A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.
(9/1209)
Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region. (+info)
Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica.
(10/1209)
Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release. (+info)
Separate effects of a classical conditioning procedure on respiratory pumping, swimming, and inking in Aplysia fasciata.
(11/1209)
We examined whether swimming and inking, two defensive responses in Aplysia fasciata, are facilitated by a classical conditioning procedure that has been shown to facilitate a third defensive response, respiratory pumping. Training consisted of pairing a head shock (UCS) with a modified seawater (85%, 120%, or pH 7.0 seawater--CSs). Animals were tested by re-exposing them to the same altered seawater 1 hr after the training. For all three altered seawaters, only respiratory pumping is specifically increased by conditioning. Swimming is sensitized by shock, and inking is unaffected by training, indicating that the conditioning procedure is likely to affect a neural site that differentially controls respiratory pumping. Additional observations also indicate that the three defensive responses are differentially regulated. First, different noxious stimuli preferentially elicit different defensive responses. Second, the three defensive responses are differentially affected by shock. Inking is elicited only immediately following shock, whereas swimming and respiratory pumping are facilitated for a period of time following the shock. Third, swimming and respiratory pumping are differentially affected by noxious stimuli that are delivered in open versus closed environments. These data confirm that neural pathways exist that allow Aplysia to modulate separately each of the three defensive behaviors that were examined. (+info)
Arrangement of radial actin bundles in the growth cone of Aplysia bag cell neurons shows the immediate past history of filopodial behavior.
(12/1209)
Filopodia that protrude forward from the lamellipodium, located at the leading edge of a neuronal growth cone, are needed to guide the extension of a nerve cell. At the core of each filopodium an actin bundle forms and grows into the lamellipodium. By using kymographs of time-lapse polarized light images we examined the relationship between the behavior of the filopodia, the actin bundles immediately proximal to the filopodia, and the shapes and composition of actin bundles in the whole lamellipodium. We find that the shapes of actin bundles, such as tilt, fork, and fused zones, originate at the leading edge and are surprisingly well preserved during retrograde transport of the actin cytoskeleton in the whole lamellipodium. The number of filaments that make up the radial actin bundles, as displayed by their birefringence retardation, also is preserved during retrograde flow over a distance of 4-8 microm from the leading edge into the lamellipodium. Thus, the disposition of the actin bundles in the lamellipodium frozen at any time point preserves and portrays a history of the past behavior of actin bundles proximal to the filopodia and the behavior of the filopodia themselves. These findings suggest that the arrangement of actin bundles in static image records, such as electron or fluorescence micrographs of fixed and stained specimens, can in fact reveal the sequence of the past history of filopodial behavior and the generation, density, fusion, etc. of the filaments in the actin bundles. (+info)
Expression and branch-specific export of mRNA are regulated by synapse formation and interaction with specific postsynaptic targets.
(13/1209)
Mechanosensory neurons (SNs) of Aplysia form synapses in culture with some targets (L7), but not others (L11), even when a SN is plated with both targets. We examined whether branch-specific net export of mRNA encoding synapse-specific molecules might contribute to branch-specific synapse formation. Single-cell RT-PCR was used to assay levels of mRNA encoding the SN-specific neuropeptide (sensorin A) and other transcripts in cell bodies and neuritic processes of SNs cultured alone or with synaptic targets. Some mRNAs are exported to neurites, but not others. Sensorin A mRNA is detected only in SN cell bodies and neurites, and expression levels correlate with the strength of the synaptic connections formed with L7 after 4 d in culture. After 4 d, more sensorin A transcripts are detected in SN neurites contacting L7 than in SN neurites contacting L11. The differential expression at 4 d is found even when a single SN contacts both targets simultaneously. By contrast, no significant difference in expression is detected in SN neurites contacting L7 versus L11 after 1 d of coculture. The results suggest that interaction and synapse formation with a specific target lead to a time-dependent change in the branch-specific accumulation of sensorin A mRNA in SNs. Because local protein synthesis at synaptic sites might contribute to synaptic function or plasticity, the results suggest that branch-specific targeting of mRNA encoding synapse-related molecules may contribute to the formation of specific synapses. (+info)
Properties of cholinergic responses in isolated parapodial muscle fibers of Aplysia.
(14/1209)
The parapodial neuromuscular junction in the marine snail Aplysia brasiliana is a model synapse for the investigation of neural modulation. The parapodial muscle fibers are innervated by cholinergic motoneurons and by serotonergic modulatory cells. The physiological properties of voltage-gated currents of the muscle membranes and the effects of serotonin on these currents have been published previously. However, the pharmacological properties of the cholinergic receptors have not been investigated. Acetylcholine (ACh) applied exogenously to dissociated muscle fibers produces a response with a reversal potential of about -52 mV; the resting membrane potential of the average muscle fiber is approximately -56 mV. ACh induces variable responses (depolarizations or hyperpolarizations) in individual cells, but the transmitter never causes a depolarization adequate to produce muscle contraction. We demonstrate that the ACh response is the result of the activation of two distinct receptors. One receptor is linked to a chloride channel and induces a hyperpolarization with a reversal potential near -70 mV. This receptor is activated selectively by suberyldicholine and by nicotine and is antagonized by curare but not by hexamethonium. The second response, presumably caused by increased conductance to mixed cations, results in muscle fiber depolarization with a reversal potential near -35 mV and does induce muscle contraction. This receptor is activated by methylcarbamylcholine and selectively blocked by hexamethonium; atypically, this receptor is not activated by nicotine nor by carbachol. The depolarizing, cation-selective receptors likely are associated with identified excitatory cholinergic motoneurons the activity of which typically results in muscle contractions because the reversal potential for this ACh response is more depolarized than the activation threshold for voltage-gated calcium channels in these fibers. The hyperpolarizing, chloride-selective receptors may be associated with inhibitory motoneurons; such motoneurons have yet to be identified, but their presence is inferred because of the occurrence of spontaneous inhibitory junctional potentials recording from muscle fibers in situ. Muscle fiber responses to exogenously applied ACh reflect the relative contribution of each receptor type in each muscle fiber. (+info)
Multiple memory processes following training that a food is inedible in Aplysia.
(15/1209)
In many organisms, memory after training can be separated into a number of processes. We now report that separable memory processes are also initiated by a training procedure affecting Aplysia feeding behavior, a model system for examining the neural mechanisms underlying the regulation of a complex behavior. Four distinct memory process were identified: (1) a very short-term memory that declines within 15 min, (2) a short-term memory that persists for 0.5-1.0 hr, (3) an intermediate-term memory, observed 4 hr after training, and (4) a long-term memory that is seen only after a 12- to 24-hr delay. The four memory processes can be distinguished by the different training procedures that are required to elicit them. A single 5-min training session is sufficient to elicit the very short-term memory. However, a longer training session that continues until the animal stops responding to food is needed to elicit short-term memory. Intermediate-term memory is observed only after a spaced training procedure (three 5-min training sessions separated by 30-min intervals). A single 5-min training session that does not cause either short-term or intermediate-term memory is sufficient to induce long-term memory, indicating that short- and long-term memory are independent, parallel processes. Short- and long-term memory can also be separated by the effects of a post-training experience. Long-term, but not short-term, memory can be attenuated by cooling animals immediately after training. Cooling before the training does not affect either the training or the subsequent short- or long-term memory. (+info)
Differential induction of long-term synaptic facilitation by spaced and massed applications of serotonin at sensory neuron synapses of Aplysia californica.
(16/1209)
Serotonin (5HT)-induced facilitation of synaptic transmission from tail sensory neurons (SNs) to motor neurons (MNs) in the marine mollusc Aplysia provides a cellular model of short- and long-term memory for behavioral sensitization of the tail withdrawal reflex. Synaptic facilitation at these synapses occurs in three temporal phases: short-term (STF, lasting minutes), intermediate-term (ITF, lasting more than an hour), and long-term (LTF, lasting >24 hr). STF, ITF, and LTF differ in their induction requirements: A single brief exposure of 5HT induces STF, whereas five applications are required for ITF and LTF. Moreover, STF and LTF can be induced independently. Different forms of memory often show differential sensitivity to the pattern of training trials. To begin to explore this effect at a cellular level, we examined ITF and LTF induced by one of two patterns of 5HT application: a spaced pattern (five 5-min exposures with an interval of 15 min) or a massed pattern (one continuous 25-min application). The spaced and massed patterns both induced ITF; however, spaced 5HT application was significantly more reliable at inducing LTF than was massed application. Thus, whereas induction of ITF and LTF require similar amounts of 5HT, the cellular mechanisms underlying the induction of LTF are more sensitive to the pattern of the induction trials. In the massed group, further analysis revealed a relationship between the expression of ITF and the subsequent expression of LTF, suggesting that these two processes may be mechanistically related. (+info)