Identified Snail Neurons Research Articles

P33. Nitrite hyperactivation of identified snail neurons

P33. Nitrite hyperactivation of identified snail neurons

Correction: Onset Dynamics of Action Potentials in Rat Neocortical Neurons and Identified Snail Neurons: Quantification of the Difference

Correction: Onset Dynamics of Action Potentials in Rat Neocortical Neurons and Identified Snail Neurons: Quantification of the Difference

Reflections

Reflections

Onset Dynamics of Action Potentials in Rat Neocortical Neurons and Identified Snail Neurons: Quantification of the Difference

The generation of action potentials (APs) is a key process in the operation of nerve cells and the communication between neurons. Action potentials in mammalian central neurons are characterized by an exceptionally fast onset dynamics, which differs from the typically slow and gradual onset dynamics seen in identified snail neurons. Here we describe a novel method of analysis which provides a quantitative measure of the onset dynamics of action potentials. This method captures the difference between the fast, step-like onset of APs in rat neocortical neurons and the gradual, exponential-like AP onset in identified snail neurons. The quantitative measure of the AP onset dynamics, provided by the method, allows us to perform quantitative analyses of factors influencing the dynamics.

Intracellular localization of the HCS2 gene products in identified snail neurons in vivo and in vitro.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

Caspase‐like activity is essential for long‐term synaptic plasticity in the terrestrial snail Helix

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase-3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase-3-like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase-3-immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase-3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase-3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase-3 with the irreversible and cell-permeable inhibitor of caspase-3 N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone prevented development of the long-term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase-3-like activity is essential for long-term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long-term memory.

A cGMP-dependent cascade enhances an l-type-like Ca 2+ current in identified snail neurons

We studied the impact of an NO-cGMP dependent signalling pathway on the high-voltage-activated (HVA) Ca 2+ current in identified neurons of the pulmonate snail, Helix pomatia, using Ba 2+ as charge carrier. The 3′,5′-cyclic guanosine monophosphate (cGMP) analogues, dibutyryl-cGMP and 8-bromo-cGMP, consistently induced a biphasic response, consisting of an increase superseded by a decline of the Ba 2+ current. The NO donor, sodium nitroprusside (SNP), modulated only in a minority of neurons the Ba 2+ current. Blockade of protein kinase activity with 1-[5-isoquinolinesulfonyl]-2 methyl piperazine (H 7), a nonselective protein kinase inhibitor, or Rp-8-pCPT-cGMP, a selective protein kinase G (PKG) inhibitor, decreased, whereas Rp-cAMP, a selective protein kinase A (PKA) inhibitor, increased the Ba 2+ current upon application of cGMP analogues or SNP. Okadaic acid or calyculin, inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), augmented the Ba 2+ current. Under these conditions, cGMP analogues or SNP had an additive-enhancing effect on the Ba 2+ current. When neurons were exposed to the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), cGMP analogues induced a persistent increase of the Ba 2+ current, whereas SNP induced a biphasic response. These data suggest coexistence of cGMP-PKG and cGMP-PDE pathways as well as crosstalk between cGMP and 3′,5′-cyclic adenosine monophosphate (cAMP) pathways, which converge on HVA Ca channels in Helix neurons. In this model, augmentation of the Ba 2+ current through HVA Ca channels is accomplished by PKA and PKG, whereas attenuation is mediated by PDEs, which prevent activation of protein kinases via hydrolysis of cyclic nucleotides.

The phosphorylation state of neuronal processes determines growth cone formation after neuronal injury.

Growth cones are essential for neuronal pathfinding during embryonic development and again after injury, when they aid in neuronal regeneration. This study was aimed at investigating the role of kinases in the earliest events in neuronal regeneration, namely, the formation of new growth cones from injured neuronal processes. Neurites of identified snail neurons grown in vitro were severed, and the formation of growth cones was observed from the ends of such transected processes. Under control conditions, all neurites formed a new growth cone within 45 min of transection. In contrast, growth cone formation in the presence of a general kinase inhibitor, K252a, was significantly inhibited. Moreover, decreasing the phosphorylation state of neurites by activating protein phosphatases with C2-ceramide also reduced growth cone formation. Pharmacological analysis with specific kinase inhibitors suggested that targets of protein kinase C (PKC) and tyrosine kinase (PTK) phosphorylation control growth cone formation. Inhibition of PKC with calphostin C and cerebroside completely blocked growth cone formation, whereas the inhibition of PTK with erbstatin analog significantly reduced growth cone formation. In contrast, inhibitors of protein kinase A, protein kinase G, CaM-kinase II, myosin light-chain kinase, Rho kinase, and PI-3 kinase had little or no effect 45 min after transection. These results suggest that the transformation underlying the formation of a growth cone from an injured (transected) neurite stump is highly sensitive to the phosphorylation state of key target proteins. Therefore, injury-induced signaling events will determine the outcome of neuronal regeneration through their action on kinase and phosphatase activities.

Selective blockade of gene expression in a single identified snail neuron

In the present study, the applicability of antisense morpholino oligos for loss-of-function experiments in neurobiology was investigated. The identified withdrawal interneurons of the parietal ganglia expressing helix command neuron-specific 2 (HCS2) gene were pressure injected with HCS2 antisense or control morpholino oligo solution at a final concentration 1–4 μM. No toxic or side effects for the neural functioning were noted immediately or several hours after injection. The changes in the concentration of HCS2-encoded protein in neurons after injection were monitored by two methods, Western blotting and immunostaining of the brain. The amount of the peptide immunoreactive with the HCS2 antibody started to decline in the injected cells at day 2 post-injection, decreased four- to five-fold at day 4, and stayed at this low level thereafter. Similar results obtained by both methods suggest significant selective blockade of production of the HCS2-encoded peptide. In contrast, no substantial decrease of the HCS2-encoded polypeptide was observed after injection with control oligos. Due to the high stability of the morpholino oligos in the cell, they represent a highly efficient tool for a specific long-term blockade of gene expression in molluscan neurons.

Membrane and synaptic effects of anti-S-100 are prevented by the same antibodies in low concentrations.

Effects of antiserum to S-100 protein (AS-100) in high (HC, corresponding to antiserum dilution 1:5 - 1:50) and low (LC, 1:10(12)) concentrations were studied in identified snail neurons and rat hippocampal slices. HC-AS-100 changed the frequency of action potential generation in spontaneously active neurons and blocked formation of long-term potentiation in mossy fiber synapses. LC-AS-100 alone did not affect any characteristic measured in our experiments, but 20 min pre-incubation of snail ganglia or hippocampal slices with LC-AS-100 abolished the effects of the same antibodies in HC. Simultaneous addition of both LC and HC did not prevent the development of HC-AS-100 effects. It seems possible that LC-AS-100 is capable of interaction with neuronal cells modifying their reaction to HC-AS-100.

Dependence of synaptic facilitation postsynaptically induced in snail neurones on season and serotonin level.

The problem of stability of long-lasting synaptic facilitation postsynaptically induced by intracellular current pulses without concomitant presynaptic activation was addressed. A short (15-20 min) phase of synaptic facilitation induced by intracellular tetanization in identified snail neurones was stable and present in all experiments, while a long-term phase (lasting > or = 50 min) was observed only some experiments. Data analysis revealed dependence of the long-term phase on season. Dependence of the long-term phase of facilitation on serotonin concentration in hemolymph, which is known to change with season, was shown using the selective neurotoxin 5,7-dihydroxytriptamine. Dependence of habituation rate in the same synaptic connection on season and serotonin level was shown.

A biphasic dopaminergic modulation of the high voltage-activated Ba2+ current of identified snail neurons

We have investigated the intracellular mechanisms by which dopamine induced a biphasic modulation of the Ba2+ current amplitude through the high voltage-activated Ca2+ channel (HVA-IBa) in identifiedHelix aspersa neurons. We used the two electrode voltage clamp technique on a group of identified neurons of the right parietal ganglionin situ, and the whole cell patch clamp technique on these same neurons in primary culture. Brief application of dopamine induced an initial fast reduction of the HVA-IBa followed by a slower enhancement of HVA-IBa. This enhancement was not due to a shift of the current-voltage curve. Repetitive application of dopamine did not attenuate this phase of the response. During longer application, the inhibition began to ‘sag’ and returned towards control levels. These results indicate that the enhancement was not due to a desensitization of the receptor or a relief from tonic G-protein mediated inhibition of the current. Manipulations of the levels of intracellular second messengers such as Ca2+, cGMP, cAMP, and arachidonic acid, as well as inhibition of protein kinases and phosphatases, had no effect on the dopamine induced biphasic effect on HVA-IBa. Pertussis toxin added to the patch pipette had a slow but simultaneous blocking effect on both phases of the dopamine action on HVA-IBa. Since our results show that pertussis toxin affects both phases of the dopamine action on this current, we suggest that both phases of the dopamine action on HVA-IBa are mediated by a pertussis toxin-sensitive G-protein. If a second messenger is implicated, it is none of the ‘classical’ second messenger systems.

Suppression by calmodulin of glutamate-induced potassium current in identified snail neurons

Using the voltage-clamp technique combined with pressure injection, we examined the inhibitory action of glutamate (Glu) in identified snail neurons. Calimidazolium and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), calmodulin inhibitors, enhanced a slow outward current (Glu current) induced by Glu in a dose-dependent manner. This Glu current was suppressed by intracellular injection of calmodulin. However, no significant change in the Glu current was observed when either CaCl 2 or EGTA was intracellularly applied. These results suggest a novel calmodulin-mediated process, through which Glu induces an outward current.

Evidence that Ca 2+/calmodulin-dependent protein phosphorylation is involved in the opening process of potassium channels in identified snail neurons

The effect of Ca 2+/calmodulin-dependent protein phosphorylation on K + channels was examined in snail neurons, using several pharmacological agents, the voltage clamp method and the pressure injection technique. H-7, a general protein kinase inhibitor, reduced the delayed outward K + current ( I KD) which was suppressed by tetraethylammonium. Ca 2+/calmodulin-dependent protein kinase II, when injected into neurons which had been treated with H-7, transiently restored the reduced I KD nearly to the pre-H-7 level. However, this restoration was blocked by W-7, a calmodulin inhibitor. In contrast, the catalytic subunit of cAMP-dependent protein kinase or protein kinase C injected into the H-7-treated neurons had little effect on the current. These findings suggest that Ca 2+/calmodulin-dependent protein phosphorylation is involved in the opening process of K +channels.

Muscarinic enhancement of the voltage‐dependent calcium current in an identified snail neuron.

1. In the F1 neuron of the snail Helix aspersa bathed in a Ba2+ and 4-aminopyridine-containing saline, carbamylcholine (CCh) enhanced the inward current carried by Ba2+ through the voltage-dependent Ca2+ channels. 2. This effect of CCh on the F1 neuron was not affected by the nicotinic antagonists (+)-tubocurarine and hexamethonium, but it was mimicked by oxotremorine and blocked by both atropine and pirenzepine. 3. The intracellular injection of GTP gamma S (guanosine 5'-O-(3- thiotriphosphate] into the F1 neuron caused both a decrease in Ca2+ current and a blockade of the CCh-induced enhancement of the Ca2+ current. 4. Neither cyclic AMP, cyclic GMP nor arachidonic acid mimicked the effect of CCh on the Ca2+ current in the F1 neuron. In contrast, the intracellular injection of EGTA blocked the CCh-induced enhancement of the Ca2+ current thus suggesting that cytosolic Ca2+ is involved in the CCh-induced response. 5. We then investigated the possible role of inositol 1,4,5-trisphosphate (InsP3) and Ca(2+)-dependent protein kinases in the CCh-induced enhancement of the Ca2+ current. The intracellular injection of InsP3 in the F1 neuron elicited no consistent change in the Ca2+ current. Diacylglycerol analogues (OAG and DOG) decreased the Ca2+ current amplitude, i.e. an effect opposite to that produced by CCh. This effect of the diacylglycerol analogues resulted from the activation of protein kinase C (PKC) since it was blocked by staurosporine. In addition, staurosporine did not affect the CCh-induced increase in Ca2+ current. 6. The intracellular injection of either Ca(2+)-calmodulin-dependent protein kinase II (Ca(2+)-CaM-PK) or a peptide inhibitor of this enzyme into the F1 neuron affected neither the Ca2+ current nor its enhancement by CCh. 7. We conclude that the CCh-induced enhancement of the Ca2+ current in the snail F1 neuron involves the activation via muscarinic receptors of an intracellular transduction mechanism in which cytosolic Ca2+ plays a key role. However, InsP3, protein kinase C and Ca(2+)-CaM-PK do not appear to be directly involved in this CCh-induced response.

Cyclic AMP-dependent protein phosphorylation is involved in the development of negative slope resistance and a reduction of the potassium current induced by pentylenetetrazole in identified snail neurons

The role of cAMP-dependent protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. The cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and isoquinolinesulfonamide, inhibited the PTZ-induced negative slope resistance (NSR) in the steady state I– V curve. These inhibitors also suppressed the action of PTZ on the delayed outward potassium current ( I KD). This suppression was transiently abolished by intracellular injection of the catalytic subunit of cAMP-dependent protein kinase. These findings suggest that cAMP-dependent protein phosphorylation may be involved in both the development of the NSR and a reduction of the I KD by PTZ, leading to depolarizing phase of a bursting cycle.

Serotonin Induces a Cyclic AMP-Mediated Outwardly Rectifying Slow K+ Current in a Single Identified Snail Neuron.

In the F2 neuron of the parietal ganglion of the snail Helix aspersa either bath or iontophoretic application of serotonin (5-HT) induces an outward current. This current has a long latency (10 - 60 s) and a slow time course, a 500 ms iontophoretic application of 5-HT evoking a response lasting 3 - 5 min. This slow outward current reverses at -80 mV, a value equal to EK. After doubling the extracellular K+ concentration the reversal potential of the 5-HT response is shifted by 19 mV, as predicted by the Nernst equation. The I-V curves reveal that the 5-HT-induced slow outward current is outwardly rectifying. This 5-HT response is blocked by intracellular Cs+ and tetraethylammonium (TEA+) and by extracellular TEA+ and Ba2+, but is not affected by the removal of extracellular Ca2+ or the intracellular injection of ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-acetic acid (EGTA). These results indicate that the outwardly rectifying slow outward current induced by 5-HT in the F2 neuron is carried by K+ and is Ca2+-independent. In the single isolated F2 neuron, 5-HT induces a 2.5-fold stimulation of the adenylate cyclase activity. In addition, both the intracellular injection of 3',5'-adenosine monophosphate (cAMP) and the application of forskolin mimic the effect of 5-HT on the F2 neuron. The phosphodiesterase inhibitor isobutylmethylxanthine also induces a slow outward current and potentiates the 5-HT response. The intracellular injection of a synthetic 20-residue peptide inhibitor of the cAMP-dependent protein kinase blocks the slow outward K+ current induced by 5-HT. These results show that in the F2 neuron, 5-HT elicits a slow K+ current via the stimulation of adenylate cyclase, an increase in intracellular cAMP and the activation of the cAMP-dependent kinase which probably phosphorylates a population of outwardly rectifying K+ channels or some protein/s associated with these channels.

Oxytocin-induced sodium current is mediated by cAMP-dependent protein phosphorylation in an identified snail neuron

The intracellular biochemical process underlying oxytocin-induced change of membrane properties was analyzed in an identified neuron of Achatina fulica Férussac, using pressure injection technique and pharmacological tools. Oxytocin dose-dependently enhanced the negative slope resistance (NSR) region on the current—voltage relation. The oxytocin-induced current was attenuated by a reduction of extracellular Na + and not influenced by the addition of 100 μM tetrodotoxin (TTX) to the medium, suggesting that this current is predominantly due to the activation of TTX-resistant Na + channels. In the Ca 2+-free state, substituted by an equivalent amount of Co 2+, the amplitude of oxytocin-induced current was somewhat reduced at the NSR region but it was not influenced at less than −60 mV. Application of 100 μM isobutylmethylxanthine, a phosphodiesterase inhibitor, augmented the oxytocin-induced current. Pressure injection of 10 mM adenosine 3′,5′-cyclic monophosphate (cAMP) elicited a Na +-dependent inward current similar to the oxytocin response. The further role of cAMP linked with the oxytocin-induced current was investigated using two kinds of cAMP-dependent protein kinase inhibitors, isoquinolinesulfonamide (H-8) and protein kinase inhibitor (PKI). Extracellular application of H-8 or pressure injection of PKI, prior to oxytocin application, both blocked the oxytocin-induced current. Based on these results, oxytocin-elicited inward currents may mediate cAMP-dependent protein phosphorylation mainly by activation of Na + channels.

Decrease of free calcium concentration at the outer surface of identified snail neurons during paroxysmal depolarization shifts

Changes of free calcium concentration at the outer neuronal surface during paroxysmal depolarization shifts elicited by pentylenetetrazol were measured. Investigations were performed on the identified neuron B3 of the buccal ganglion of Helix pomatia. Extracellular calcium concentration was recorded by calcium-selective microelectrodes. The extracellular calcium concentration steeply decreased with the commencement of paroxysmal depolarization and started to reincrease when the paroxysmal depolarization had reached its plateau level. It is concluded that an influx of calcium ions takes place during paroxysmal depolarization shifts.