherence of mutans streptococci.

The present study evaluated the chemical composition and the antimicrobial activity of the extracts and fractions of Mikania laevigata and Mikania glomerata on growth and cell adherence of mutans streptococci. Ethanolic extract, hexane and ethyl acetate fractions of Mikania laevigata and Mikania glomerata were chemically identified by chromatographic methods and tested on mutans streptococci from culture collection and clinical isolates. Twenty-two compounds were identified in both Mikania extracts, including coumarin, 1-octadecene, and diterpenic, cupressenic and kaurenoic acids. Antimicrobial activity was assessed by determination of the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibition of cell adherence to a glass surface. Hexane fraction from both plant extracts was the most effective in inhibiting the growth of the bacterial strains tested (MIC values between 12.5 microg/ml and 400 microg/ml, and MBC values between 25 microg/ml and 400 microg/ml). In addition, sub-MIC levels of the crude extracts and their hexane fractions significantly inhibited the adherence of the microorganisms to a glass surface. The data indicate that the biologically active compounds are present mostly in the hexane fraction of both Mikania species, which showed remarkable inhibitory activities against mutans streptococci. Mikania genus plant is a promising source for novel antimicrobial agents against oral pathogens.

Antiulcerogenic activity of a crude hydroalcoholic extract and coumarin isolated from Mikania laevigata Schultz Bip.

The leaves of Mikania (Asteraceae) species are used in folk medicine as antispasmodic, antiulcerogenic and antirheumatic agents. Phytochemical screening of the crude hydroalcoholic 70% extract (CHE) of Mikania laevigata Shultz Bip. revealed coumarins, terpenes and organic acids. Antiulcerogenic activity of CHE was evaluated, employing different experimental models in rats, to discern the pharmacological mechanism of action. Both the antisecretory and the cytoprotection hypothesis were evaluated. The crude hydroalcoholic extract (1000 mg/kg body wt., vo) decreased the ulcerative lesion index produced by indomethacin, ethanol, stress and reserpine in rats by 85%, 93%, 82% and 50%, respectively. In the pyloric ligation model, a decrease of hydrogenionic concentration (53%) was observed, suggesting that the pharmacological mechanism has a relationship to antisecretory activity. The antisecretory mechanisms of CHE and the coumarin isolated from M. laevigata were confirmed by acid hypersecretion induced by histamine, pentagastrin and bethanechol. Duodenal administration of CHE (1000 mg/kg body wt.) and coumarin (100 mg/kg body wt.) inhibited only the gastric acid secretion produced by bethanecol. These results suggest that both CHE and coumarin may influence the secretion control mediated by the parasympathetic system.

Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L.

The present study evaluated the central activity of the essential oil and the ethanolic extract from Artemisia annua L. in animals as a part of a psychopharmacological screening of this plant. The extract was prepared with fresh leaves in ethanol (AEE) and the essential oil (AEO) was obtained by hidrodestilation. The ED(50) and the LD(50) obtained for the essential oil were 470mg/kg (correlation coefficient r=0.97333 and linear regression y=-26.52x+0.158) and 790mg/kg, and for the extract, 450mg/kg (correlation coefficient r=0.99266 and linear regression y=-27.34+0.156) and more than 2g/kg, respectively. The doses increased the latency time to convulsions induced by picrotoxin and pilocarpine but prevented the onset of pentylenotetrazol and strychnine induced seizures. In addition to, the products have caused marked inhibition in the Rota-rod assay. According to the results, the AEO has a high acute toxicity and a possible cholinergic action, and the AEE showed a possible central activity as dopaminergic and cholinergic agents, and did not present a significant acute toxicity. These differences should be due to chemical substances present in each product. These products had no significant effect as an anticonvulsant, while exhibited a strong depressant activity on the CNS.

Regulation of neuronal growth cone filopodia by nitric oxide depends on soluble guanylyl cyclase.

Nitric oxide has been proposed to play an important role in neuronal development. We have previously shown that growth cones from an identified neuron, B5, in the snail Helisoma trivolvis, respond to nitric oxide (NO) donors by increasing the length of their filopodia within minutes of application (Van Wagenen and Rehder, 1999). This effect was mediated through a cGMP-induced increase in [Ca2+]i and resulted in an enlargement of the growth cone's action radius, suggesting that NO could function as a signaling molecule during neuronal pathfinding. We show here that NO functions as a specific rather than a general regulator of growth cone filopodia, because another identified neuron from the same ganglion, B19, failed to respond to NO with an increase in filopodial length. We found that, contrary to B5 neurons, B19 growth cones contained little or no soluble guanylyl cyclase (sGC) immunoreactivity, presumably preventing their response to NO. This hypothesis was supported by the finding that the sGC activator YC-1 (10 microM) had no effect on B19 filopodia but induced elongation of B5 filopodia. These results indicate that the effects of NO can be quite specific, and raise the interesting possibility that neurons could selectively tune in to NO by differentially expressing the target enzyme sGC in the appropriate cellular location during critical developmental stages. In addition, our NADPH-diaphorase staining and anti-NOS immunohistochemisty suggest that growth cones of B5 neurons, but not of B19 neurons, could be a source of NO, making NO a potential intra- and transcellular messenger.

Filopodial behavior is dependent on the phosphorylation state of neuronal growth cones.

Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.

Stimulation-induced changes in filopodial dynamics determine the action radius of growth cones in the snail Helisoma trivolvis.

Filopodia on neuronal growth cones constantly extend and retract, thereby functioning as both sensory probes and structural devices during neuronal pathfinding. To better understand filopodial dynamics and their regulation by encounters with molecules in the environment, we investigated filopodial dynamics of identified B5 neurons from the buccal ganglion of the snail Helisoma trivolvis before and after treatment with nitric oxide (NO). We have previously demonstrated that treatment with several NO-donors caused a transient, cGMP-mediated elevation in [Ca(2+)](i), which was causally related to an increase in filopodial length and a reduction in the number of filopodia on growth cones. We demonstrate here that these effects were the result of distinct changes in filopodial dynamics. The NO-donor SIN-1 induced a general increase in filopodial motility. Filopodial elongation after treatment with SIN-1 resulted from a significant increase in the rate at which filopodia extended, as well as a significant increase in the time filopodia spent elongating. The reduction in filopodial number was caused by a significant decrease in the frequency with which new filopodia were inserted into the growth cone. With the exception of the back where filopodia appeared less motile, filopodial dynamics appeared to be mostly independent of the location on the growth cone. These results suggest that NO can regulate filopodial dynamics on migrating growth cones and might function as a messenger to adjust the action radius of a growth cone during pathfinding.

Regulation of chick dorsal root ganglion growth cone filopodia by protein kinase C.

Growth cone filopodia function both as structural and sensory devices during neuronal pathfinding and their presence is important for correct growth cone navigation. It is assumed that a growth cone can adjust the area of the environment it can explore by regulating the length and number of its filopodial sensors, and in several cell types, these parameters are controlled by the intracellular calcium concentration ([Ca(2+)](i)). In the present report, we address the question whether [Ca(2+)](i) is a general regulator of growth cone filopodia, or whether different cell types utilize different second-messenger systems for this purpose. We show that increasing [Ca(2+)](i) in growth cones of chick dorsal root ganglion (DRG) neurons does not affect average filopodial length in this cell type, suggesting that this parameter is not controlled by [Ca(2+)](i) in chick DRG neurons. Further, we demonstrate that the second-messenger protein kinase C (PKC) is involved in the regulation of filopodial length in chick DRG neurons. Activation of PKC with the phorbol ester, phorbol myristate-13-acetate (PMA), caused filopodial shortening, whereas inhibition of PKC with either bisindolylmaleimide I or calphostin C caused a significant elongation of filopodia. Although the pathway through which PKC mediates its effect on growth cone filopodia in chick DRG neurons remains to be identified, our results indicate that filopodial regulation by [Ca(2+)](i), though clearly important in several other neuronal cell types in vitro, appears to be less important in chick DRG neurons. Rather, we find that in chick DRG neurons, filopodial parameters are controlled by PKC.

Regulation of neuronal growth cone filopodia by nitric oxide.

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.

Autonomous regulation of growth cone filopodia.

The fan-shaped array of filopodia is the first site of contact of a neuronal growth cone with molecules encountered during neuronal pathfinding. Filopodia are highly dynamic structures, and the "action radius" of a growth cone is strongly determined by the length and number of its filopodia. Since interactions of filopodia with instructive cues in the vicinity of the growth cone can have effects on growth cone morphology within minutes, it has to be assumed that a large part of the signaling underlying such morphological changes resides locally within the growth cone proper. In this study, we tested the hypothesis that two important growth cone parameters-namely, the length and number of its filopodia-are regulated autonomously in the growth cone. We previously demonstrated in identified neurons from the snail Helisoma trivolvis that filopodial length and number are regulated by intracellular calcium. Here, we investigated filopodial dynamics and their regulation by the second-messenger calcium in growth cones which were physically isolated from their parent neuron by neurite transection. Our results show that isolated growth cones have longer but fewer filopodia than growth cones attached to their parent cell. These isolated growth cones, however, are fully capable of undergoing calcium-induced cytoskeletal changes, suggesting that the machinery necessary to perform changes in filopodial length and number is fully intrinsic to the growth cone proper.

Functional compartmentalization of the neuronal growth cone: determining calcium's place in signaling cascades.

The growth cone is generally regarded as the basic unit of neuronal organization concerned with development of connections within the nervous system. The discussion below illustrates that the growth cone itself can be subdivided into distinct units of organization. It is this functional compartmentalization which enables the growth cone to read the molecular terrain it traverses and to convert this information into precise motor events. Our discussion will focus on the flow of information from the environment to the growth cone. In particular, we will follow signaling events from their remote processing within filopodia to the biological equivalent of a central processing unit in the core of the growth cone.

The sensory-motor role of growth cone filopodia.

Neuronal growth cones, the motile tips of elongating axons and dendrites, respond very precisely to cues encountered during pathfinding. During the past year, our knowledge about their sensory function, their integrative properties, and their motor function has advanced significantly. In particular, growth cone filopodia are currently being recognized for their prominent roles as sensors, transducers, and autonomous motor structures important for growth cone steering.

Calcium wave fronts that cross gap junctions may signal neuronal death during development.

Embryonic anterior pagoda (AP) neurons in the leech interact with their segmental homologs in adjacent ganglia through transient axons that overlap during a critical period of development and then retract. However, when an AP neuron is ablated mechanically or by irradiation during this period, an adjacent homolog responds by reinitiating growth of its overlapped axon and thereby taking over vacated territory (Gao and Macagno, 1987b; Gao, 1989). The death of an AP cell is therefore communicated to its homolog, but the mechanism underlying this signaling is presently unknown. Since it was recently found that AP homologs are electrically and dye coupled through their transient axons (Wolszon et al., 1994), we investigated the possibility that gap junctions may mediate the cell death signal that could occur between developing neurons. Among several candidate intercellular signals, we began by studying calcium dynamics in embryonic AP cells, in situ, since calcium is known to cross gap junctions and is implicated in cell death in many systems. We found that elements that usually increase [Ca2+]i in adult neurons, such as releasable internal stores or voltage-dependent calcium channels, were not present at the critical period. Instead, mechanisms that reduce free calcium, such as buffering and pumping, were the most robust. When a large, focal calcium rise was produced in an AP axon by making a lesion with a UV microbeam (leading to eventual death of these neurons), calcium did not rise quickly throughout the cell, but rather moved in a slow (0.05-0.25 micron/sec) wave front away from the lesion site, into other processes of the damaged cell. Furthermore, when a calcium wave front reached the growth cone of a transient axon, it crossed at the gap junctions into the coupled axon of the neighboring AP neuron, but went no further. Since it is known that an AP responds to a neighbor's death by reinitiating growth only in that axon that contacts the dying cell (Gao and Macagno, 1987b; Gao, 1989), these observations are consistent with calcium playing a role in the signaling of cell death to homologs that are coupled to a dying cell.

Self-recognition: a constraint on the formation of electrical coupling in neurons.

Electrical coupling between specific neurons is important for proper function of many neuronal circuits. Identified cultured neurons from the snail Helisoma show a strong correlation between electrical coupling and presence of gap junction plaques in freeze-fracture replicas. Gap junction plaques, however, were never seen between overlapping neurites from a single neuron, even though those same neurites formed gap junctions with neurites from another essentially identical identified neuron. This observation suggests that a form of self-recognition inhibits reflexive gap junction formation between sibling neurites. When one or both of those growth cones had been physically isolated from the neuronal cell body, both electrical coupling and gap junction plaques, between growth cones from the same neuron, were observed to form rapidly (within 30 min). Thus, inhibition of electrical coupling between sibling neurites apparently depends on cytoplasmic continuity between neurites, and not the molecular composition of neurite membrane. The formation of gap junctions is not likely due to the isolation process; rather, the physical isolation appears to release an inhibition of reflexive gap junction formation. These data demonstrate the existence of a previously unknown constraint on the formation of electrical synapses.

A sensory role for neuronal growth cone filopodia.

The dynamic nature of neuronal growth cone filopodia led to the suggestion that the primary function of filopodia is to sample their immediate environment, responding to and transducing environmental signals that affect growth cone behaviour and shape. Filopodia seem well suited to serve as antenna-like sensors, their broad span allows sampling of information over a greatly enhanced radius, and forward-projecting filopodia encounter potential cues in the molecular terrain long before the advancing growth cone itself. Filopodia in culture can serve structural roles, exert mechanical tension and selectively adhere to their surrounding. Whether or not filopodia have a general sensory role has not been tested directly, largely because of their small size, which limits an electrophysiological approach, and their integral relationship with the parent growth cone, which prevents resolution of their different functions. Here we use surgical procedures to isolate individual filopodia from their parent growth cone and, by monitoring their morphology and calcium second messenger systems, we show that neuronal growth cone filopodia contain signal transduction mechanisms that allow autonomous responses and the transmission of distant environmental information to their parent growth cone.

The initial stages of neural regeneration are dependent upon intracellular calcium levels.

The earliest events of neuronal regeneration require resealing of the neurite's membrane after injury and the subsequent formation of a new growth cone. We have investigated these activities in vitro employing the large identified neurons of the snail Helisoma. Regeneration was elicited by transection of neurite processes and assessed by studying the formation of new growth cones from the proximal neurite stumps. Under normal conditions new growth cones formed rapidly in 100% of the preparations. This formation appeared to follow, however, a large rise in intracellular calcium and did not start until after the cells homeostatic machinery had re-established near baseline calcium levels. To test the hypothesis that elevated intracellular calcium levels delayed or inhibited growth cone formation, transections were performed after experimentally increasing intracellular calcium concentrations to different levels by either depolarization or by calcium ionophores. Under these conditions, regeneration was significantly retarded in a fashion dependent upon the intracellular calcium concentration. Another change in the extracellular milieu, namely lowering of the extracellular calcium concentration, also significantly retarded growth cone formation. Under these conditions neurons appeared unable to reseal their cut ends and eventually died. Taken together, these studies demonstrate the importance of both the extracellular and intracellular milieu at times immediately following neurite transection in determining whether or not the earliest stages of neuronal regeneration will occur.