In vivo recordings of spontaneous and odor-modulated dynamics in the Limax olfactory lobe

The major central site of olfactory information processing in the terrestrial slug Limax maximus is the procerebral lobe of the cerebral ganglion, which exhibits oscillatory dynamics of its local field potential and propagates activity waves from its apex to its base, as determined by multisite optical and electrical measurements in vitro. The learning-dependent uptake of Lucifer yellow into procerebral neurons suggests that the procerebral lobe may form learned representations of odors. To determine the role of the procerebral lobe in odor processing and odor learning, we developed procedures to implant fine wire electrodes in the lobe, which allowed recordings of local field potential in freely behaving slugs. The procerebral lobe displays oscillatory dynamics of its local field potential in vivo; however the amplitude and frequency of the local field potential are much more variable in vivo than in vitro. Odor presentation leads to increased frequency and amplitude of the local field potential signal. Several lines of evidence indicate that the variations in the local field potential signal recorded in vivo are not due to movement artifacts or activity in adjacent muscles. Multiple amine, gaseous, and peptide neuromodulators known to be present in the procerebral lobe provide pathways by which activity or coupling of bursting neurons in the procerebral lobe could be altered, resulting in the observed amplitude and frequency modulation of the local field potential.     

Aminergic and peptidergic amplification of intracellular cyclic AMP levels in a molluscan neural network

1. Serotonin (5-hydroxytryptamine; 5-HT), dopamine (DA), and small cardioactive peptide B (SCPB) can activate adenylate cyclase and increase the intracellular cyclic AMP (cAMP) levels in the Limax procerebrum (PC), with differing time courses and to differing extents. 5-HT and SCPB are potent stimulators of adenylate cyclase, and when both were applied simultaneously, an additive effect was observed. 2. In contrast, DA shows a great variability in the time course of cAMP synthesis and is a weak stimulator. Ergonovine, a DA antagonist, failed to inhibit cyclase activation, indicating that ergonovine-sensitive receptors are absent or ergonovine-sensitive DA receptors are not coupled to adenylate cyclase. 3. 5-HT and SCPB cause a rapid synthesis of cAMP, reaching the maximum 20- to 30-fold increase within a minute. DA's effect is slow in onset and very prolonged, reaching a maximum of only a two- to three-fold increase in the cAMP level. Reasons for variability in DA action are discussed.     

In vivo recordings of spontaneous and odor‐modulated dynamics in the Limax olfactory lobe

The major central site of olfactory information processing in the terrestrial slug Limax maximus is the procerebral lobe of the cerebral ganglion, which exhibits oscillatory dynamics of its local field potential and propagates activity waves from its apex to its base, as determined by multisite optical and electrical measurements in vitro. The learning‐dependent uptake of Lucifer yellow into procerebral neurons suggests that the procerebral lobe may form learned representations of odors. To determine the role of the procerebral lobe in odor processing and odor learning, we developed procedures to implant fine wire electrodes in the lobe, which allowed recordings of local field potential in freely behaving slugs. The procerebral lobe displays oscillatory dynamics of its local field potential in vivo; however the amplitude and frequency of the local field potential are much more variable in vivo than in vitro. Odor presentation leads to increased frequency and amplitude of the local field potential signal. Several lines of evidence indicate that the variations in the local field potential signal recorded in vivo are not due to movement artifacts or activity in adjacent muscles. Multiple amine, gaseous, and peptide neuromodulators known to be present in the procerebral lobe provide pathways by which activity or coupling of bursting neurons in the procerebral lobe could be altered, resulting in the observed amplitude and frequency modulation of the local field potential. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 126–141, 2001     

Model for Transition from Waves to Synchrony in the Olfactory Lobe of Limax

A biophysical model for the interactions between bursting (B) cells and nonbursting (NB) cells in the procerebral lobe of Limax is developed and tested. Phase-sensitivity of the NB cells is exhibited due to the strong inhibition from the rhythmically bursting B cells. Electrical and chemical junctions coupled with a parameter gradient lead to sustained periodic waves in the lobe. Excitatory interactions between the NB cells, which rarely fire, lead to stimulus evoked synchrony in the lobe oscillations. A novel calcium current is suggested to explain the effects of nitric oxide (NO) on the lobe. Gap junctions are shown both experimentally and through simulations to be required for the oscillating field potentials.     

Lateralized memory storage and crossed inhibition during odor processing by Limax

After odor conditioning intact Limax maximus and injecting LY into their haemocoel, labeled groups of neurons are found in either the right or left procerebral lobe but never in both procerebral lobes. This suggests that a competitive interaction occurs between right and left odor processing pathways of which the procerebral lobe is a part. We use the nerve discharge in the external peritentacular nerve evoked by applying a puff of conditioned odor to the nose to document crossed inhibition between left and right odor processing pathways. Responses in the external peritentacular nerve evoked by stimulating one superior nose with a conditioned odor are strongly lateralized as responses occur only on the stimulated side. Stimulating both superior noses simultaneously with the same conditioned odor yields responses in both external peritentacular nerves that resemble the sum of responses to unilateral stimulation. Simultaneously stimulating both superior noses, each with a different conditioned odor, leads to strong inhibition of both external peritentacular nerve responses. The crossed inhibition is also evident if both superior and inferior noses on the same side are stimulated simultaneously. A lateral inhibitory mechanism, situated postsynaptic to odor recognition, appears to inhibit external peritentacular nerve responses if the two noses receive conflicting sensory inputs. Accepted: 14 December 1999     

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl⁻ ions, while the late component was presumed to be mediated by K⁺ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K⁺ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K⁺ current both in the bursting and nonbursting neurons. The glutamate-induced K⁺ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity. Accepted: 10 February 1999     

Optical recording of the odor-evoked responses in olfactory structures of the brain of the terrestrial mollusk Helix

Regular oscillations were recorded in olfactory part of the brain (procerebrum) of gastropod mollusk Helix both electrographically and optically. In general, oscillations resembled those in slugs reported earlier. Odor application caused a transient change in the procerebral oscillations followed by appearance of a special pattern. For the first time the evoked potential was recorded in procerebrum and mapped in reference to the area of oscillations. The area of spreading of evoked potential roughly corresponded to the neuropil projection, while the oscillations were recorded in the projection of the cell body layer of procerebrum. The wave of the evoked potential emerged near the place of the olfactory nerve entrance into the procerebrum and propagated via the procerebrum neuropil towards the cell body layer. The evoked potential did not produce a phase-independent wave in rhythmical oscillations.     

Delayed phosphorylation of classical protein kinase C (PKC) substrates requires PKC internalization and formation of the pericentrion in a phospholipase D (PLD)-dependent manner

It was previously demonstrated that sustained activation (30-60 min) of protein kinase C (PKC) results in translocation of PKC α and βII to the pericentrion, a dynamic subset of the recycling compartment whose formation is dependent on PKC and phospholipase D (PLD). Here we investigated whether the formation of the pericentrion modulates the ability of PKC to phosphorylate substrates, especially if it reduces substrate phosphorylation by sequestering PKC. Surprisingly, using an antibody that detects phosphosubstrates of classical PKCs, the results showed that the majority of PKC phosphosubstrates are phosphorylated with delayed kinetics, correlating with the time frame of PKC translocation to the pericentrion. Substrate phosphorylation was blocked by PLD inhibitors and was not observed in response to activation of a PKC βII mutant (F663D) that is defective in interaction with PLD and in internalization. Phosphorylation was also inhibited by blocking clathrin-dependent endocytosis, demonstrating a requirement for endocytosis for the PKC-dependent major phosphorylation effects. Serotonin receptor activation by serotonin showed a similar response to phorbol 12-myristate 13-acetate, implicating a potential role of delayed kinetics in G protein-coupled receptor signaling. Evaluation of candidate substrates revealed that the phosphorylation of the PKC substrate p70S6K kinase behaved in a similar manner. Gradient-based fractionation revealed that the majority of these PKC substrates reside within the pericentrion-enriched fractions and not in the plasma membrane. Finally, proteomic analysis of the pericentrion-enriched fractions revealed several proteins as known PKC substrates and/or proteins involved in endocytic trafficking. These results reveal an important role for PKC internalization and for the pericentrion as key determinants/amplifiers of PKC action.     

Gaseous Oxides and Olfactory Computation

The gaseous neurotransmitters nitric oxide (NO) and carbon monoxide(CO) are prominent and universal components of the array ofneurotransmitters found in olfactory information processingsystems. These highly mobile communication compounds have effectson both second messenger signaling and directly on ion channelgating in olfactory receptors and central synaptic processingof receptor input. Olfactory systems are notable for the plasticityof their synaptic connections, revealed both in higher-orderassociative learning mechanisms using odor cues and developmentalplasticity operating to maintain function during addition ofnew olfactory receptors and new central olfactory interneurons.We use the macrosmatic terrestrial mollusk Limax maximus toinvestigate the role of NO and CO in the dynamics of centralodor processing and odor learning. The major central site ofodor processing in the Limax CNS is the procerebral (PC) lobeof the cerebral ganglion, which displays oscillatory dynamicsof its local field potential and periodic activity waves modulatedby odor input. The bursting neurons in the PC lobe are dependenton local NO synthesis for maintenance of bursting activity andwave propagation. New data show that these bursting PC interneuronsare also stimulated by carbon monoxide. The synthesizing enzymefor carbon monoxide, heme oxygenase 2, is present in the neuropilof the PC lobe. Since the PC lobe exhibits two forms of synapticplasticity related to both associative odor learning and continualconnection of new receptors and interneurons, the use of multiplegaseous neurotransmitters may be required to enable these multipleforms of synaptic plasticity.     

Serotonin activates angiogenic phosphorylation signaling in human endothelial cells

Serotonin, a known neurotransmitter, also functions as an angiokine to promote angiogenesis. The majority of serotonin in the human body is stored in platelets, and platelet aggregation leads to significant release of serotonin in thrombotic tumor environment. We have investigated serotonin signaling in human endothelial cells. Through G-protein-coupled receptors, serotonin at physiologically relevant concentrations activated Src/PI3K/AKT/mTOR/p70S6K phosphorylation signaling, and this activation was similar to that seen with VEGF. This finding provides insight into the overlapping angiogenic signaling pathways stimulated by serotonin in tumor environment, and suggests one of the mechanisms underlying resistance to current VEGF-targeting antiangiogenic therapy against cancer.     

Cyclic Adenosine Monophosphate in the Nervous System of Aplysia californica : II. Effect of serotonin and dopamine

Serotonin and dopamine, both likely transmitter substances in Aplysia, stimulated formation of adenosine-3',5' monophosphate (cAMP) in ganglia, connectives, and identified nerve cell bodies. This widespread distribution suggests that receptors for the response are localized throughout the nervous system, as is adenyl cyclase. Both synthesis of cAMP-³H from precursor previously labeled in incubations with adenine-³H and total content of cAMP were stimulated up to 15-fold. The acetylcholine analogue carbachol, glutamate, norepinephrine, and histamine were inactive. Full stimulation occurred within 2–4 min of applying serotonin; the extent of the effect was half maximal at 6µ serotonin. Even in the continued presence of serotonin, the increased cAMP diminished with time. When serotonin was removed, tissue remained refractory for 15–20 min; sensitivity returned after 25 min. Serotonin stimulated cAMP after removal of extracellular Na, K, or Cl and in isotonic sucrose, with all extracellular ions removed. Elevating Mg, which blocked the stimulation of cAMP caused by synaptic activity, did not affect the response to serotonin. Thus the response appeared to be independent of transmitter release and of changes in synaptic potentials and current flow. The role of cAMP in neuronal functioning remains to be determined. Conditions which markedly increased cAMP in neurons, however, did not affect the rate of RNA synthesis, nor did they alter the distribution of phosphorylated adenine or uridine nucleotides.     

Neurogenesis in the procerebrum of the snail Helix aspersa: a quantitative analysis

The procerebrum, a specialized structure for olfaction in terrestrial pulmonate molluscs, contains 20,000 to 50,000 small, uniformly sized neurons that increase in number with age. Here I show the likely source of neurons added to the procerebrum of Helix aspersa and that the rate of neuron addition depends on snail weight. After hatching, during the initial exponential growth phase, H. aspersa adds neurons to the procerebral apex by mitosis and from a cerebral tube. In the logistic growth phase beginning 30-40 days post-hatch, neurons also seem to be added to the procerebrum from the peritentacular and olfactory nerves, causing the rate of neuron addition to approximately double; but as in the earlier exponential growth phase, this rate remains a function of snail weight. This neuron addition throughout the life of the snail can be predicted by snail weight. In the two growth phases, the number of neurons in the procerebrum is given by logarithmic functions of snail weight. The results here for H. aspersa provide the basis for experiments to determine the peripheral origin and destination of neuronal precursors that are added to the procerebrum and to determine how neuron addition affects the function of the procerebrum.     

GABAergic effects on the slow oscillatory neural activities in the procerebrum of Limax valentianus. Short communication

We examined GABAergic modulation on "slow" oscillation (<1.0 Hz) of the procerebrum in the terrestrial mollusk, Limax valentianus. Short application of GABA-receptor agonists slightly increased the frequency of a periodic oscillation in the procerebrum, whereas persistent application decreased it. GABA-receptor antagonists decreased the oscillatory frequency. The GABA-like immunoreactivities were found in the neuropil and the cell body layers of the procerebrum. Because GABAergic inhibition is known to be essential for the generation of "fast" synchronous neuronal oscillation in the CNSs in othre many animals, our present findings are first evidence suggesting that GABA modulates 'slow' oscillation in the CNS.     

Heat-shock response in Fonsecaea pedrosoi, a pathogenic fungus.

Using two-dimensional electrophoresis we have investigated the heat-shock response in a pathogenic fungus, Fonsecaea pedrosoi. Fungal cultures were transferred from 37 to 45 degrees C for either 30 or 90 min and then returned back to the initial temperature. Analysis of the total proteins resolved on two-dimensional gels indicated important changes in the accumulation of several peptides according to the duration of treatment and the temperature. The 30-min incubation at 45 degrees C resulted in the induction of several new proteins, whereas other proteins were either increased or decreased. These inductions and repressions of proteins (called heat-shock and heat-stroke proteins, respectively) were either specific to this time period or still present after a 90-min incubation. In addition, the 90-min incubation period led to the enhancement of several proteins, which were therefore called late heat-shock proteins to distinguish them from the early ones detected after 30 min. Finally, when cultures were shifted back to 37 degrees C most of the heat-shock proteins decreased or disappeared; in parallel, most of the heat-stroke proteins were reinduced at this time. These results are in good agreement with previous studies on the heat-shock response and provide additional evidence that this phenomenon is highly conserved among species.     

Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR

The role of the AMP-activated kinase (AMPK) as a metabolic sensor in skeletal muscle has been far better characterized for glucose and fat metabolism than for protein metabolism. Therefore, the studies presented here were designed to examine the effects of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of mRNA translation and protein synthesis in cultures of C(2)C(12) myotubes. The findings show that, following AICAR (2 mM) treatment, AMPK phosphorylation was increased within 15 min and remained elevated throughout a 60-min time course. In association with the increase in AMPK phosphorylation, global rates of protein synthesis declined to 90, 70, and 63% of the control values at the 15-, 30-, and 60-min time points, respectively. By 60 min, polysomes disaggregated into free ribosomal subunits, suggesting an inhibition of initiation of mRNA translation. However, phosphorylation of eukaryotic elongation factor 2 was increased at 15 and 30 min but then declined to control values by 60 min, suggesting a transient inhibition of translation elongation. The decline in protein synthesis and changes in mRNA translation were associated with a repression of the mammalian target of rapamycin (mTOR) signaling pathway, as indicated by increased association of Hamartin with Tuberin, increased association of regulatory associated protein of mTOR with mTOR, and dephosphorylation of the downstream targets ribosomal protein S6 kinase-1 and eukaryotic initiation factor 4E-binding protein-1. They were also associated with activation of the MAPK signaling pathway, as indicated by increased phosphorylation of MEK1/2 and ERK1/2 and the downstream target eIF4E. Overall, the data support the conclusion that AICAR-induced AMPK activation suppresses protein synthesis through concurrent repression of mTOR signaling and activation of MAPK signaling, the combination of which modulates transient changes in the initiation and elongation phases of mRNA translation.     

Protein synthesis in germinating Saccharomyces cerevisiae ascospores

The uptake and incorporation of macromolecular precursors in germinating Saccharomyces cerevisiae ascospores were investigated. Addition of cycloheximide at various times during germination revealed that protein synthesis can occur within 20 min after the spores are shifted to glucose-containing media. The time of initiation of uptake and incorporation of several amino acids differed; this can be attributed to differing amino acid pool levels in the spores, as well as differing transport activities. Two-dimensional gel electrophoresis of proteins labeled with [35S]methionine for various 20-min periods after germination began showed at least one protein whose synthesis begins well after the bulk of the proteins.     

Quantitative phosphoproteome profiling of Wnt3a-mediated signaling network: indicating the involvement of ribonucleoside-diphosphate reductase M2 subunit phosphorylation at residue serine 20 in canonical Wnt signal transduction

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of beta-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling.     

Intraduodenal serotonin elicits non-propagating spike potentials in the small intestine of the rat

Serotonin (5-hydroxytryptamine, 5-HT) is an endogenous signalling molecule capable of altering small intestinal motility. Serotonin is normally present in the intestinal lumen and released by enterochromaffin cells of the mucosal epithelium. We found that intraduodenal infusion of exogenous serotonin causes a dose-dependent myoelectric response in the smooth muscle of the small intestine in the conscious rat. The response consists of repetitive bursts of action potentials (RBAP) that are characterized as short bursts of non-propagative myoelectric spiking. RBAP occur intermittently and only during the first 15 min after intralumenal serotonin infusion. After the initial 15 min period, the frequency of RBAP declines, and the myoelectric pattern shifts to prolonged and continuous spiking, eliminating the interdigestive migrating myoelectric pattern. The effects of intralumenal serotonin are not replicated by parenteral or intraperitoneal infusion nor by intralumenal infusion of 5-hydroxytryptophan or 5-hydroxyindoleacetic acid. The response to intralumenal serotonin was eliminated by several specific 5-HT receptor antagonists. On repeated intralumenal administration of serotonin, the RBAP response decreased demonstrating a decreased sensitivity of the muscle contraction on re-exposure to serotonin. We conclude that intralumenal infusion of serotonin can temporarily initiate specific small intestinal muscle events that are not generated by serotonin from other non-lumenal administration sites. We speculate that an afferent neuro-pathway is necessary for the induction of RBAP, since RBAP are not observed from in vitro muscle preparations.     

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.