Role of activity in the selection of new electrical synapses between adult Helisoma neurons

Our aim was to determine whether neural activity in the form of sodium-dependent action potentials play a role in the formation, maintenance and specificity of electrical synapses between regenerating neurons. We axotomized buccal neurons of the mollusc, Helisoma trivolvis, and placed ganglia into organ culture in the absence or presence of tetrodotoxin (TTX), a specific sodium channel blocker. Electrical coupling was measured using intracellular microelectrodes positioned within the soma of identified neurons. Neurite outgrowth was assessed by epifluorescence microscopy after filling neurons by iontophoresis with Lucifer yellow. Previous studies found that two days after axotomy transient electrical synapses form between heterologous neurons (e.g. buccal neurons 4 and 5). Five days after axotomy these transient connections disappeared and a new electrical synapse was stabilized between the paired buccal neurons 5. To determine whether blocking neural activity with TTX affected the specificity and formation of new electrical synapses, we examined electrical coupling between the heterologous neurons 4 and 5 two days after axotomy, and the paired buccal neurons 5 five days after axotomy. Our electrophysiological recordings indicated that different neurons in the buccal ganglion varied in their sensitivity to TTX (i.e. sensitivity of buccal neurons 19 greater than 5 greater than 4), but spontaneous activity was abolished in all 3 neurons by 2 x 10(-5) M TTX. Furthermore, the inhibitory effects of TTX occurred within seconds of superfusion and persisted for at least 6 days. Inhibition of activity by TTX could be reversed after superfusion with normal saline. Neurite outgrowth from axotomized neurons was not appreciably altered in the presence of TTX. Furthermore, no differences in the incidence of electrical coupling or the coupling resistance were detected between neurons 4 and 5 two days after axotomy and organ culture in the presence of TTX. However, electrical coupling between the symmetrically paired neurons 5 was elevated in the presence of TTX after 5 days. We conclude from these results that neural activity in the form of sodium-dependent action potentials does not play an important role in the formation or breaking of transient electrical synapses during neuronal regeneration in the mollusc Helisoma trivolvis.     

Role of activity in the selection of new electrical synapses between adult Helisoma neurons

Our aim was to determine whether neural activity in the form of sodium-dependent action potentials plays a role in the formation, maintenance and specificity of electrical synapses between regenerating neurons. We axotomized buccal neurons of the mollusc, Helisoma trivolvis, and placed ganglia into organ culture in the absence or presence of tetrodotoxin (TTX), a specific sodium channel blocker. Electrical coupling was measured using intracellular microelectrodes positioned within the soma of identified neurons. Neurite outgrowth was assessed by epifluorescence microscopy after filling neurons by iontophoresis with Lucifer yellow. Previous studies found that two days after axotomy transient electrical synapses form between heterologous neurons (e.g. buccal neurons 4 and 5). Five days after axotomy these transient connections disappeared and a new electrical synapse was stabilized between the paired buccal neurons 5. To determine whether blocking neural activity with TTX affected the specificity and formation of new electrical synapses, we examined electrical coupling between the heterologous neurons 4 and 5 two days after axotomy, and the paired buccal neurons 5 five days after axotomy. Our electrophysiological recordings indicated that different neurons in the buccal ganglion varied in their sensitivity to TTX (i.e. sensitivity of buccal neurons 19 > 5 > 4), but spontaneous activity was abolished in all 3 neurons by 2 × 10⁻⁵ M TTX. Furthermore, the inhibitory effects of TTX occurred within seconds of superfusion and persisted for at least 6 days. Inhibition of activity by TTX could be reversed after superfusion with normal saline. Neurite outgrowth from axotomized neurons was not appreciably altered in the presence of TTX. Furthermore, no differences in the incidence of electrical coupling or the coupling resistance were detected between neurons 4 and 5 two days after axotomy and organ culture in the presence of TTX. However, electrical coupling between the symmetrically paired neurons 5 was elevated in the presence of TTX after 5 days. We conclude from these results that neural activity in the form of sodium-dependent action potentials does not play an important role in the formation or breaking of transient electrical synapses during neuronal regeneration in the mollusc Helisoma trivolvis.     

Formation of electrical synapses between isolated, cultured Helisoma neurons requires mutual neurite elongation

Our previous experiments have suggested the hypothesis that conjoint active neuronal outgrowth may be necessary for formation of new electrical synapses between identified neurons of adult Helisoma buccal ganglia. This growth dependence hypothesis now has been tested by examining the responses of individual pairs of neurons in isolation from the influences of the ganglionic environment. Isolated cell culture of identified neurons (neuron 5) showed that: (i) neurons growing in cell culture undergo a predictable sequence of morphological changes culminating in a stable morphological state (i.e., growth stops); (ii) contact between actively growing neurons in cell culture results in the formation of electrical connections, just as in ganglia; and (iii) when an actively growing neuron encounters a neuron that is morphologically stable, electrical connections do not form or are very weak, even though strong connections are made between pairs of actively growing neurons in the same culture. These results establish that processes closely associated with growth are required for formation of electrical synapses between these neurons.     

Neurite outgrowth in individual neurons of a neuronal population is differentially regulated by calcium and cyclic AMP

In identified Helisoma neurons, intracellular calcium can regulate neurite elongation and growth cone motility. Neurotransmitters such as 5-HT suppress both neurite elongation and the filopodial and lamellipodial movements of growth cones by causing increases in intracellular calcium (Haydon et al., 1984; Cohan et al., 1987; Mattson and Kater, 1987). Since an additional second messenger, cyclic AMP (cAMP), is known to mediate many physiological effects of neurotransmitters, we tested (1) the possible involvement of cAMP in the regulation of neurite outgrowth from Helisoma buccal neurons and (2) calcium-cAMP interrelationships in the regulation of outgrowth. The cAMP-elevating agents forskolin (5 x 10(-6)-10(-4) M) and dibutyryl cAMP (dbcAMP; 5 x 10(-3)-10(-2) M) suppressed neurite elongation and growth cone movements in identified neurons B19 (5-HT sensitive) and B5 (5-HT insensitive); the suppression was reversible. Exposure of these particular identified neurons to the calcium channel blocker La3+ (10(-5) M) or a culture medium with reduced calcium prevented and reversed the suppressive effects of forskolin and dbcAMP. In order to determine if the results on neurons B5 and B19 were representative of all neurons or only a subset, we examined a larger population of neurons. Calcium ionophore A23187 suppressed outgrowth from all neurons in mass dissociate cultures of buccal neurons, while forskolin or dbcAMP plus IBMX suppressed outgrowth from only one-half of buccal neurons. Finally, we found that 2 subpopulations exist among the neurons whose outgrowth is suppressed by cAMP: One subpopulation requires calcium influx for cAMP to act, while the other does not. Thus, even within the relatively small population of neuronal types comprising the buccal ganglion of Helisoma, second messengers within different neurons can act and interact in different ways to regulate outgrowth.     

Release of neurite outgrowth promoting factors by Helisoma central ganglia depends on neural activity.

Identified buccal neurons B5 and B19 from the mollusc, Helisoma trivolvis, were plated into cell culture in order to assay for neurite outgrowth promoting factors released from central ring ganglia. The release and attachment of neurite promoting factors to the substratum of poly-lysine coated dishes could be inhibited by blocking spontaneous bioelectric activity in central ring ganglia used to condition the medium and dishes. Bioelectric activity within neurons in central ring ganglia was assayed by intracellular recording and found to be inhibited by exposure to the sodium channel blocker, tetrodotoxin (TTX; 2 x 10(-5) M), or CoCl2 (10 mM). Neither of these agents appeared to be toxic over a three day period since activity within neurons in central ring ganglia was restored following superfusion with saline. To examine the effect of blocking neural activity on the ability of central ring ganglia to release neurite outgrowth promoting factors, we compared the percentage of neurons that extended processes under 5 different conditions: (1) dishes containing conditioned medium and substrate attached growth factors (Super SAM); (2) dishes with substrate attached growth factors only and defined medium (SAM); (3) dishes containing substrate attached growth factors prepared in the presence of TTX; or (4) CoCl2; and (5) dishes containing unconditioned defined medium. The percentage of neurons extending processes under the 5 conditions were: (1) 71% (n = 32); (2) 51% (n = 33); (3) 14% (n = 37); (4) 15% (n = 47); (5) 0% (n = 40), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements

The neurotransmitter 5-HT alters growth cone motility and neurite elongation in neuron B19, isolated from the buccal ganglion of Helisoma trivolvis (Haydon et al., 1984). The effects of 5-HT are mediated by increases in intracellular calcium levels within the growth cones (Cohan et al., 1987). 5-HT causes a receptor-mediated depolarization of the membrane, which results in the opening of voltage-sensitive calcium channels. The resulting calcium influx decreases both the elongation rate and the total outgrowth of neurites. However, the mechanism(s) mediating these calcium-dependent changes is unclear. As many of the intracellular effects of calcium in eukaryotic cells are mediated by the calcium-binding protein calmodulin, we tested the involvement of such an interaction in the regulation of neurite outgrowth. In these experiments, a new, potent calmodulin antagonist with increased selectivity, CGS 9343B (CGS; Norman et al., 1987), was used to inhibit calmodulin activity during the application of 5-HT to neuron B19. The addition of 100 microM 5-HT to the culture medium resulted in a significant decrease in the rate of neurite elongation and total neurite outgrowth. Administration of CGS to the culture medium at a concentration (1.8 microM) equivalent to its IC50 for calmodulin inhibition completely blocked the inhibitory effects of 100 microM 5-HT, on both neurite elongation and total neurite outgrowth. CGS alone caused a slight decrease in elongation rate but had no significant effect on total outgrowth. CGS did not block 5-HT-induced electrical activity, indicating that it was not acting as a 5-HT receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axonal regeneration of an identified Helisoma neuron depends on the site of axotomy.

Axotomy of molluscan neurons usually results in axonal regeneration. In this study, we tested the axotomy response of an identified neuron of the pond snail Helisoma trivolvis (buccal neuron 4, B4). This neuron has two primary axonal branches, the ipsilateral axon and the contralateral axon, each innervating one of the paired salivary glands. The ipsilateral axon projects via the ipsilateral esophageal nerve trunk whereas the contralateral axon crosses both buccal ganglia and projects via the contralateral esophageal nerve trunk. We tested various procedures of axotomy: injury to one axon or both axons, close to the ganglion or more distal. Surprisingly, we found that proximal axotomy of the ipsilateral axon was not usually followed by axonal regeneration. By contrast, all other procedures of axotomy (e.g., distal ipsilateral, or proximal bilateral) resulted in robust axonal regeneration and target reinnervation. Thus, in this preparation, axotomy may or may not result in axonal regeneration, depending on the site(s) of axotomy. To the best of our knowledge, such a differential result has not yet been found in any other preparation. We conclude that axotomy is not always a sufficient condition for axonal regeneration of molluscan neurons. We hypothesize that a damaged axonal stump may be a necessary condition for the initiation of regeneration. An alternative hypothesis is that neurite outgrowth is inhibited in normal mature neurons by a target-derived factor. This hypothetical factor would be transported retrogradely, but not anterogradely, along axons.     

‘Zap axotomy’: Localized fluorescent excitation of single dye-filled neurons induces growth by selective axotomy

The response of populations of neurons to axotomy has traditionally been studied by crushing or sectioning whole nerve trunks. The present communication describes a technique by which single neurons can be reliably and selectively axotomized in the absence of damage to other axons and non-neuronal cells within the nerve. To obtain selective axotomy, identified neurons of the buccal ganglia of the snail, Helisoma, were first filled with fluorescent dye. Next, the preparation was positioned in a restricted beam of blue light using low light video fluorescence microscopy. Finally, the selected region of axon was briefly exposed to light levels normally employed for fluorescence microscopy.Shortly after irradiation of the identified neuron 5, antidromic action potentials no longer propagated past the region of exposure in the dye-filled cell, whereas adjacent axons were physiologically intact. Several days after exposure, profuse neurite outgrowth was observed from the proximal region of axon of neuron 5, but never in neighboring axons which were not filled with dye at the time of irradiation. When the axons of both neurons 5R and 5L were spot irradiated neurite outgrowth resulted in the formation of a novel electrical connection between these cells. These changes in growth and connectivity which were induced by selective axotomy of single axons were indistinguishable from the changes which are produced by crushing entire nerve trunks.     

Evidence for the presence, synthesis, immunoreactivity, and uptake of GABA in the nervous system of the snail Helisoma trivolvis.

In the present study several techniques were employed to test the hypothesis that gamma-aminobutyric acid (GABA) is a neurotransmitter in the central nervous system (CNS) of the pond snail Helisoma trivolvis (Mollusca, Pulmonata). First, by using chromatographic techniques, the presence of GABA and its differential distribution among the ganglia constituting the CNS was demonstrated. Second, de novo synthesis of 3H-GABA from 3H-glutamate was shown by the CNS. Levels of both endogenous and newly synthesized GABA were greatest in the buccal, cerebral, and pedal ganglia. Third, indirect immunohistochemistry of wholemounts revealed a central network of GABA-like immunoreactive neurons. With the possible exceptions of two pairs of fibers in nerve trunks, all projections from GABA-immunoreactive neurons were confined to the CNS, suggesting a predominantly central role for GABA. Stained neurons were found on the dorsal surface of the buccal ganglia and throughout the cerebral and pedal ganglia. No GABA-immunoreactive cell bodies were observed in the parietal, pleural, or visceral ganglia. Finally, uptake of 3H-GABA was examined autoradiographically in sectioned ganglia. A pattern of radiolabelled cells was observed that closely resembled the distribution of GABA-immunoreactive neurons. The data described above fulfill several criteria necessary to establish GABA as a transmitter in the nervous system of Helisoma. Taken together with previously obtained pharmacological evidence demonstrating that GABA acts on Helisoma central neurons, GABA is considered to be a strong candidate for a neurotransmitter in Helisoma.     

Dopamine and serotonin inhibition of neurite elongation of different identified neurons

This study demonstrates that a second classical neurotransmitter, dopamine, can act to suppress regenerative neurite outgrowth. Single identified neurons were dissected from two central ganglia of the snail Helisoma, and growth cone motility was studied as neurites regenerated in cell culture. Both dopamine and serotonin inhibited growth cone motility and elongation of neurites. Outgrowth inhibition ranged from sustained arrest to a similar but transient response. The effects of dopamine and serotonin are neuron-selective. Specific neurons affected by dopamine and serotonin represent distinct sets. One neuron was found that responds to both agents. The implications of neurotransmitter regulation of the dynamics of neuronal morphology are discussed.     

Nerve growth-promoting factor produced in culture media conditioned by specific CNS tissues of the snailHelisoma

Medium conditioned by tissue from the CNS of the snail, Helisoma, is capable of promoting neurite outgrowth in isolated neurons from adult central ganglia. The conditioning factor(s) (CF), contained in conditioned medium (CM), is produced only by central ganglionic rings and buccal ganglia and not by other tissues, including hemolymph. CF requires a minimum of 24 h to be produced or released into the medium. At 12 h growth-promoting activity was not detectable. CF binds tightly to the polylysine substratum and its activity is not mimicked by addition of various sera, NGF or fibronectin. CF activity is abolished by chymotrypsin, trypsin or heating to 100 degrees C, but is stable to DNase and RNase treatment. The percentage of cells exhibiting neurite outgrowth is approximately linear with the amount of neural tissue used to condition the medium up to 2 ganglionic rings/ml. Addition of more ganglia fails to stimulate a greater response. This apparent plateau of CM activity appears to be a function of production and/or release of CF, rather than a saturation effect on plated cells, since dose-response curves for dilutions of CM are approximately linear regardless of the number of ganglia used for conditioning. In addition, anisomycin inhibits 35% of CF appearance under conditions of over 90% protein synthesis inhibition in the ganglia used to produce the CM. Under these conditions anisomycin has no apparent effect on the maintenance of electrical excitability. The inhibitor data suggest that 65% of CF is derived from a pre-existing storage pool and that the remainder is synthesized during the 72 h conditioning period.     

Differential synapse formation and neurite outgrowth at two branches of the metacerebral cell of Aplysia in dissociated cell culture

The metacerebral cell (MCC) of Aplysia californica was isolated with its bifurcate axon from the cerebral ganglion and maintained in vitro under three conditions: (a) with no targets, (b) with identified buccal ganglion neurons B1 or B2 placed near the stump of the large diameter cerebral-buccal connective (CBC) branch, and (c) with B1 or B2 placed near the stump of the small diameter posterior lip nerve (PLN) branch. After 5 days in culture, the two branches differed significantly in the formation of chemical connections and in the extent of neurite outgrowth. Chemical connections characteristic of MCC-B1(B2) connections in vivo were observed in more than 90% of the cultures in which the buccal neuron was contacted by neurites emerging from the CBC branch, but in only 20% of the cultures in which the buccal neuron was contacted by neurites extending from the PLN branch. Neurite outgrowth from the CBC stump was always greater than growth from the PLN and was not affected significantly by the presence of a buccal neuron target at either branch. In contrast, neurite outgrowth from the PLN decreased significantly when the target was contacted by neurites from the CBC branch. These results suggest that two branches of a single neuron can differ in their capacities to form chemical connections. In addition, the two branches show differential growth as a result of target interaction at one of the branches. This simple in vitro system may therefore be useful in exploring the ways in which individual neurons control neurite extension from different branches as they seek to form chemical connections with their targets.     

Interleukin-17A increases neurite outgrowth from adult postganglionic sympathetic neurons

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.     

Sprouting by isolated Helisoma neurons: enhancement by glutamate

We tested glutamate for its ability to modulate neurite outgrowth from isolated neurons of the adult snail, Helisoma trivolvis. Although glutamate did not induce neurite outgrowth from neurons maintained in defined medium, nevertheless it showed a dose-dependent ability to enhance the activity of conditioned medium. We concluded that glutamate can enhance the release and/or activity of CNS derived sprouting factor(s) present in conditioned medium. The general conclusion to be drawn from this study is that the ability of a neurotrophic factor(s) to promote neurite outgrowth can be regulated by a neurotransmitter. This mechanism may be important in the regulation of trophic factors in the adult nervous system.     

Estrogen increases sensory nociceptor neuritogenesis in vitro by a direct, nerve growth factor-independent mechanism

Estrogen affects many aspects of the nervous system, including pain sensitivity and neural regulation of vascular function. We have shown that estrogen elevation increases sensory nociceptor innervation of arterioles in Sprague-Dawley rat mammary gland, external ear and mesentery, suggesting widespread effects on sensory vasodilatory innervation. However, it is unclear whether estrogen elicits nociceptor hyperinnervation by promoting target release of neurotrophic factors, or by direct effects on sensory neurons. To determine if estrogen may promote axon sprouting by increasing release of target-derived diffusible factors, dorsal root ganglia explants were co-cultured with mesenteric arterioles for 36 h in the absence or presence of 17beta-estradiol (E2). Mesenteric arteriolar target substantially increased neurite outgrowth from explanted ganglia, but estrogen had no effect on outgrowth, suggesting that estrogen does not increase the availability of trophic proteins responsible for target-induced neurite outgrowth. To assess the direct effects of estrogen, dissociated neonatal dorsal root ganglion neurons were cultured for 3 days in the absence or presence of E2 and nerve growth factor (NGF; 1-10 ng/mL), and immunostained for the nociceptor markers peripherin or calcitonin gene-related peptide. NGF increased neuron size, survival and numbers of neurons with neurites, but did not affect neurite area per neuron. Estrogen did not affect neuron survival, size or numbers of neurons with neurites, but did increase neurite area per neuron. The effects of these agents were not synergistic. We conclude that estrogen exerts direct effects on nociceptor neurons to promote axon outgrowth, and this occurs through an NGF-independent mechanism.     

Triiodothyronine Exerts a Trophic Action on Rat Sensory Neuron Survival and Neurite Outgrowth through Different Pathways

Apart from several growth factors which play a crucial role in the survival and development of the central and peripheral nervous systems, thyroid hormones can affect different processes involved in the differentiation and maturation of neurons. The present study was initiated to determine whether triiodothyronine (T₃) affects the survival and neurite outgrowth of primary sensory neurons in vitro. Dorsal root ganglia (DRG) from 19-day-old embryos or newborn rats were plated in explant or dissociated cell cultures. The effect of T₃ on neuron survival was tested, either in mixed DRG cell cultures, where neurons grow with non-neuronal cells, or in neuron-enriched cultures where non-neuronal cells were eliminated at the outset. T₃, in physiological concentrations, promoted the growth of neurons in mixed DRG cell cultures as well as in neuron-enriched cultures without added nerve growth factor (NGF). Since neuron survival in neuron-enriched cultures cannot be promoted by endogenous neurotrophic factors synthesized by non-neuronal cells, the increased number of surviving neurons was due to a direct trophic action of T₃. Another trophic effect was revealed in this study: T₃ sustained the neurite outgrowth of sensory neurons in DRG explants. The stimulatory effect of T₃ on nerve fibre outgrowth was considerably reduced when non-neuronal cell proliferation was inhibited by the antimitotic agent cytosine arabinoside, and was completely suppressed when the great majority of non-neuronal cells were eliminated in neuron-enriched cultures. These results indicate that the stimulatory effect of T₃ on neurite outgrowth is mediated through non-neuronal cells. It is conceivable that T₃ up-regulates Schwann cell expression of a neurotrophic factor, which in turn stimulates axon growth of sensory neurons. Together, these results demonstrate that T₃ promotes both survival and neurite outgrowth of primary sensory neurons in DRG cell cultures. The trophic actions of T₃ on neuron survival and neurite outgrowth operate under two different pathways.     

Effects of serotonin on intracellular calcium in embryonic and adult Helisoma neurons.

The neurotransmitter serotonin has been shown to regulate neurite outgrowth in many embryonic and adult Helisoma neurons. To determine whether intracellular calcium concentration is also regulated by serotonin in large numbers of neurons, the calcium indicator Fura 2 was used to measure intracellular calcium in mass-dissociated cultures of embryonic and adult neurons. Comparisons between embryonic and adult neurons revealed that embryonic neurons have a narrow population distribution of rest intracellular calcium levels around relatively low values. In contrast, the population distribution for adult neurons covered a much wider range of rest calcium concentrations. In both embryonic and adult cultures, serotonin induced a shift in the population distribution of calcium concentrations to higher levels, and increased the mean and median calcium concentrations. Analysis of individual adult neurons prior to and following the addition of serotonin revealed that approximately 50% of the neurons responded with an increase in calcium concentration. In contrast, there was no evidence of a serotonin-induced decrease in calcium concentration in any neurons. Since the percentage of neurons responding to serotonin in this study is very similar to the percentage that responded in previous studies on neurite outgrowth, these data support the hypothesis that an increase in intracellular calcium is a common intermediate step in the regulation of neurite outgrowth by serotonin throughout the Helisoma nervous system.     

FMRFamide peptides in Helisoma: identification and physiological actions at a peripheral synapse

Previous reports have demonstrated powerful neuromodulatory actions of the molluscan tetrapeptide FMRFamide in both the central and peripheral nervous systems of the freshwater snail Helisoma. The present study was designed to examine both the nature of the FMRFamide-like peptides in Helisoma and to define their physiological actions at a peripheral synapse. We report that, as determined by HPLC/RIA and mass spectrometry, Helisoma contains both FMRFamide and 2 of its analogs, FLRFamide and GDPFLRFamide. Whereas whole animals contain about 100 pmol/gm of these peptides, they were enriched in the nervous system (3000 pmol/gm) and in a peripheral target organ, the salivary glands (500 pmol/gm). For histochemical and physiological studies we examined the salivary glands, which are known to be innervated by neuron 4 of the buccal ganglion. We confirmed the presence of FMRFamide-like fibers on the salivary gland by immunohistochemistry using a polyclonal antiserum. These fibers appear to be largely derived from somata located in the central ring ganglia. For physiological tests we examined the neuron 4-gland synapse, at which presynaptic action potentials normally evoke a suprathreshold EPSP in gland cells. Bath application of FMRFamide, FLRFamide, or GDPFLRFamide at micromolar concentration to a buccal ganglia/salivary gland preparation completely suppressed spontaneous rhythmic activity. The sites of action of these peptides were examined by iontophoretic application of FMRFamide to neuron 4 or the salivary gland. Application of the peptide to the soma of neuron 4 caused a hyperpolarization that suppressed spontaneously generated action potentials. When applied to the salivary gland, FMRFamide caused a hyperpolarization that reduced the EPSPs evoked by neuron 4 to below spike threshold. The latter observation implies a postsynaptic locus of action for FMRFamide, and this possibility was tested by direct depolarization of the gland with iontophoresis of ACh (the putative transmitter of neuron 4). Such depolarizations were also reduced by FMRFamide. We conclude that Helisoma contains FMRFamide and 2 of its analogs, these peptides being enriched in the nervous system and salivary glands. Furthermore, these peptides can suppress activation of the salivary glands by actions both directly on gland cells and on the effector neuron.     

Vasopressin promotes neurite growth in cultured embryonic neurons

Vasopressin (AVP) has been identified as a neural peptide which may influence memory function. Because of this action, we investigated the effect of AVP on neurons growing in culture. Vasopressin was found to markedly increase neurite outgrowth from cultured embryonic neurons and to also accelerate the rate of neuritic growth. Maximal stimulation of neurite production occurred after 24-hour incubation in the presence of 1 microM AVP. In AVP-treated cultures the profuse neuritic arborization was characterized by numerous microspikes along the neuritic shafts and at the perimeters of growth cones. These data provide strong evidence for a neurotrophic effect of AVP which, we suggest, may be relevant to neuronal development as well as to morphological changes which occur in the mature nervous system, possibly during memory formation.     

体外低频电刺激促进背根神经元突起的生长：上调钙离子介导的脑源性神经营养因子表达可为其途径

摘要 背景：短时低频电刺激已被证明可显著促进周围神经系统损伤后轴突的再生,目前对电刺激是如何促进其突起生长还有待证实。 目的：体外培养背根神经元,观察短时低频电刺激对神经元突起生长的影响,探讨电刺激发挥作用可能的细胞信号分子。 设计、时间及地点：体外培养背根神经元及离体电刺激处理,于2007-05/2008-10在上海交通大学医学院完成。 材料：新生48h Sprague-Dawley大鼠20只(中科院上海生命科学研究所动科所)。 方法：体外培养背根神经元,随机分为两组,正常对照组(n = 6)及电刺激组(n = 8)。电刺激组施予离体电刺激(20Hz, 100μs, 3V),持续作用1h。为探讨电刺激发挥作用经由的细胞信号分子,在施予电刺激前预先加入钙离子通道阻滞剂Nifedipine孵育4小时,再给予电刺激,再次检测各组神经元突起的生长情况。 主要观察指标：β-tubulin染神经元,测量各组神经元突起的长度。RT-PCR、 western blot和ELISA分别检测神经元BDNF的表达和分泌。 结果：短时低频电刺激促进神经元突起的生长,增强其表达和分泌BDNF (P < 0.05)。Nifedipine的使用削弱了电刺激对神经元突起生长及BDNF合成的促进作用 (P < 0.05)。 结论：短时低频电刺激促进体外培养的背根神经元突起的生长及BDNF的合成,初步认为电刺激对神经元突起生长的促进作用,至少通过促发钙内流所致BDNF表达和分泌增多所致。 关键词：电刺激;背根神经元;突起生长;BDNF;Ca2+